Agnès Bouchez

Responses of chronically contaminated biofilms to short pulses of diuron. An experimental study simulating flooding events in a small river.

An experimental study was undertaken to highlight the potential ecotoxicological impact of the herbicide diuron on biofilms during flooding events in a small river (Morcille) in the Beaujolais vineyard area (France). We investigated the responses of chronically contaminated biofilms exposed to short-term pulses (3 h) of diuron. Biofilms were grown in indoor microcosms that were either non-contaminated or exposed to low-level chronic contamination, and not exposed, or exposed to single or double pulses of two environmental concentrations (7 and 14 microg L(-1)) of diuron. Exposure to pollution and its impact on biofilms were assessed by measuring pesticide concentrations in biofilms, biomass parameters (chl a, AFDW), community structure (using 18S and 16S rDNA gene analysis by DGGE, and HPLC pigment analysis to target eukaryotes, bacteria and photoautotrophs, respectively) and by performing a physiological test. Control biofilms displayed very low diuron concentrations, whereas the herbicide was found in the contaminated biofilms. Nevertheless, diuron concentrations were not higher in the pulsed biofilms than in the non-pulsed ones. AFDW and chl ain vivo fluorescence increased in both microcosms during the experiment and biomass was higher in chronically exposed biofilms than in control ones. The impact on biomass was higher for the control double-pulsed biofilms than for the non-pulsed ones. Carbon incorporation by the chronically exposed biofilms was greater during the first 28 days of growth than during the first 28 days of growth in the control biofilms. Both single and double pulses inhibited carbon incorporation of all biofilm communities, especially of the control ones. Short-term inhibition of photosynthesis was never significantly different in exposed and non-exposed biofilms. Few differences in the pigment structure were found between chronically exposed and control biofilms, but pulses impacted on the pigment structure of all biofilm communities. Bacterial structural differences were observed between single-pulsed and non-pulsed biofilms, but not between double-pulsed and non-pulsed biofilms. The different pulses affected the eukaryotic community structures of the control biofilms, but not of the chronically exposed ones. Unlike the bacterial communities, the control eukaryotic communities were structurally different from the chronically exposed ones. This preliminary experimental study indicates that exposure to environmental concentrations of diuron and other agricultural contaminants and further exposure to diuron can have measurable effects on small river biofilm communities. The effects of a pulsed acute exposure to diuron on biofilms depended on whether the biofilms had previously been exposed to the same stressors or not.

Using diatom life-forms and ecological guilds to assess organic pollution and trophic level in rivers: a case study of rivers in south-eastern France

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.

Next-generation sequencing to inventory taxonomic diversity in eukaryotic communities: a test for freshwater diatoms

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.

Pioneer marine biofilms on artificial surfaces including antifouling coatings immersed in two contrasting French Mediterranean coast sites.

Marine biofilm communities that developed on artificial substrata were investigated using molecular and microscopic approaches. Polystyrene, Teflon® and four antifouling (AF) paints were immersed for 2 weeks at two contrasting sites near Toulon on the French Mediterranean coast (Toulon military harbour and the natural protected area of Porquerolles Island). Biofilms comprising bacteria and diatoms were detected on all the coatings. The population structure as well as the densities of the microorganisms differed in terms of both sites and coatings. Lower fouling densities were observed at Porquerolles Island compared to Toulon harbour. All bacterial communities (analysed by PCR-DGGE) showed related structure, controlled both by the sites and the type of substrata. Pioneer microalgal communities were dominated by the same two diatom species, viz. Licmophora gracilis and Cylindrotheca closterium, at both sites, irrespective of the substrata involved. However, the density of diatoms followed the same trend at both sites with a significant effect of all the AF coatings compared to Teflon and polystyrene.

Influence of slight differences in environmental conditions (light, hydrodynamics) on the structure and function of periphyton

Small streams are ecosystems mainly controlled by physical factors. Minor differences in these factors can affect periphyton, which are key functional communities in these ecosystems. Eight different environmental conditions combining two types of current, two flow velocities and two light intensities were produced and controlled in artificial channels. Their impact on young and mature periphyton was investigated during a 6-week exposure period. The two different levels of light intensity produced early effects on the algal community. In young periphyton, the lower level of light intensity enhanced the number of algal cells, and this community appeared to be significantly structured by light. As the periphyton matured, the effects of physical factors became more marked. At this later stage, both the bacterial and algal communities began to be affected. Both function (primary production) and structure began to respond to differences in light and in flow velocity. Small differences in low-level environmental factors, such as light and flow, had an effect both on the structure of periphyton and its functional capacities. Keeping in mind the close link between diversity and function in microbial communities, periphyton confronted to various environmental stresses (pollution, flooding) in the field may behave differently due to minor differences in physical factors.

In situ assessment of periphyton recovery in a river contaminated by pesticides.

