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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.

Tire and road wear particles (TRWP) have been shown to represent a large part of anthropogenic particles released into the environment. Nevertheless, the potential ecological risk of TRWP in the different environmental compartments and their potential toxic impacts on terrestrial and aquatic organisms remain largely underinvestigated. Several heavy metals compose TRWP, including Zn, which is used as a catalyst during the vulcanization process of rubber. This study investigated the solubilization potential of metals from cryogenically milled tire tread (CMTT) and TRWP in simulated gastric fluids (SFGASTRIC) and simulated intestinal fluids (SFINTESTINAL) designed to mimic rainbow trout (Oncorhynchus mykiss) gastrointestinal conditions. Our results indicate that the solubilization of heavy metals was greatly enhanced by gastrointestinal fluids compared to that by mineral water. After a 26 hin vitrodigestion, 9.6 and 23.0% of total Zn content of CMTT and TRWP, respectively, were solubilized into the simulated gastrointestinal fluids. Coingestion of tire particles (performed with CMTT only) and surrogate prey items (Gammarus pulex) demonstrated that the animal organic matter reduced the amount of bioavailable Zn solubilized from CMTT. Contrastingly, in the coingestion scenario with vegetal organic matter (Lemna minor), high quantities of Zn were solubilized fromL. minorand cumulated with Zn solubilized from CMTT., Environmental Science & Technology, 55 (23), ISSN:0013-936X, ISSN:1520-5851

International audience; Surface sediments can accumulate contaminants that affect microorganisms and invertebrates and disturb benthic ecological functions. However, effects of contaminants on ecological functions supported by sediment communities are understudied. Here, we tested the relevance of two simple tools to assess the ecotoxicological effects of metal contamination on natural sediment communities using particulate organic matter breakdown and decomposition as a functional descriptor. To this aim, we performed a 21-day laboratory microcosm experiment to assess the individual and combined effects of Cu and As (nominal concentration of 40 mg kg−1 dw each) using the bait-lamina method (cellulose, bran flakes, and active coal in PVC strips) as well as artificial tablets (cellulose, bran flakes and active coal embedded in an agar matrix). Sediment toxicity was also evaluated using the standardized ostracod toxicity test. Both the bait-lamina and artificial tablet methods showed low effects of As on organic matter breakdown and decomposition but strong effects of Cu on this important ecological function. Both also showed that the presence of Cu and As in mixture in the sediment induced total inhibition of organic matter breakdown and decomposition. The ostracod toxicity test also showed high toxicity of Cu-spiked and Cu-plus-As-spiked sediments and low toxicity of As-spiked sediments. Besides confirming that artificial organic matter substrates are relevant and useful for assessing the functional effects of contaminants on sediment micro-and macro-organism communities, these results suggest that the proposed methods offer promising perspectives for developing tools for use in assessing functional ecotoxicology in the sediment compartment.

A preparatory study was performed to develop a sediment quality and risk assessment strategy for Switzerland, addressing the following questions: the sediment fraction to be analyzed chemically (

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)

The authors would like to correct the published article [...]

Chromium is widely used as a tanning agent and can become a contaminant of concern in aquatic ecosystems receiving discharges from industrial or artisanal tanning activities. In a companion study, we showed that Cr discharged by tanneries was bioavailable to indigenous chironomids with accumulation via sediment ingestion likely to represent the predominant exposure route. However, Cr accumulation by chironomids did not directly reflect the degree of sediment contamination and the potential adverse effects of Cr accumulation on chironomids were not evaluated. In the present study, chironomids were exposed to homogenised, field-collected sediments in the laboratory and to intact sediments in situ using a customized caging system. Chromium concentrations were assessed in sediments, exposed larvae of laboratory-reared Chironomus riparius and overlying waters of in situ cages. Experimental results of Cr bioaccumulation were compared with expected Cr body burden in chironomids calculated using biodynamic modelling. Our data provided strong support to the hypothesis that Cr bioaccumulation in the field is specifically controlled by the deposition of contaminated suspended particulate matter (SPM) containing a pool of Cr readily bioavailable to surface deposit feeders. Considering freshly deposited SPM as an additional route of exposure for surface deposit feeders leads to a good agreement between the modelling and experimental results. Additionally, a Cr body burden of about 77 mu g g(-1) d.w. was identified as a tentative threshold above which effects on the growth of C. riparius may appear. While both laboratory and in situ experiments provide evidence for the availability of Cr in aquatic systemimpacted by tannery wastewaters, standard laboratory exposure conditions may miss additional exposure routes in the field and underestimate possible adverse effects on benthic organisms. (c) 2018 Elsevier B.V. All rights reserved.

An ever-increasing diversity of potentially toxic chemical compounds are being developed and released into the environment as a result of human activities (e.g. agriculture, drugs, and cosmetics). Among these, pesticides have been shown to affect non-targeted wildlife since the 1960s. A range of ecotoxicological tests are used to assess the toxicity of pesticides on various model organisms. However most model organisms are metazoans, while the majority of Eukaryotes are unicellular microorganisms known as protists. Protists are ubiquitous organisms of key functional roles in all ecosystems but are so far little studied with respect to pesticide impact. To fill this gap, we developed a new ecotoxicological test based on Euglypha rotunda, a common soil amoeba, grown in culture flask with Escherichia coli as sole food source. We tested this assay with the herbicide S-metolachlor, which is known to affect cell division in seedling shoots and roots of weeds. Reproducible growth conditions were obtained for E. rotunda. The growth of E. coli was not affected by the herbicide. The growth of E. rotunda was affected by the herbicide in a non-linear way, growth being significantly reduced at ca. 15 μg/L, but not at 150 μg/L. Our results show the potential for using soil protists in ecotoxicology and adds to the growing body of evidence for non-linear impacts of pesticides on non-target organisms. With the acquisition of additional data, the protocol should be suitable for standard ecotoxicological tests.

