Use of the MicroResp™ method to assess pollution-induced community tolerance to metals for lotic biofilms.

Understanding the ecological status of aquatic ecosystems and the impact of anthropogenic contamination requires correlating exposure to toxicants with impact on biological communities. Several tools exist for assessing the ecotoxicity of substances, but there is still a need for new tools that are ecologically relevant and easy to use. We have developed a protocol based on the substrate-induced respiration of a river biofilm community, using the MicroResp™ technique, in a pollution-induced community tolerance approach. The results show that MicroResp™ can be used in bioassays to assess the toxicity toward biofilm communities of a wide range of metals (Cu, Zn, Cd, Ag, Ni, Fe, Co, Al and As). Moreover, a community-level physiological profile based on the mineralization of different carbon substrates was established. Finally, the utility of MicroResp™ was confirmed in an in-situ study showing gradient of tolerance to copper correlated to a contamination gradient of this metal in a small river.

Transplantation of microbenthic algal assemblages to assess structural and functional recovery after diuron exposure.

The potential of microbenthic algal assemblages to recover after diuron exposure was investigated. Microbenthic algal assemblages (periphyton) were grown on glass slides in correspondence to a diuron-polluted and adiuron-free sampling site of a river. After 5 weeks of colonization, the impacted periphyton was transferred by translocating the colonized glass slides to the unpolluted site. To monitor the changes in functional and structural parameters and to assess recovery, both the transferred and the local reference periphyton were sampled at the day of transfer (t0) and 1, 3, and 5 weeks after the transfer (t1, t3,t5). Structural transitions of eukaryotic communities were characterized by denaturing gradient gel electrophoresis (DGGE) and functional ones were characterized by short-term incubation toxicity tests with diuron. As shown by Bray­Curtis similarity values based on DGGE band patterns, almost total structural recovery of the transferred periphyton took place 5 weeks after transfer. For the transferred periphyton, previous diuron exposure at the contaminated site induced the development of diuron tolerance,which decreased after 1 week and became very similar to the tolerance of the nonimpacted community after 5 weeks. It is concluded that 5-week-old eukaryotic periphyton communities are capable of restoring their structural and functional attributes after 5 weeks within a noncontaminated environment.

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.

Comparison of the structure and composition of bacterial communities from temperate and tropical freshwater ecosystems.

We used a partial 16S rRNA sequencing approach to compare the structure and composition of the bacterial communities in three large, deep subalpine lakes in France with those of communities in six shallow tropical reservoirs in Burkina Faso. Despite the very different characteristics of these ecosystems, we found that their bacterial communities share the same composition in regard to the relative proportions of the different phyla, suggesting that freshwater environmental conditions lead to convergence in this composition. In the same way, we found no significant difference in the richness and diversity of the bacterial communities in France and Burkina Faso. We defined core and satellite operational taxonomic units (OTUs) (sequences sharing at least 98% identity) on the basis of their abundance and their geographical distribution. The core OTUs were found either ubiquitously or only in temperate or tropical and subtropical areas, and they contained more than 70% of all the sequences retrieved in this study. In contrast, satellite OTUs were characterized by having a more restricted geographical distribution and by lower abundance. Finally, the bacterial community composition of these freshwater ecosystems in France and Burkina Faso was markedly different, showing that the history of these ecosystems and regional environmental parameters have a greater impact on the relative abundances of the different OTUs in each bacterial community than the local environmental conditions.

Spatial heterogeneity of periphytic microbial communities in a small pesticide-polluted river.

Spatial variability in the microbial community composition of river biofilms was investigated in a small river using two spatial scales: one monitored the upstream-downstream pesticide contamination gradient, referred to as the 'between-section variability', and the other monitored a 100-m longitudinal transect (eight sampling sites per section) within each sampling section, referred to as the 'within-section variability'. Periphyton samples were collected in spring and winter on artificial substrates placed in the main channel of the river. Denaturing gradient gel electrophoresis (DGGE) was used to assess the prokaryotic and eukaryotic community richness and diversity, and HPLC pigment analysis to assess the global taxonomic composition of the photoautotrophic community. In order to try to reduce the biological variability due to differences in flow velocity and in light conditions within each sampling section, and consequently to take into account only the changes due to water chemistry, nine plates (three per sampling section) subjected to similar physical conditions were chosen, and the results for these plates were compared with those obtained for all 24 plates. As shown by DGGE and by HPLC analyses, using these three substrate plates exposed to similar environmental conditions did indeed reduce the within-section variability and maximize the between-section variability. This sampling strategy also improved the evaluation of the impact of pollutants on the periphytic communities, measured using short-term sensitivity testing.

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.

Spatial variability in the abundance and composition of the free-living bacterioplankton community in the pelagic zone of Lake Bourget (France).

