Annette Bérard

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.

Comparison of the ecotoxicological impact of the triazines Irgarol 1051 and atrazine on microalgal cultures and natural microalgal communities in Lake Geneva

The antifouling herbicide Irgarol 1051 has been detected in recent years in numerous estuaries, marinas, harbors and coastal areas, and in some harbors on Lake Geneva, but so far only a few studies have investigated the ecotoxicological effects of this compound on microalgae. The purpose of this study was to assess the ecotoxicological impact of Irgarol 1051 on the algal communities of Lake Geneva, and to compare its phytotoxicity to that of the common triazine herbicide, atrazine. We investigated the response of phytoplanktonic and periphytonic algal communities and single-species isolates collected from the lake, to the PS II inhibitor Irgarol 1051 (growth, proxy of photosynthetic activity and community structure). A short-term bioassay was developed based on in vivo fluorescence, together with nanocosm experiments with natural algal communities, and single-species tests on algal strains isolated from the lake. The toxicity of Irgarol 1051 towards periphyton and phytoplankton was shown to be higher than that of atrazine. Indications of the tolerance induced by this triazine in the algal communities of Lake Geneva, suggests that even at the levels of contamination reported in some parts of the lake, Irgarol 1051 is already exerting selection pressure. Information about sensitivities, selection and tolerance from laboratory experiments are used to explain the observations in natural microalgal communities from the lake.

PO43- dependence of the tolerance of autotrophic and heterotrophic biofilm communities to copper and diuron.

Pollution-induced community tolerance (PICT) concept is based on the assumption that the toxicant exerts selection pressure on the biological communities when exposure reaches a critical level for a sufficient period of time and therefore sensitive species are eliminated. However, induced tolerance of microbial biofilm communities cannot be attributed solely to the presence of toxicants in rivers but also to various environmental factors, such as amount of nutrients. An experimental study was undertaken to highlight the potential impact of a phosphorus gradient on the sensitivity of periphytic microbial community to Cu and diuron. Biofilms were exposed to real-world levels of chronic environmental contamination of toxicants with a phosphorus gradient. Biofilm sensitivity to Cu and diuron was assessed by performing short-term inhibition tests based on photosynthetic efficiency to target photoautotrophs, extracellular enzyme activity (beta-glucosidase and leucine-aminopeptidase) and substrate-induced respiration activity to target heterotrophs. The impact of P-gradient associated to pollution was evaluated by measuring pesticide concentrations in biofilms, biomass parameters (chla, AFDW), bacterial cell density, photosynthetic efficiency and community structure (using 18S and 16S rDNA gene analysis to target eukaryotes and DGGE and HPLC pigment analysis to target bacteria and photoautotrophs). The obtained results show that depending on the studied toxicant and the used structural or functional parameter, the effect of the phosphorus gradient was variable. This highlights the importance of using a range of parameters that target all the biological communities in the biofilm. The PICT method can be regarded as a good tool for assessing anthropogenic environmental contamination, but it is necessary to dissociate the real impact of toxicants from environmental factors.

Toxic effects of Irgarol 1051 on phytoplankton and macrophytes in Lake Geneva

Irgarol 1051 is a recent herbicidal compound, inhibitor of photosynthesis, used in antifouling paints. This toxic is persistent in aquatic environments, with low abiotic and biotic degradation, highly phytotoxic, and has already been detected in estuaries and coastal areas, with suspected negative impacts on non-target organisms (aquatic plants and algae). We measured the toxicity of Irgarol 1051 to macrophytes and phytoplankton from Lake Geneva (between Switzerland and France) by determining chlorophyll fluorescence yield, and phytoplankton primary production. Long-term toxicity for phytoplankton was estimated in a microcosm study, and growth inhibition tests were performed with isolated algal strains. The concentration of Irgarol 1051 was analysed in the water, and the most polluted site showed a higher level (up to 135 ng/L) than the lowest observed effect concentration for phytoplankton (8-80 ng/L), while the macrophytes appeared to be more tolerant to Irgarol 1051 in short-term tests. The microcosm study showed that phytoplankton structure might be even more sensitive to Irgarol 1051.

Pollution-induced community tolerance (PICT) and seasonal variations in the sensitivity of phytoplankton to atrazine in nanocosms

Algae communities exposed to a herbicide like atrazine (PS II inhibitor) are expected to be selected and to be more tolerant to the herbicide than unexposed communities (pollution-induced community tolerance, PICT). The PICT may be an ecotoxicological tool for detecting this selective action of chronic pollution, and this method has been applied to several toxicants in experimental systems and in field studies. But the detection of PICT with PS II inhibitors has sometimes been variable. This work was done to study the long-term effects of exposure to atrazine (10 microg/l) and the PICT responses of phytoplankton communities in repeated outdoor nanocosms. Phytoplankton communities were sampled in Lake Geneva at different periods of the year and the effects of atrazine were analysed by studying community structure, biomass and primary production, and by measuring tolerance to atrazine in a short-term physiological test based on 14C incorporation. We find that PICT is a sensitive method for measuring effects. Even atrazine concentrations causing little restructuring induced tolerance in most of our experiments. But the short- and long-term responses of phytoplankton to atrazine varied between experiments, probably due to the initial compositions of the communities and environmental factors associated with seasonal parameters. The selection and detection steps of PICT to atrazine thus vary greatly with environmental conditions and the physiological adaptations of algae to the herbicide. To monitor risk assessment in aquatic systems, PICT studies applied to algae, must be investigated in the light of seasonal contaminations and seasonal events and successions.

A risk assessment of pollution: induction of atrazine tolerance in phytoplankton communities in freshwater outdoor mesocosms, using chlorophyll fluorescence as an endpoint

We investigated the validity and sensitivity of assessments of the induction of atrazine tolerance in freshwater outdoor mesocosmic phytoplankton communities, using the in vivo fluorescence of chlorophyll a as an endpoint, for monitoring ecotoxicology and for risk assessment programs applied to phytoplankton contaminated by photosystem II herbicides. Atrazine inhibits the photosynthetic process, and so the rise in in-vivo fluorescence could be used as a physiological manifestation of acute toxicity. Short-term tests (1 h) were used, in which increasing concentrations of the herbicide were applied to phytoplankton samples taken every two days from the mesocosms, and used to plot dose-response curves. The concentration at which atrazine increased the fluorescence by 25% relative to control samples was used to demonstrate the sensitivity of the phytoplankton, and the values found were compared for samples from different mesocosms (contaminated and non-contaminated). The taxonomic composition of the phytoplankton was also determined. The data showed that chronic exposure (25 days) to 30 microg/L of atrazine significantly increased the apparent tolerance of the phytoplankton to further contamination by the same compound. The use of in vivo fluorescence of chlorophyll a appears to be a reliable and effective parameter for monitoring the effects of atrazine pollution, and detecting the changes in community tolerance driven by pollution selection pressure.

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.

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.

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.

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.