In Switzerland, surface waters are protected by the Swiss Water Protection Ordinance (OEaux; OFEV 1998), which stipulates that the water quality shall be such that the water, suspended matter, and sediments contain no persistent synthetic substances to ensure the protection of aquatic life. Local agencies are in charge of water quality monitoring, using a set of validated methods. Several lists of priority substances have been developed for aquatic microcontaminants for surface water monitoring but not for sediments. Some local agencies have established sediment monitoring programs, but to date, there exists no harmonized methodology for sediment quality assessment in Switzerland. Within the main goal of developing and providing methodologies for monitoring sediment quality in Switzerland, a screening was performed to help prioritize sediment-relevant microcontaminants. The screening approach was largely based on the NORMAN (network of reference laboratories, research centers, and related organizations for monitoring emerging environmental substances) system and was carried out in four steps: (1) identification of candidate substances, (2) selection of sediment relevant substances, (3) classification of substances into different categories based on identified data gaps and envisaged actions, and (4) ranking within each action category. This paper describes the methodology used in the prioritization process for sediment-relevant substances and provides recommendations for monitoring strategies in Switzerland.

International audience; In freshwater environments, microbial assemblages attached to submerged substrates play an essential role in ecosystem processes such as primary production, supported by periphyton, or organic matter decomposition, supported by microbial communities attached to leaf litter or sediments. These microbial assemblages, also called biofilms, are not only involved in nutrients fluxes but also in contaminants dynamics. Biofilms can accumulate metals and organic contaminants transported by the water flow and/or adsorbed onto substrates. Furthermore, due to their high metabolic activity and their role in aquatic food webs, microbial biofilms are also likely to influence contaminant fate in aquatic ecosystems. In this review, we provide (1) a critical overview of the analytical methods currently in use for detecting and quantifying metals and organic micropollutants in microbial biofilms attached to benthic substrata (rocks, sediments, leaf litter); (2) a review of the distribution of those contaminants within aquatic biofilms and the role of these benthic microbial communities in contaminant fate; (3) a set of future challenges concerning the role of biofilms in contaminant accumulation and trophic transfers in the aquatic food web. This literature review highlighted that most knowledge on the interaction between biofilm and contaminants is focused on contaminants dynamics in periphyton while technical limitations are still preventing a thorough estimation of contaminants accumulation in biofilms attached to leaf litter or sediments. In addition, microbial biofilms represent an important food resource in freshwater ecosystems, yet their role in dietary contaminant exposure has been neglected for a long time, and the importance of biofilms in trophic transfer of contaminants is still understudied.

Aquatic oligochaete communities are valuable indicators of the biological quality of sediments in streams and lakes, but identification of specimens to the species level based on morphological features requires solid expertise in taxonomy and is possible only for a fraction of specimens present in a sample. The identification of aquatic oligochaetes using DNA barcodes would facilitate their use in biomonitoring and allow a wider use of this taxonomic group for ecological diagnoses. Previous approaches based on DNA metabarcoding of samples composed of total sediments or pools of specimens have been proposed for assessing the biological quality of ecosystems, but such methods do not provide precise information on species abundance, which limits the value of resulting ecological diagnoses. Here, we tested how a DNA barcoding approach based on high-throughput sequencing of sorted and genetically tagged specimens performed to assess oligochaete species diversity and abundance and the biological quality of sediments in streams and lakes. We applied both molecular and morphological approaches at 13 sites in Swiss streams and at 7 sites in Lake Geneva. We genetically identified 33 or 66 specimens per site. For both approaches, we used the same index calculations. We found that the ecological diagnoses derived from the genetic approach matched well with those of the morphological approach and that the genetic identification of only 33 specimens per site provided enough ecological information for correctly estimating the biological quality of sediments in streams and lakes.

Waste materials from coal mining and consumer products can pose significant risks to the environment. Residual coal deposits lead to the formation of acid drainage and release of contaminants, causing negative changes in soil and aquatic systems. Low density polyethylene (LDPE) polymers are an environmental concern due to their high useage, and slow degradation in the environment. In this study both waste materials were used to develop a composite to mitigate the environmental impacts of coal mining waste (CMW). The composite material was produced in different formulations (0–80 % wt CMW), and samples were tested for formation of acid drainage and release of contaminants. Chemical characterisation of the CMW and leachate of the composite materials was performed by X-ray fluorescence and atomic absorption spectrometry. Ecotoxicological effects in soil and water were investigated using standard tests with the earthworm, Eisenia fetida, the collembolan, Folsomia candida and the bacterium, Aliivibrio fischeri. Composites with 20 % wt LDPE showed a 50% increase in the pH value of the leachate compared to the CMW leachate. Iron, aluminium and sulfate concentrations were lower in leachates of the composite materials, and a reduction in the ecotoxicological impact on the tested organisms was observed. The hydrophobic nature of the composite's polymeric matrix as well its physical properties contributed to a better coating of the coal residue particles, blocking the contact with water and reducing the environmental risks of CMW. These results show that the production of composite material is a viable alternative route for treating coal and LDPE waste.

Gammarids were exposed to sediments from a deposition site located on the Rhône River (France) downstream of a fluoropolymer manufacturing plant. Gammarids accumulated to various extents four long-chain perfluoroalkyl carboxylic acids (PFCAs) from C9 to C13, one sulfonate, perfluorooctane sulfonate (PFOS) and three of its precursors (the perflurooctane sulfonamide (FOSA), the N-methyl perfluorooctane sulfonamidoacetic acid (MeFOSAA), the N-ethyl perfluorooctane sulfonamidoacetic acid (EtFOSAA) and the 6:2 fluorotelomer sulfonic acid (6:2 FTSA). Whatever the compound, the steady state was not achieved after a 3-week exposure; elimination was almost complete after a 3-week depuration period for perfluorononanoic acid (PFNA), PFOS, the three precursors and the 6:2FTSA. However, this was not the case for long-chain PFCAs, whose elimination rates decreased with increasing chain length. PFAS accumulation in gammarids occurred via the trophic and respiratory pathways, in proportions varying with the carbon chain length and the terminal moiety.