Recovery of bacterial and eukaryotic communities in biofilms naturally grown on stones was studied for 9 weeks after transferring them from a pesticide polluted downstream site of the river Morcille (Beaujolais, France) to a non-contaminated upstream site. Site-specific periphyton present on stones at both the down- and the upstream sampling site were collected to analyze the site-specific colonization. Throughout the experiment, structural and functional parameters were analyzed for the periphyton transferred and for the site-specific up- and downstream periphyton. Comparison between these three communities allowed quantifying recovery of the transferred one. Denaturing Gradient Gel Electrophoresis of PCR-amplified 16S and 18S rRNA gene fragments were used to assess prokaryotic and eukaryotic microbial community composition, respectively. Microscopy counts allowed characterizing the diatom taxa abundances. The sensitivity of the microalgal communities towards diuron and copper was investigated at the laboratory by short-term photosynthesis inhibition assays. The functional reaction of the bacterial communities towards copper was assessed by short-term respiration inhibition assays. The structure of transferred eukaryotic, bacterial and diatom communities was more similar to the structure of the downstream communities than to upstream ones even after 9 weeks acclimatization in particular for the bacterial community. In the same way, the community tolerance towards diuron and copper, as estimated by the EC50 values, was intermediate for the transferred biofilms compared to the local up- or downstream biofilm, even after 9 weeks of acclimatization. These results strongly suggest slow recovery, likely to be linked to long lasting exposure of pesticides and in particular copper adsorbed to the biofilm matrices and to the toughness for pioneer microorganisms to invade mature biofilms.

A next-generation sequencing approach to river biomonitoring using benthic diatoms

Abstract: 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.

Using Bioassays and Species Sensitivity Distributions to Assess Herbicide Toxicity towards Benthic Diatoms

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.

Genotoxic pressure of vineyard pesticides in fish: Field and mesocosm surveys

The present study deals with the genotoxicity assessment of vineyard pesticides in fish exposed in the field or in mesocosm conditions. Primary DNA damage was quantified as strand breaks using the single cell gel electrophoresis assay (Comet assay) applied to fish erythrocytes. In a first experiment, a significant genotoxic effect was observed following an upstream-downstream gradient in early life stages of brown trout (Salmo trutta fario) exposed in the Morcille River contaminated by a mixture of vineyard pesticides during three consecutive years. The pronounced response in terms of DNA damage reported in the present study could argue for a high sensitivity of fish early life stage and/or a high level of exposure to genotoxic compounds in the Morcille River. This stresses the interest in using trout larvae incubated in sediment bed to assess genotoxic compounds in the field. In a second experiment, adult European topminnow (Phoxinus phoxinus) were exposed in water running through artificial channels to a mixture of diuron and azoxystrobin, two of the main pesticides detected in the Morcille watershed. As compared with the unexposed channel, a 3-5-fold increase in the DNA damage was observed in fish exposed to chronic environmental pesticide concentrations (1-2 microg L(-1) for diuron and 0.5-1 microg L(-1) for axoxystrobin). A single 6h pulse of pesticide (14 microg L(-1) of diuron and 7 microg L(-1) of azoxystrobin) was applied to simulate transiently elevated chemical concentrations in the river following storm conditions. It did not increase genotoxicity. After a 1-month recovery period, DNA damage in exposed fish erythrocytes recovered to unexposed level, suggesting possible involvement of both repair mechanisms and cellular turnover in this transient response. This work highlights that vineyard treatment by pesticides and in particular diuron and azoxystrobin can represent a genotoxic threat to fish from contaminated watershed rivers.

Impact of chronic and acute pesticide exposures on periphyton communities

Aquatic ecosystems face variable exposure to pesticides, especially during floodings which are associated with short bursts of high contaminant concentrations that influence biological systems. A study was undertaken to highlight the impact of the herbicide diuron applied in mixture with the fungicide tebuconazole on natural periphyton during flooding events. Periphyton were grown in two series of two lotic outdoor mesocosms: one series was non-contaminated while the other was exposed to chronic contamination. After 4weeks, one channel of each series was exposed to three successive pulses, with each pulse followed by one week of recovery. Impacts on periphyton were assessed by using Denaturing Gel Gradient Electrophoresis to characterize eukaryotic community structure. At a functional scale, photosynthetic efficiency was quantified during each pulse, and the induced tolerance to diuron was estimated by performing short-term inhibition tests based on photosynthetic efficiency. Moreover, pesticide concentrations in the water column and periphyton matrix were measured. Diuron was adsorbed in the periphyton during each pulse and desorbed 13h after pulse end. The different pulses affected the eukaryotic community structures of the control biofilms, but not of the chronically exposed ones. During the first pulse, photosynthetic efficiency was correlated with pesticide concentration in the water phase, and there was no difference between periphyton from chronically contaminated channels and control channels. However, during the second and third pulses, the photosynthetic efficiency of periphyton chronically exposed to pesticides appeared to be less impacted by the acute pulsed exposure of pesticide. These changes were consistent with the acquisition of induced tolerance to diuron since only after the third pulse that periphyton from chronic channel became tolerant to diuron. Our experimental study indicates that the effects of pulsed acute exposures to pesticides on periphyton depended on whether the communities had previously been exposed to the same stressors or not.