Bernard Montuelle

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.

In situ relationships between spatio-temporal variations in diuron concentrations and phototrophic biofilm tolerance in a contaminated river

Microbial biofilms are useful indicators for monitoring the ecological state of rivers. When assessing the effects of toxicants on microbial communities in natural ecosystems, special attention must be paid to the distinction between such effects and those resulting from other environmental variables. The pollution-induced community tolerance (PICT) approach offers the means to partially isolate effects of individual toxicants within a complex ecosystem by studying shifts in community sensitivity. To validate PICT for site-specific risk assessment, we investigated how closely diuron tolerance acquisition by photoautrophic biofilm communities could reflect their previous in situ exposure to this herbicide. To this end, a field survey was conducted for 9 months in a French river contaminated by diuron inputs from adjacent vineyards. Spatio-temporal variations in diuron tolerance capacities of photoautotrophic communities were estimated monthly using short-term photosynthetic bioassays. The biological survey was coupled with intensive physico-chemical monitoring to analyze the environmental factors influencing tolerance induction. We revealed a possible influence of three co-varying environmental variables (nitrates, conductivity and temperature), but statistical analysis clearly demonstrated that the main factor explaining variation in diuron sensitivity was the mean in situ diuron exposure level during biofilm colonization periods. A remarkable exponential correlation between EC(50) values and in situ diuron concentrations (R(2)=0.90, p<0.0001) was recorded, confirming that PICT can be a powerful tool for the environmental monitoring of rivers.

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.

Response of spring and summer riverine microbial communities following glyphosate exposure.

Seasonal variation in the response of riverine microbial communities to an environmentally relevant exposure to glyphosate (about 10 microgl(-1)) was assessed on natural communities collected in spring and summer, using two 14-day microcosm studies. The two experiments showed no major effect of glyphosate on algal biomass (chlorophyll a concentrations), bacterial activity ([(3)H]thymidine incorporation), or bacterial community diversity (16S PCR-TTGE detection). Effects on algal community composition (genus-level taxonomic identification) and eukaryotic community diversity (18S PCR-DGGE on <100 microm organisms) were only detected on the samples collected in summer. This work demonstrates that even if the effects of a short pulse of glyphosate (10 microgl(-1)) on riverine microorganisms seem to be limited, the responses of natural microbial communities to glyphosate exposure (and probably to other pesticide exposures) can clearly vary between the experiments, and can be seasonally dependent.

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.

Use of multivariate analyses to investigate the contribution of metal pollution to diatom species composition: search for the most appropriate cases and explanatory variables

The aim of this study was to elucidate how the spatial scale and the set of variables included influence our ability to detect the effects of different types of pollution on the biota. Using variance partitioning analysis, we assessed the individual importance of a set of environmental factors (eutrophication and organic pollution) versus metal level pollution on the community structure of diatom assemblages at different spatial scales. At regional scale, environmental factors did not explain more of the variance compared to the watershed study. The results of the watershed scale field survey indicate that diatom community composition was influenced by low metal concentrations, but this pattern was only observed by the inclusion of biofilm metal concentration data. We recommend the analysis of metal traces in the water phase and the biota (fluvial biofilms) as well as the use of the Diffusive Gradient in Thin films (DGT) technique to characterize low metal level pollution in freshwater systems.

Combining polar organic chemical integrative samplers (POCIS) with toxicity testing to evaluate pesticide mixture effects on natural phototrophic biofilms

Polar organic chemical integrative samplers (POCIS) are valuable tools in passive sampling methods for monitoring polar organic pesticides in freshwaters. Pesticides extracted from the environment using such methods can be used to toxicity tests. This study evaluated the acute effects of POCIS extracts on natural phototrophic biofilm communities. Our results demonstrate an effect of POCIS pesticide mixtures on chlorophyll a fluorescence, photosynthetic efficiency and community structure. Nevertheless, the range of biofilm responses differs according to origin of the biofilms tested, revealing spatial variations in the sensitivity of natural communities in the studied stream. Combining passive sampler extracts with community-level toxicity tests offers promising perspectives for ecological risk assessment.