Aquatic oligochaetes are important bioindicators of sediment quality in watercourses and lakes, but their morphological identification to the species level is challenging and sometimes impossible. The use of DNA barcoding and metabarcoding could greatly facilitate the identifications of specimens and improve ecological diagnoses based on oligochaete communities. The aim of this study was to test how well metabarcoding approaches based on high throughout sequencing (HTS) could perform in assessing oligochaete species diversity and the biological quality of sediments. We analysed oligochaete communities at several sites in Swiss rivers, comparing morphological data to metabarcoding of mixed specimen, sieved sediment and total sediment samples. We amplified the cytochrome c oxidase (COI) marker using universal primers and/or specific primers to metazoans. Our results showed that metabarcoding analysis of mixed specimens allowed a more representative assessment of the biodiversity than the metabarcoding of sieved or total sediments. Although community structures obtained with the morphological and metabarcoding analyses were different in term of presence/absence of species and species abundances, the ecological diagnoses based on these two approaches largely agreed.

The tanning industry uses large quantities of Cr whose contribution to the contaminant burden of aquatic organismsis not yet fully understood. The present study investigated Cr bioaccumulation by indigenous chironomids in a freshwater ecosystem impacted by tannery effluents. Total Cr content in sediments and in chironomidswas determined on several occasions. Chromium distribution among sediments and pore waters, and Cr speciation in overlying and pore waters were studied in detail to understand possible factors controlling Cr bioavailability to chironomids. Total chromium concentration ranged from 69 to over 3000 mu g g(-1) dry weight in sediments and fromnegligible to over 300 mu g g(-1) dryweight in chironomids (values corrected for sediment gut content). Filterable (

International audience; The occurrence at different trophic levels of 17 poly- and perfluoroalkyl substances (PFASs), including perfluoroalkyl acids (PFAAs) and some of their precursors (e.g., perfluoroalkane sulfonamides, 6:2 fluorotelomer sulfonate), were investigated in riverine freshwater food webs in Southeastern France. Two fish species (Barbus barbus and Squalius cephalus) and various invertebrate taxa were collected at five locations to assess the spatial variability of trophic magnification factors (TMFs). Particular attention was devoted to sample and data processing to minimize potential biases associated with the TMF determination. Fish were significantly more contaminated than invertebrates (ΣPFAS = 7-1811 vs. 0.9-213 ng g-1 wet weight (ww)). Those from the Rhône River presented significantly higher levels due to high concentrations of perfluoroundecanoic acid (406 ng g-1 ww) and perfluorotridecanoic acid (566 ng g 1 ww) ascribed to an industrial point source. Perfluorooctane sulfonate (PFOS) was dominant at the other sites (range concentration = 3.6-134 ng g-1 ww). Two linear regression models were compared (i.e., Kendall regression vs. Generalized Linear Mixed-Effect Model, GLMM). Results showed that TMFs calculated using the non-weighted Kendall regression were higher than those obtained using the GLMM approach. GLMM-based TMFs were consistently > 1 for C9-C14 perfluorocarboxylic acids (PFCAs), PFOS and perfluorodecane sulfonate (PFDS), indicating their apparent biomagnification in the investigated food webs. Comparatively, 6:2 FTSA and N-ethylperfluorooctane sulfonamidoacetic acid (N EtFOSAA) were less often detected and were not significantly biomagnified, probably because of metabolization. TMF estimates were generally consistent across sites although some PFASs (in particular C9, C10 and C13 PFCAs) displayed higher variability, due to a unique extreme value that may have resulted from the contribution of unattributed precursor biotransformation.

Aquatic oligochaetes, comprising a large number of species showing various degrees of resistance to chemical pollution, are recognized as valuable bioindicators of sediments&rsquo, quality. In the Geneva area (Switzerland), oligochaete tools were previously tested for assessing the biological quality of stream sediments, and effect thresholds of combined metals (quotients) in sediments were defined. The aims of the present study were to update this previous work with new data acquired in different cantons of Switzerland and to establish effect thresholds on oligochaete communities for individual metals and for combined metals. The oligochaete metrics &ldquo, Oligochaete index of sediment bioindication (IOBS)&rdquo, &ldquo, oligochaete density&rdquo, and &ldquo, percentage of tubificids without hair setae&rdquo, proved pertinent for assessing the effects of metals and organic matter in sediments. We established a threshold effect level (TELoligo) and probable effect level (PELoligo) for eight metals in sediments (Cr, Ni, Zn, Cu, Pb, Cd, Hg and As) as well as a probable effect level for these metals combined (mPELoligo-Q). These thresholds could be used directly to screen for alteration of in situ communities restricted to sediments and/or for establishing sediment quality standards based on a combination of different biological and ecotoxicological tools.

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.

The fate of two trivalent chromium salts (nitrate and chloride) in ISO algal culture medium was followed over 72 h; i.e., the typical duration of algal toxicity tests. Fifty percent of the initial Cr spikes was lost from the solutions by 24 h, with losses up to 90% after 72 h. Monitoring of the temporal variability of Cr(III) concentrations in algal culture media appears necessary to better characterize the toxicity of trivalent chromium to algae.

The aims of this study were to determine depuration rates for a range of per- and polyfluoroalkyl substances (PFASs) using Chironomus riparius, and to test a concentration-dependency hypothesis for the long-chain perfluorotridecanoic acid (PFTrDA) for this species. Midge larvae were exposed to field sediments collected downstream of a fluorotelomer plant, and to the same sediment spiked with PFTrDA. Elimination kinetics results indicated complete elimination of all PFASs by chironomids after 42 h. These data were used to develop two PFTrDA bioaccumulation models accounting for chironomid growth and for compound concentration dependency or not. There was much better agreement between observed and simulated data under the concentration-dependency hypothesis than under the alternative one (passive diffusion). The PFTrDA uptake rate derived from the concentration-dependency model equaled 0.013 ± 0.008 goc gww h−1, and the depuration rate 0.032 ± 0.009 h−1.

