Frédéric Rimet

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.

Regional distribution of diatom assemblages in the headwater streams of Luxembourg

The aim of this study was to determine the influence of environmental variables on the structure of benthic diatom assemblages, and to propose type assemblages of diatoms characterizing unpolluted headwater streams of Luxembourg. A total of 289 diatom samples were collected in the headwater streams of Luxembourg. At each sampling site, physical and chemical variables were also measured. The relationships between environmental variables and the distribution of the taxa were estimated using univariate and multivariate statistical analyses. On the basis of geology and stream water chemistry, Luxembourg could be separated in two regions. The streams in the schistose northern part of Luxembourg presented a low carbonate hardness (median 3.5° F); the streams in the southern part of Luxembourg presented a high carbonate hardness (median 21.8° F) notably connected to the presence of sandstone and limestone substrata. A Twinspan classification carried out on diatom assemblages defined two groups of samples corresponding to these two regions. The carbonate hardness that is related to the nature of the geological substratum appeared to be the major structuring variable for the assemblage composition. Anthropogenic pollution was a secondary structuring variable for diatom assemblages since each group could be subdivided in subgroups presenting statistically different nutrient and organic matter concentrations. The diatom assemblages of these subgroups were characterised by differences of saprobic and trophic preferences. On the basis of these results, two type assemblages of diatoms are proposed for the unpolluted headwater streams in the two regions of Luxembourg. The presence of these different type assemblages in Luxembourg shows the necessity to adapt diatom bioindication to the different regions of the country.

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.

Response of diatom indices to simulated water quality improvements in a river

Various diatom indices are routinely used in European countries to monitor water quality in waterways. In order to assess their sensitivities and their integration interval after a sudden and lasting environmental change, epilithic diatom biofilms were transferred from several polluted rivers to an unpolluted stream. To monitor the changes of the index values, the biofilms were sampled in a first experiment 20 and 40 days after transfer, and in a second experiment 30 and 60 days after transfer. Sensitivities of the indices to the water quality improvement were assessed calculating the differences between the index values of the reference and the transferred assemblages. Some indices have intermediate sensitivities (BDI, GDI, ILM, SLA), others higher sensitivities (CEE, EPI, ROT, SPI, TDI). The integration interval of these indices was 40–60 days. Some differences were observed between the indices, but their results were homogeneous when compared to those obtained with other metrics such as Bray-Curtis or Chord distances, used to assess the difference between the transferred and the reference diatom assemblages. These other metrics showed that even after 60 days, the transferred assemblages still differed from the reference. This underlines that metrics do not have the same integration intervals and do not assess the same stresses; the choice of the metric used to assess water quality is of prime importance.

Diatom communities and water quality assessment in Mountain Rivers of the upper Segre basin (La Cerdanya, Oriental Pyrenees)

Epilithic diatoms of mountain rivers from the upper Segre catchment (Oriental Pyrenees) were studied in 1998, during three different seasons: March, July and September. Four rivers, the river Segre and its three most important tributaries, Duran, Molina and Querol, were sampled in upstream and downstream stretches. The diatom communities were comparable in all upstream stretches of these mountain rivers draining siliceous substrates. Dominant taxa were Achnanthidium subatomus, Diatoma mesodon, Encyonema cf. minutum, E. silesiacum, Fragilaria arcus, F. capucina, Gomphonema calcifugum, G. pumilum, Meridion circulare and Nitzschia pura. Changes in water quality in the downstream stretches lead to the appearance of pollution tolerant taxa, such as Eolimna minima, Gomphoneis minuta, Navicula gregaria, and Nitzschia inconspicua. As a result, the values obtained with the diatom water quality indices (IPS Specific Polluosensitivity Index, CEE and IBD Biological Diatom Index) decreased. The diatom community composition and the derived water quality values did not change in the upstream stretches over the year. In contrast, significant changes were observed in the downstream stretches with best water␣quality in July, during high flows due to melting snow, and worst values in September, during low␣discharge. The diatom indices, especially the IPS, showed a good performance in these mountain rivers.

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.

Effects of atrazine and nicosulfuron on freshwater microalgae

Growth modifications caused by various concentrations of atrazine and nicosulfuron were monitored in closed and continuous culture of Chlorella vulgaris (chlorophyta), Navicula accommoda (diatomophyta), and Oscillatoria limnetica (cyanophyta). The concentration at which algal growth rate was reduced twofold (EC50) was determined in the three species for both herbicides. Comparatively, the two toxicants were applied at 10 microg/l level in microcosms inoculated with natural phytoplankton from Lake Geneva. The relative abundances of major phytoplanktonic species were measured by algal cell count at the beginning and at the end of each experiment. Atrazine and nicosulfuron have different targets in plant metabolism, respectively, photosystem II (PSII) and acetolactate synthase (ALS), and the expected effects were different. Generally, the cultured phytoplankton exhibited various sensitivities, depending on species or herbicide. In the microcosms, the major taxa of natural phytoplanktonic samples exhibited various patterns, from acute toxicity to growth enhancement. For example, the diatoms inside the community were not affected by atrazine and nicosulfuron, except for Stephanodiscus minutulus that was sensitive to both, and Asterionella/formosa that was sensitive only to nicosulfuron. The specific physiology and the relationships among the phytoplanktonic communities have to be carefully considered when one would try to predict the extent of herbicide action on natural phytoplankton using in vitro tests. There is a need to test the toxic effect on various cultured strains, representative of most of the taxonomic composition of natural communities, to take into account the wide range of sensitivities and reaction to herbicide contamination. But this is not enough to give a solid frame when transposing the results to the field, and the use of more ecologically relevant systems is recommended.