Spatial variations in the abundance and diversity of the free-living bacterioplankton community of a large Alpine lake, Lake Bourget (France), were investigated in the pelagic zone by means of two two-dimensional samplings taken in 2003. Lake-water samples were collected in winter during water mixing, and in early summer during stratification. The population abundance in each sample was determined by flow cytometry. Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments from organisms measuring less than 2 mum was used to assess eubacterioplankton community composition. In winter, no obvious differences were observed in either the abundance or the diversity of the bacterial community, on either the horizontal or the vertical scales. The only influence detected was that of river water input, but this was at a very minor scale relative to the surface area of the lake. In early summer, changes were found in the community composition on the vertical scale related to the thermal stratification of the water column. There were also marked differences on the horizontal scale at 15 m depth due to internal waves. The implications of these findings for sampling strategies are very important from the perspective of comparative studies of free-living bacterial community diversity and functioning in large and deep lakes.

Molecular approaches to the assessment of biodiversity in aquatic microbial communities.

For the past 20 years, the increased development and routine application of molecular-based techniques has made it possible to carry out detailed evaluations of the biodiversity of aquatic microbial communities. It also offers great opportunities for finding out how this parameter responds to various environmental stresses. Most of these approaches involve an initial PCR amplification of a target, which is generally located within the ribosomal operon. The amplification is achieved by means of primers that are specific to the organisms of interest. The second step involves detecting sequence variations in the PCR fragments either by a cloning/sequencing analysis, which provides a complete characterization of the fragments, or by an electrophoretic analysis, which provides a visual separation of the mixture of fragments according to sequence polymorphism (denaturing or temperature gradient gel electrophoresis, single strand conformation polymorphism) or length polymorphism (terminal-restriction fragment length polymorphism, automated ribosomal intergenic spacer analysis). Other non-PCR-based methods are also commonly used, such as fluorescence in-situ hybridization and DNA re-association analysis. Depending on the technique used, the information gained can be quite different. Moreover, some of these analyses may be rather onerous in terms of time and money, and so not always suitable for screening large numbers of samples. The most widely used techniques are discussed in this paper to illustrate the principles, advantages and shortcomings of each of them. Finally, we will conclude by evaluating the techniques and discussing some emerging molecular techniques, such as real-time PCR and the microarray technique.

Cyanophage diversity, inferred from g20 gene analyses, in the largest natural lake in France, Lake Bourget.

The genetic diversity of the natural freshwater community of cyanophages and its variations over time have been investigated for the first time in the surface waters of the largest natural lake in France. This was done by random screening of clone libraries for the g20 gene and by denaturing gradient gel electrophoresis (DGGE). Nucleotide sequence analysis revealed 35 distinct cyanomyovirus g20 genotypes among the 47 sequences analyzed. Phylogenetic analyses showed that these sequences fell into seven genetically distinct operational taxonomic units (OTUs). The distances between these OTUs were comparable to those reported between marine clusters. Moreover, some of these freshwater cyanophage sequences were genetically more closely related to marine cyanophage sequences than to other freshwater sequences. Both approaches for the g20 gene (sequencing and DGGE analysis) showed that there was a clear seasonal pattern of variation in the composition of the cyanophage community that could reflect changes in its biological, chemical, and/or physical environment.

Seasonal changes in the sensitivity of river microalgae to atrazine and isoproturon along a contamination gradient.

A study was undertaken to investigate the environmental impact of herbicides on natural communities of freshwater periphyton and phytoplankton in the river Ozanne and in related nearby water reservoirs, including both pristine and pesticide- (atrazine and isoproturon) contaminated stations. The microalgal toxicity of both herbicides was investigated by short-term studies, using the in vivo fluorescence pattern to perform dose-effect experiments. The taxonomic composition of the communities sampled was assessed by microscopy and by HPLC pigment analysis. The EC50 (periphyton) or EC125 (phytoplankton) values, calculated using in vivo fluorescence endpoints, increased with the herbicide concentration found in the water. In contrast, the structure of the algal communities (periphyton) inhabiting the contaminated stations seemed to be permanently affected when compared to the reference community. A 'memory effect' could be detected, both in herbicide sensitivity and in the structure of periphytic communities that persisted even when peak contaminations had disappeared. This study shows that the response of algal communities is likely to reflect past selection pressures, and suggests that the function and structure of a community could both be modified by the persistent or repeated presence of microcontaminants in natural environments. We could use short-term ecotoxicological tests on freshwater microalgae to assess the effects of past temporary contaminations by agricultural pesticides, and combining this with diversity indices could make it possible to assess the ecotoxicological risk of herbicide contamination even when a complete chemical analysis of the contamination is not feasible.