The first French-Swiss workshop on ecotoxicology of freshwater sediment communities was co-organized by the French Research Institute of Science and Technology for Environment and Agriculture (Irstea) and the Swiss Centre for Applied Ecotoxicology (Ecotox Centre EAWAG-EPFL) in Villie-Morgon (Beaujolais Region, France) on April 27–28, 2017. The workshop brought together scientists working in different fields of expertise (ecotoxicologists, ecologists, environmental chemists…), environmental stakeholder groups and managers, as well as economic players (start-ups and consultancies) to better connect research needs of potential end-users with research outputs. The objectives of this workshop were (i) to establish the state of the art of research in the characterization of sediment contamination and in the evaluation of the effects on sediment-associated biological communities and ecosystem functioning and (ii) to give an overview of the French and Swiss regulations dealing with the assessment of contaminated sediments in freshwater ecosystems. The ultimate goal was to collectively identify research needs and knowledge gaps, as well as to highlight ways to improve the ecotoxicological assessment of sediments in freshwater environments by further considering the structure and functions of associated microbial and invertebrate communities.

WOS:000392786400024; International audience; Further progress in the development of reliable biomonitoring strategies requires to better link effects in aquatic ecological systems to ambient concentrations of chemical contaminants. Among existing tools, in situ bioassays using caging method represent an interesting way to achieve this challenge. However, elaboration of adapted exposure chambers and suitable operating procedures is still required, particularly to assess ecological relevant traits such as those related to the reproduction. In such context, we developed a new device (Emergence board E-Board) which allows assessing in rivers the development of the Chironpmus riparius species from the early fourth instar larvae to the adult stage. The system acts as a suspended matter trap floating in the subsurface of the water equipped of an emergence trap for catching adults. The system was tested in actual field conditions. Its easy handling allowed obtaining data which demonstrated its applicability for assessing the development of the chironomids. Moreover, by adapting energy-based models (DEB) specifically developed in the laboratory for the species C. riparius, we were able to predict the growth pattern and the emergence of chironomids in real environmental conditions. The E-Board represents thus a promising new in situ tool in perspective of evaluation of the quality of the ecosystems.

Introduction Aquatic oligochaetes represent valuable indicators of the quality of sediments of watercourses and lakes, but their difficult identification based on morphological criteria compromises their more common use for eco-diagnostic analyses. This issue could be overcome by using DNA barcodes for species identification. A 10% threshold of cytochrome c oxidase (COI) divergence was proposed for differentiating between oligochaete species based on molecular and morphological data. A Swiss database of COI sequences of aquatic oligochaetes was initiated in 2012. The aim of this study is to complement the Swiss oligochaete database of COI sequences and to confirm the relevance of this threshold for species delimitation. Methods We sequenced the COI sequence of 216 specimens collected in different regions of Switzerland and ITS2 region of some lineages whose delimitation with COI data was doubtful. Results We distinguished 53 lineages, among which 34 were new for Switzerland and 17 sequenced for the first time. All the lineages were separated by more than 10% of COI variation, with the exception of some species within Nais and Uncinais. In these two genera, the threshold was lowered to 8% to be congruent with the morphological analysis. The total number of lineages reported so far for Switzerland is 75, including 59 morphospecies or unidentified species and 16 cryptic species. Discussion Our study shows that the threshold of 10% of COI divergence is generally appropriate to distinguish aquatic oligochaete lineages, but that it must be adjusted for some species. The database reported here will be complemented in the future in parallel to the development of genetic oligochaete indices.

Pools of aquatic plants and benthic invertebrates were collected along with 47 individuals from three cyprinid fish species (Barbus barbus, Gobio gobio, Rutilus rutilus) at a site in the Rhone River (France). Carbon and nitrogen isotopic ratios (delta C-13 and delta N-15) and a wide range of per-and poly-fluorinated chemicals (PFASs) were analysed in all samples. The sum of PFAS concentrations (Sigma PFAS) increased from aquatic plants to fish dorsal muscles; molecular profiles were dominated by C9-C13 perfluorocarboxylic acids (PFCAs), while perfluorooctane sulfonate (PFOS) and perfluorooctane sulfonamide (FOSA) were detected in all samples at lower concentrations. SPFAS and especially Sigma PFCAs were higher in barbels (B. barbus) than in other species, while roaches (R. rutilus) were less contaminated by PFOS than barbels and gudgeons (G. gobio). Gudgeons accumulated significantly higher FOSA concentrations. Young (small) barbels displayed significantly higher PFOS, perfluorononanoic acid (PFNA) and perfluorodecanoic acid (PFDA) concentrations than did large specimens; conversely, perfluorotetradecanoic acid (PFTeDA) concentrations were significantly higher in large barbels. Multiple linear regressions were performed on the whole set of fish samples with size, mass and isotopic ratios as explicative variables, and several single compounds as explained variables. Regardless of the compound, the regressions did not explain much of the contamination variability. However, adding species as a qualitative variable, i.e. performing analyses of covariance (ANCOVAs) improved the fit greatly, while adding sex did not. Diet (i.e. delta C-13 and delta N-15) was the main factor explaining interspecific differences. Biotransformation was assessed by comparing concentration ratios of PFOS or FOSA to their precursors in the food-web compartments. These ratios increased from invertebrates to fish, and differed among fish species, suggesting that biotransformation occurred but was species-specific. Biomagnification factor calculations showed that C11-C13 PFCAs, PFOS and FOSA were apparently biomagnified in barbels and gudgeons. (C) 2017 Elsevier B.V. All rights reserved.