Effects of flow regime and pesticides on periphytic communities: Evolution and role of biodiversity

The effects of chemical and physical factors on periphyton structure, diversity and functioning were investigated in an outdoor mesocosm experiment. Stream biofilms were subjected to a pesticide mix (diuron and azoxystrobin) under two different hydraulic regimes. The hydraulic regimes differed by spatial variations of flow conditions (turbulent with high variations vs. laminar with low variations). The effects of the hydraulic regime and pesticides were assessed at the level of the periphytic communities. We focused on the change in the biodiversity of these communities under the two hydraulic regimes, and on the role of these biodiversity changes in case of pesticide contamination. Changes in structural (biomass, cell density), diversity (community composition assessed by PCR-DGGE and microscopic analysis) and functional (bacterial and algal production, sensitivity to the herbicide) parameters were monitored throughout a 2-month experiment. The results showed that exposure to pesticides affected the phytobenthic community targeted by the herbicide, impacting on both its growth dynamics and its primary production. Conversely, the impact of the flow regime was greater than that of pesticides on the non-target bacterial community with higher bacterial density and production in laminar mesocosms (uniform regime). An interaction between flow and pollution effects was also observed. Communities that developed in turbulent mesocosms (heterogeneous regime) were more diversified, as a result of increased microhabitat heterogeneity due to high spatial variations. However, this higher biodiversity did not increase the ability of these biofilms to tolerate pesticides, as expected. On the contrary, the sensitivity of these communities to pesticide contamination was, in fact, increased.

Potential for microbial diuron mineralisation in a small wine-growing watershed: from treated plots to lotic receiver hydrosystem

BACKGROUND Since biological degradation processes are known to be a major driver of the natural attenuation of pesticide residues in the environment, microbial communities adapted to pesticide biodegradation are likely to play a key environmental role in reducing pesticide exposure in contaminated ecosystems. The aim of this study was to assess the diuron-mineralising potential of microbial communities at a small-scale watershed level, including a diuron-treated vineyard (pollution source), its associated grass buffer strip (as a river protection area against pesticide runoff) and the lotic receiver hydrosystem (sediments and epilithon), by using radiorespirometry. RESULTS Comparison of results obtained at different sampling sites (in both soil and aquatic systems) revealed the importance of diuron exposure in the adaptation of microbial communities to rapid diuron mineralisation in the vineyard but also in the contaminated grass strip and in downstream epilithic biofilms and sediments. CONCLUSION This study provides strong suggestive evidence for high diuron biodegradation potential throughout its course, from the pollution source to the final receiving hydrosystem, and suggests that, after microbial adaptation, grass strips may represent an effective environmental tool for mineralisation and attenuation of intercepted pesticides.

Enhanced co-tolerance and co-sensitivity from long-term metal exposures of heterotrophic and autotrophic components of fluvial biofilms

Understanding the interactive effects of multiple stressors on ecosystems has started to become a major concern. The aim of our study was therefore to evaluate the consequences of a long-term exposure to environmental concentrations of Cu, Zn and As on the pollution-induced community tolerance (PICT) of lotic biofilm communities in artificial indoor channels. Moreover, the specificity of the PICT was assessed by evaluating the positive and negative co-tolerance between these metals. Photosynthetic efficiency and substrate-induced respiration (SIR), targeting the autotrophic and heterotrophic communities respectively were used in short-term inhibition bioassays with Cu, Zn and As to assess sensitivities of pre-exposed biofilms to the metals tested. Diversity profiles of a phototrophic, eukaryotic and prokaryotic community in biofilms following the different treatments were determined and analyzed with principal component analysis. The results demonstrated that pre-exposure to metals induced structural shifts in the community and led to tolerance enhancements in the phototrophic and heterotrophic communities. On the other hand, whatever the functional parameter used (i.e. photosynthesis and SIR), communities exposed to Cu were more tolerant to Zn and vice versa. Furthermore, only phototrophic communities pre-exposed to As developed tolerance to Cu but not to Zn, whereas no co-tolerance between Cu and As was observed in the heterotrophic communities. Finally, phototrophic and heterotrophic communities exposed to Cu and Zn became more sensitive to As, reflecting a negative co-tolerance between these metals. Overall, our findings support the fact that although the mode of action of the different metals is an important driver for the structure and thus the tolerance of the communities, it appears that the detoxification modes are the most important factors for the occurrence of positive or negative co-tolerance.