Dredging generates remobilisation of sediments contaminated by non-degradable compounds such as metals, to which aquatic organisms can be exposed. This study aims at assessing the environmental impact of sediments remobilised in the Rhine River (France) during the dredging of Marckolsheim dam by pumping/dilution in 2013 on metal speciation and organisms' exposure. The monitoring coupling chemical and biological tools was performed 2 years before dredging operation on 2 sampling sites, upstream and downstream from the discharge of pumping/dilution, to acquire data on the natural variability of labile (DGT as passive samplers), dissolved and particulate concentrations of Cd, Co, Cr, Cu, Ni, Mn, Pb and Zn in Rhine during full hydrological cycles. In parallel, size-calibrated zebra mussels were transplanted at both sites to monitor continuously metal bioavailability from particulate and dissolved fractions. This long-term monitoring allowed the establishment of reference baselines of Rhine water and mussels' contamination levels and subsequently, the detection of averred environmental changes due to the dredging. Indeed, Co and Mn accumulations in mussels exposed to the discharge were consistent with increasing labile species in Rhine whereas ones of Cr and Pb were likely due to an enhanced particulate bioavailability. Whatever the exposure route, the mussels recovered their basal metal contents 2 weeks after the end of dredging, suggesting a transient impact of sediment remobilisation on bioaccumulation. This long-term monitoring highlights the interest of coupling chemical and biological time-integrated tools for a better assessment of environmental risks because metallic exchanges between organisms and their media are complex and metal-specific., Une opération de dragage engendre la remobilisation de sédiments contaminés par des composés non dégradables tels que les métaux, auxquels les organismes aquatiques peuvent être exposés. Cette étude vise à évaluer l'impact environnemental de la remobilisation de sédiments chargés en métaux dans le Rhin lors du dragage du barrage de Marckolsheim par pompage/dilution en 2013 sur la spéciation et l'exposition des organismes aux métaux. La surveillance chimique et biologique a été effectuée 2 ans avant l'opération de dragage sur 2 sites d'échantillonnage, en amont et en aval de la zone de rejet, pour acquérir des données sur l'évolution naturelle des concentrations labiles, dissoute et particulaires en Cd, Co, Cr, Cu, Ni, Mn, Pb et en Zn dans le Rhin pendant des cycles hydrologiques complets. En parallèle, des moules zébrées calibrées ont été transplantées sur les deux sites pour surveiller la biodisponibilité particulaire et dissoute des métaux. Cette surveillance à long terme a permis d'établir des niveaux de contamination de référence du Rhin et des moules, et ainsi de détecter des changements environnementaux dus au dragage. En effet, les accumulations de Co et de Mn dans les moules exposées au rejet concordaient avec l'augmentation de leurs formes labiles dans l'eau du Rhin alors que celles de Cr et Pb étaient probablement liées à une biodisponibilité particulaire favorisée par la remobilisation de sédiments. Quelle que soit la voie d'exposition, les moules ont récupéré leur teneur basale en métaux 2 semaines après la fin du dragage, suggérant un impact transitoire de la remobilisation des sédiments sur la bioaccumulation. Cette surveillance à long terme met aussi en évidence l'intérêt de coupler des outils chimiques et biologiques pour une meilleure évaluation des risques environnementaux car les échanges métalliques entre les organismes et leurs milieux sont complexes et dépendent du métal considéré.

Background Oligochaetes are valuable bioindicators of the quality of watercourse and lake sediments. The morphological identification of aquatic oligochaetes is difficult, prompting the development of new molecular oligochaete indices based on DNA barcoding and Next-generation sequencing of sorted specimens. In general, the samples for DNA barcoding are fixed in absolute ethanol. However, in the case of aquatic oligochaetes, this medium is not appropriate as it can induce a modification of specimen abundances and of the composition of communities. Therefore, we investigated the possibility to amplify and sequence aquatic oligochaetes fixed in formalin for a short time. We performed guanidine extraction and polymerase chain reaction (PCR) amplification/sequencing of the cytochrome c oxydase I (COI) gene on tissue fragments fixed in formalin for different periods of time (from 1 h to 1 week) and in ethanol. Results The large majority of aquatic oligochaete specimens fixed in formalin for up to 1 week could be successfully amplified and all obtained sequences were of high quality. The amplification and sequencing success rate of formalin-fixed samples and ethanol-fixed samples was similar. These results suggest that formalin fixation of aquatic oligochaete tissues for a short time does not cause serious damages to DNA and inhibit PCR amplification. Conclusion The possibility to fix aquatic oligochaetes with formalin before genetic analyses is very promising for diversity monitoring, for construction of a comprehensive DNA barcode library and for development of an index based on Next-generation sequencing analysis of samples composed of sorted specimens. Electronic supplementary material The online version of this article (doi:10.1186/s13104-016-2140-1) contains supplementary material, which is available to authorized users.

International audience; Human activities can disturb the natural dynamics of exchanges between surface water and groundwater in rivers. Such exchanges contribute to the self-purification of the environment and an excess of infiltration can lead to contamination of groundwater. In addition, the porous matrix (coarse surface sediments and hyporheic zone), through which water exchanges occur, is a sink for pollutants. For environmental monitoring programs, it is therefore essential to take into account both the dynamics of vertical hydrological exchanges and the biological quality of this matrix. The functional trait (FTR) method, which is based on the study of oligochaete communities in coarse surface sediments and the hyporheic zone, was proposed as a tool to simultaneously assess the dynamics of vertical hydrological exchanges and the effects of pollutants present in the porous matrix. Here, we applied this method during two different periods (in March and September 2016), upstream and downstream of locations affected by discharges from wastewater treatment plants (WWTP) located in Switzerland. The biological quality of surface sediments and the hyporheic zone was shown to be better upstream of the WWTP in both campaigns. In addition, results suggested that the capacity for self-purification was lower downstream of the WWTP, and that groundwater at these locations was vulnerable to pollution by surface water. The FTR method proved valuable as a field method for detecting the effects of point source contamination on receiving streams. In the near future, this community-based approach will benefit from advances in the use of DNA barcodes for oligochaete species identification.

Along with the growing body of evidence that total internal concentration is not a good indicator of toxicity, the Critical Body Residue (CBR) approach recently evolved into the Tissue Residue Approach (TRA) which considers the biologically active portion of metal that is available to contribute to the toxicity at sites of toxic action. For that purpose, we examined total mercury (Hg) bioaccumulation and subcellular fractionation kinetics in fourth stage larvae of the midge Chironomus riparius during a four-day laboratory exposure to Hg-spiked sediments and water. The debris (including exoskeleton, gut contents and cellular debris), granule and organelle fractions accounted only for about 10% of the Hg taken up, whereas Hg concentrations in the entire cytosolic fraction rapidly increased to approach steady-state. Within this fraction, Hg compartmentalization to metallothionein-like proteins (MTLP) and heat-sensitive proteins (HSP), consisting mostly of enzymes, was assessed in a comparative manner by two methodologies based on heat-treatment and centrifugation (HT&C method) or size exclusion chromatography separation (SECS method). The low Hg recoveries obtained with the HT&C method prevented accurate analysis of the cytosolic Hg fractionation by this approach. According to the SECS methodology, the Hg-bound MTLP fraction increased linearly over the exposure duration and sequestered a third of the Hg flux entering the cytosol. In contrast, the HSP fraction progressively saturated leading to Hg excretion and physiological impairments. This work highlights several methodological and biological aspects to improve our understanding of Hg toxicological bioavailability in aquatic invertebrates. (C) 2015 Elsevier B.V. All rights reserved.

International audience; Sediments are an essential component of fluvial ecosystems. They are also quite often a cause of disturbance for fluvial works, such as dams or locks. In order to manage these disturbances without causing adverse impacts downstream, managers should have in hand an efficient assessment framework. The DIESE project (tools for sediment ecotoxicity assessment) aimed to elaborate and test a tiered assessment framework for freshwater sediment sites. We present hereafter the proposed framework, and a pilot test on 17 sites. Basically, the framework intends to provide answers to 2 questions, namely (a) does the sediment contamination present a hazard to benthic invertebrates, and (b) if bioaccumulative/biomagnified contaminants are present, is their concentrations worrying? The assessment process can end at any tier, provided the data are conclusive. Sediment quality benchmarks or contamination indices were used to interpret chemical analyses; this assessment was then compared to the results of ecotoxicity tests. Overall, the framework appears consistent; this test also helped selecting the best options for interpretation. Nevertheless, the test included only 17 sites, with a limited array of contaminants; complementary tests including more organic contaminants at a wider range of concentrations would help to demonstrate the validity of this assessment framework.; Composante essentielle des écosystèmes fluviaux, les sédiments constituent aussi souvent une source de perturbations des ouvrages tels que barrages ou écluses. Pour gérer ces perturbations sans entrainer de dommages à l'environnement, les maitres d'ouvrage concernés doivent donc disposer d'une démarche d'évaluation écotoxicologique. Le projet DIESE (Outils de DIagnostic de l'Ecotoxicité des SEdiments) avait pour objectifs d'élaborer et tester un schéma d'évaluation par étapes pour des sites d'accumulation de sédiments. Nous présentons ici la démarche proposée, structurée en 3 étapes, et les résultats d'un test de celle-ci sur 17 sites. Il s'agit de répondre à deux questions : « les contaminants présents dans les sédiments (a) présentent-ils un danger probable pour les invertébrés benthiques (effet toxique direct), ou (b) ceux qui sont reconnus comme bioaccumulables ou bioamplifiables sont-ils présents à un niveau préoccupant » ? Chaque étape peut se conclure soit par l'arrêt de l'évaluation, si les données obtenues sont concluantes. La confrontation des résultats d'analyses chimiques interprétés à l'aide de seuils de toxicité ou d'indices de contamination aux résultats des tests de toxicité a permis d'illustrer la validité de la démarche, et de proposer des choix pour l'interprétation (entre indices ou classification sur la base du paramètre le plus pénalisant). Ce test a permis de montrer une bonne cohérence d'ensemble des éléments de preuve de toxicité rassemblés pour les 17 sites. Ce résultat mériterait d'être vérifié sur un plus grand nombre de sites, incluant des substances organiques plus variées et atteignant des niveaux de contamination plus élevés.

Environmental managers need suitable technological methods to use in optimization studies to improve management of hazardous waste. One of the challenges to achieving a reliable hazardous waste classification is the improvement of procedures used forthe ecotoxicological characterization of solid waste leachates. Indeed, this step requires data that meet levels of acceptable quality if scientifically based decisions are to be made. In this study, we illustrate how the variability associated with the successive steps of a procedure used to assess ecotoxicological hazard of solid waste (i.e., primary sampling, laboratory sampling, toxicity measurements) can contribute to the overall variability of the ecotoxicity results. To this end, a municipal solid waste incinerator bottom ash and a slag from a second smelting of lead were studied using a nested experimental design. The results showed that the waste sampling design is of major importance for limiting the final variability of toxicity test parameters. At the opposite, increasing the number of replicates at the toxicity test level has negligible impact on this variability. Our approach could be of great practical interest in ecotoxicological studies not only for ensuring a safe classification for these materials, but also for improving sampling protocols and facilitating less time-consuming and less expensive ecotoxicological evaluations.

Triplicate aqueous leachates of a municipal solid waste incineration bottom ash (MSWIBA) were produced according to a European standardised method. Leachates analysis showed relatively low concentrations (less than 1 mgl 1 ) for four metals (iron, cadmium, lead and copper). No mutagenic activity was revealed after performing the Salmonella/microsome assay with and without microsomal activation. With the Vicia root tip micronucleus assay, a significant increase in micronucleated cells was observed between 3.4% and 100% leachate concentration. Significant and elevated antioxidant stress enzyme activities, e.g., superoxide dismutase (SOD), catalase (CAT), peroxidase (PER) and glutathione reductase (GR), were detected in Vicia root tissues even at the lowest tested leachate concentration (i.e., 0.3%), whereas this was not always the case in leaf tissues, which showed tissue specificity for the tested enzymes. At the lowest concentration (i.e., 0.3%), a higher increase was observed (respectively 197% and 45% compared to the control) for root glutathione reductase and peroxidase activities over those of other enzymes (superoxide dismutase and catalase). Our results suggest that MSWIBA aqueous leachates need to be formally tested with genotoxic sensitive tests before recycling and support the hypothesis that plant genotoxicity is related to the cellular production of reactive oxygen species (ROS). D 2004 Elsevier B.V. All rights reserved.

In four countries (France, Greece, Italy, and Sweden) occurrence in sewage treatment plant (STP) effluents and ecotoxicity of the pharmaceuticals carbamazepine, clofibric acid, and diclofenac were investigated. Bioassays were performed on bacteria, algae, microcrustaceans, and fishes in order to calculate their predicted no-effect concentrations (PNEC) and to perform a first approach of risk characterization. For this aim, risk has been estimated by the predicted environmental concentration/PNEC ratio and the measured environmental concentration/PNEC ratio. First, regarding the PNEC, carbamazepine appears to be the more hazardous compound. Second, even though it is demonstrated that carbamazepine, clofibric acid, and diclofenac have been detected in effluents, only carbamazepine have been detected in all sewage treatment plants with the greatest concentrations. Third, risk quotients greater than unity were calculated only for carbamazepine, suggesting that risk for the water compartment is expected.

(2008). Bioavailability of sedimentary chromium for chironomids in the upper Dunajec River (Southern Poland) SIL Proceedings, 1922-2010: Vol. 30, No. 4, pp. 534-536.

International audience; Effective assessment of sediment quality and associated risks requires further integration between laboratory-based studies and field observations. Tools for in situ exposure of laboratory-reared organisms are particularly suitable for this purpose. However, available tools suffer from technical drawbacks that limit their use in deep waters and their general ability to provide results linking laboratory and field observations. To overcome these limitations, we developed a new device (Sediment-Water Interface Study System - S-WISS1) for in situ exposure of fourth instar larvae of Chironomus riparius at depths up to 18 m. S-WISS1 allows minimum alteration of the sediment natural structure (upon both deployment and recovery) and, in a way analogous to laboratory tests, repeated delivery of food doses to the in situ exposed organisms. Results obtained in situ with S-WISS1 were compared with standard laboratory tests and simple caging systems (large-mouthed bottles partly filled with sand) used as field controlks. A first series of experiments (48 h, no food addition) showed that element bioavailability to chironomids was higher in laboratory settings than on-site or in situ. A second series of experiments (96 h, food addition every 24 h) confirmed that sediments rather than overlying waters acted as the principal source of contaminants and that exposure to field sediments reduced chironomids' growth compared with on-site controls. S-WISS1 represents a new family of in situ tools capable of combining the environmental realism associated with field monitoring with the controlled experimental conditions typical of laboratory testing.

This article is a revision of the previous edition article by Pawel J. Migula, volume 2, pp 133–137, © 2005, Elsevier Inc.

Midge larvae (Chironomus riparius) were exposed to sediments from a deposition sampled at a site along the Rhône River (France) downstream of an industrial site releasing various perfluorinated chemicals. This sediment is characterized by high concentrations of perfluoroundecanoic acid (PFUnA) and perfluorotridecanoic acid (PFTrDA) and a low perfluorooctane sulfonate (PFOS) concentration. Concentrations of 23 perfluoroalkyl compounds, including C4eC14 carboxylate acids, C4eC10 sulfonates, and seven precursors, were analyzed in overlying and pore water, sediment, and larvae. Midge larvae accumulated carboxylate acids (C11eC14), PFOS, and two precursors (perfluorooctane sulfonamide: FOSA and 6:2 fluorotelomer sulfonic acid, 6:2 FTSA). These substances accumulated mainly during the fourth instar larvae exponential growth phase. Accumulation of 6:2 FTSA, PFUnA, and PFOS occured via trophic and tegumentary routes. Other compounds mainly accumulated from food. Kinetics followed a partition model, from which uptake and elimination constants were derived.

Sediments are considered as a sink for metals, and the assessment of metal bioavailability for benthic organisms represents a great challenge. Diffusive Gradient in Thin films (DGT), developed to measure labile metals in aquatic media, have more recently been applied to sediment. Nevertheless, few studies have determined the relation between measurements from DGT and bioaccumulation in different benthic organisms. The aim of our work was to determine if labile metal measured by DGT in sediment is representative of bioavailable metal for benthic organisms. We focused our work on Cd and chose to use the diversity of ecological traits from different organisms to better understand the measurement given by DGT. We exposed simultaneously DGT and 3 macroinvertebrates species (the chironomid, Chironomus riparius; the amphipod, Gammarus fossarum; the mudsnail, Potamopyrgus antipodarum) to a natural sediment Cd-spiked at environmental relevant concentrations. The nature of sediment-bound Cd was also determined by means of sequential extractions in order to better interpret DGT measurements. Cadmium concentrations were determined in DGT and in the 3 organisms after one week of exposure. Results provided by DGT indicated that Cd was poorly released from particulate phase to pore water, suggesting that Cd measured by DGT was representative of the pore water labile fraction. Sequential extractions showed that the percentage of Cd bound to carbonate fraction increased simultaneously with Cd-spiking level; hence, this Cd fraction was poorly reactive to supply DGT demand. Cadmium accumulation rates were similar between DGT measurements and P. antipodarum, suggesting that labile Cd in pore waters was representative of bioavailable Cd for this species. Cadmium accumulation rates in C. riparius were higher than in DGT, demonstrating that C. riparius can mobilize Cd bound to carbonate phase. G. fossarum showed the lowest Cd accumulation rates, suggesting that they were mainly exposed to Cd from overlaying waters.

The behavior and toxicity of Cr(III) and Cr(VI) to the green algae Pseudokirchneriella subcapitata and Chlorella kessleri were studied in a standard culture medium (ISO medium) and, for P. subcapitata only, in ultrafiltered natural water enriched with all ISO components (modified ISO medium). In all solutions amended with Cr(III), initial chromium concentrations decreased by 60-90% over 72h (the duration of algal tests) indicating that protocols for testing poorly soluble substances are required to properly evaluate Cr(III) toxicity. After accounting for its behavior in test solutions, chromium(III) was 5-10 times more toxic than Cr(VI) in both media. For P. subcapitata, the average 72h EC50 of Cr(III) in ISO medium was 17.4+/-4.7 microg/L (n=9); lower than corresponding hardness-corrected Continuous Concentration Criteria of the US EPA and well within the range of Cr concentrations found in waters impacted by tannery discharges. These results follow from intrinsic chemical properties of Cr(III) in circumneutral solutions, so that the actual toxicity of Cr(III) to aquatic organisms may be generally underestimated.

The effects of Pseudokirchneriella subcapitata exudates on the acute toxicity of metals (Cd, Cu, Pb and Zn) were studied using (1 h) algal photosynthesis inhibition tests. The metal concentrations tested were 30, 60, 120, 250 and 500 μg L −1 for Cd and Zn; and 500, 1000, 2000, 3000, 4000 μg L −1 for Cu and Pb. It was established that P. subcapitata exudates markedly decreased metal toxicity. This effect was ascribed to: (i) the presence of organic ligands that bind metals and reduce the concentration of free ionic metal, and/or (ii) interaction of exudates with the cell surface with a subsequent decrease in metal uptake. The effects of the exudates on colloid aggregation were also examined using two different types of single particle counters (SPC). Exudates facilitated the colloid removal, likely via acceleration of aggregation (bridging). The results clearly demonstrate that algal exudates play an important role in the biogeochemical cycling of metals in natural surface water: (1) by reducing free metal concentrations and toxicity to living organisms and (2) by favoring colloid aggregation leading to the removal of colloid-bound metals (colloidal pumping). Such results highlight one potential application of the algae in the remediation of metal-contaminated waters. The results also suggest that current algal toxicity testing protocols, in particular long-term and static tests, may underestimate metal toxicity because of the presence of algal exudates.

The role of colloids in regulating element transport, behavior, and bioavailability in aquatic systems is now well-established. It appears that further progress in this research field is being slowed by (i) a limited integration between the geochemical and the biological aspects of the research on colloids and (ii) a persistent gap between well-controlled laboratory studies and real field situations. This paper presents a simultaneous evaluation of the role of colloids in controlling element environmental fate and bioavailability at the confluence between a major river and a polluted tributary. Fractionation of trace elements among suspended particulate matter, colloids, and true solution suggests that colloids may play a role in the removal of trace elements from the water column to bed sediments during the mixing of the two rivers. Toxicity testing of water samples indicates that, in this specific system, contaminants associated with colloids can contribute to water toxicity for the rotifer Brachionus calyciflorus but not for the green alga Pseudokirchneriella subcapitata. To the best of our knowledge, the results for B. calyciflorus are the first ones pointing to the possible contribution of colloid-bound contaminants to water toxicity in environmental samples. Despite the uncertainties associated with field variability, the results of chemical analysis and toxicitytesting show several points of convergence. Following these observations, a few innovative research approaches are suggested to improve the understanding of trace element biogeochemistry in real field situations.

In this study, acute and chronic data of 6 human pharmaceuticals (Carbamazepine, Clofibric acid, Diclofenac, Ofloxacin, Propranolol and Sulphamethoxazole) have been collected, using our own experimental data and literature data. From this data collection, the 2-tiered European draft guideline on the environmental risk assessment of such substances was tested. Firstly, measured environmental concentrations (MEC) in effluents from France and from published data obtained within effluents and surface waters in Germany were compared to the predicted environmental concentrations (PEC) respectively in both countries. Secondly, in a similar manner, using the compiled toxicity data on aquatic organisms, predicted no effect concentrations (PNEC) derived from chronic data were compared to PNEC derived from acute data. Globally, results demonstrated that (a) all environmental concentrations (predicted or measured) for each considered pharmaceutical exceeded the 10 ng/L cut-off value, which requires the second tier assessment based on ecotoxicity data, (b) the 6 pharmaceuticals showed a relatively limited acute toxicity and (c) Carbamazepine and Propranolol were inaccurately identified as having negligible risks under the current European draft procedure. Such results should help to provide further sound considerations for discussing the actual procedure on pharmaceuticals, especially on the need of appropriate ecotoxicity tests.

IntroductionOligochaetes are recognized as valuable bioindicators of sediment quality in streams and lakes. The development of an oligochaete index based on the identification of specimens using DNA barcodes requires a method for simultaneously preserving the DNA quality and information on the specimen density and oligochaete community composition. Absolute ethanol optimally preserves DNA but fixation of freshwater oligochaetes with this medium can cause disintegration and fragmentation of specimens. Here, we investigated the possibility to preserve oligochaete specimens in low-pH formalin and in neutral buffered formalin for up to four weeks before genetic analyses and tested if the addition of absolute ethanol to formalin-fixed oligochaetes resulted in a loss of specimens and/or species.MethodsWe performed guanidine extraction and polymerase chain reaction (PCR) amplification/sequencing of a fragment of the cytochrome c oxidase I (COI) gene on tissue fragments preserved in low-pH formalin for up to 3 weeks and in neutral buffered formalin for up to 4 weeks. In addition, we compared the density and taxonomic composition of formalin-fixed oligochaetes of several sieved sediment samples before and after the addition of absolute ethanol.ResultsThe COI fragment of all oligochaete specimens preserved in neutral buffered formalin for up to 28 days was successfully amplified by PCR and obtained sequences were complete and of high quality. The amplification success rate for low-pH formalin fixed specimens declined after 7 days of storage. The addition of absolute ethanol to formalin-fixed oligochaete communities did not alter density or diversity estimates.DiscussionOur results indicate that sediment samples can be stored in neutral buffered formalin for up to 4 weeks and the sieved material can then be transferred to absolute ethanol, without affecting DNA quality, density and community composition of oligochaetes. Based on these results, a protocol for preserving freshwater oligochaetes, describing all the steps from collection of sediments to preservation of the biological material in absolute ethanol, is proposed. This method of fixation/preservation is of relevance for establishing DNA barcode reference databases, inventories of genetic diversity and developing genetically based biological indices.