Ahmed Tlili

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.

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.

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.

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.

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.

Chronic exposure effects of silver nanoparticles on stream microbial decomposer communities and ecosystem functions

With the accelerated use of silver nanoparticles (AgNP) in commercial products, streams will increasingly serve as recipients of, and repositories for, AgNP. This raises concerns about the potential toxicity of these nanomaterials in the environment. Here we aimed to assess the impacts of chronic AgNP exposure on the metabolic activities and community structure of fungal and bacterial plant litter decomposers as central players in stream ecosystems. Minimal variation in the size and surface charge of AgNP indicated that nanoparticles were rather stable during the experiment. Five days of exposure to 0.05 and 0.5 μM AgNP in microcosms shifted bacterial community structure but had no effect on a suite of microbial metabolic activities, despite silver accumulation in the decomposing leaf litter. After 25 days, however, a broad range of microbial endpoints, as well as rates of litter decomposition, were strongly affected. Declines matched with the total silver concentration in the leaves and were accompanied by changes in fungal and bacterial community structure. These results highlight a distinct sensitivity of litter-associated microbial communities in streams to chronic AgNP exposure, with effects on both microbial functions and community structure resulting in notable ecosystem consequences through impacts on litter decomposition and further biogeochemical processes.

Light History Influences the Response of Fluvial Biofilms to Zn Exposure

Fluvial biofilms are subject to multistress situations in natural ecosystems, such as the co‐occurrence of light intensity changes and metal toxicity. However, studies simultaneously addressing both factors are rare. This study evaluated in microcosm conditions the relationship between short‐term light intensity changes and Zn toxicity on fluvial biofilms with long‐term photoacclimation to different light conditions. Biofilms that had long‐term photoacclimation to 25 μmol photons · m−2 · s−1 (low light [LL] biofilms), 100 μmol photons · m−2 · s−1 (medium light [ML] biofilms), and 500 μmol photons · m−2 · s−1 (high light [HL] biofilms) were characterized by different structural (Chlorophyll‐a [Chl‐a], total biomass‐AFDW, EPS, algal groups, and diatom taxonomy) and physiological attributes (ETR‐I curves and photosynthetic pigments). HL biofilms showed higher light saturation intensity and a higher production of xanthophylls than LL biofilms. In contrast, LL biofilms had many structural differences; a higher proportion of diatoms and lower AFDW and EPS contents than ML and HL biofilms. A clear effect of light intensity changes on Zn toxicity was also demonstrated. Zn toxicity was enhanced when a sudden increase in light intensity also occurred, mainly with LL biofilms, causing higher inhibition of both the Φ′PSII and the ΦPSII. A decoupling of NPQ from de‐epoxidation reaction (DR) processes was also observed, indicating substantial damage to photoprotective mechanisms functioning in biofilms (i.e., xanthophyll cycle of diatoms) due to Zn toxicity. This study highlights the need to take into account environmental stress (e.g., light intensity changes) to better assess the environmental risks of chemicals (e.g., metals).

Antioxidant enzyme activities in biofilms as biomarker of Zn pollution in a natural system: an active bio-monitoring study.

This study aimed to explore the use of antioxidant enzyme activities (AEA) and biofilm metal accumulation capacity in natural communities as effect-based indicator of metal exposure in fluvial systems. To achieve these objectives, an active biomonitoring using fluvial biofilm communities was performed during 5 weeks. Biofilm was colonized over artificial substrata in a non-polluted site. After 5 weeks, biofilms were translocated to four different sites with different metal pollution in the same stream. The evolution of environmental parameters as well as biofilm responses was analysed over time. Physicochemical parameters were different between sampling times as well as between the most polluted site and the less polluted ones, mainly due to Zn pollution. In contrast, AEA and metal accumulation in biofilms allowed us to discriminate the high and moderate metal pollution sites from the rest. Zn, the metal with the highest contribution to potential toxicity, presented a fast and high accumulation capacity in biofilms. According to the multivariate analysis, AEA showed different responses. While catalase (CAT) and ascorbate peroxidase (APX) variability was mainly attributed to environmental stress (pH, temperature and phosphate concentration), glutathione-S-transferase (GST) changes were related to metal pollution. Glutathione reductase (GR) and superoxide dismutase (SOD) responses were related to both stress factors. AEA and metal accumulation are proposed as sensitive effect-based field methods, to evaluate biofilm responses after acute metal exposure (e.g. an accidental spill) due to their capacity to respond after few hours, but also in routinely monitoring due to their persistent changes after few weeks of exposure. These tools could improve the Common Implementation Strategy (CIS) of the Water Framework Directive (WFD) as expert group request.

The use of antioxidant enzymes in freshwater biofilms: Temporal variability vs. toxicological responses

This study aims to investigate the potential of antioxidant enzyme activities (AEA) as biomarkers of oxidative stress in freshwater biofilms. Therefore, biofilms were grown in channels for 38 days and then exposed to different concentrations (0-150 μg L(-1)) of the herbicide oxyfluorfen for 5 more weeks. Under control conditions, the AEA of biofilms were found to change throughout time with a significant increase in ascorbate peroxidase (APX) activity during the exponential growth and a more important role of catalase (CAT) and glutathione reductase (GR) activities during the slow growth phase. Chronic exposure to oxyfluorfen led to slight variations in AEA, however, the ranges of variability of AEA in controls and exposed communities were similar, highlighting the difficulty of a direct interpretation of AEA values. After 5 weeks of exposure to oxyfluorfen, no clear effects were observed on chl-a concentration or on the composition of other pigments suggesting that algal group composition was not affected. Eukaryotic communities were structured clearly by toxicant concentration and both eukaryotic and bacterial richness were reduced in communities exposed to the highest concentration. In addition, during acute exposure tests performed at the end of the chronic exposure, biofilms chronically exposed to 75 and 150 μg L(-1) oxyfluorfen showed a higher CAT activity than controls. Chronic exposure to oxyfluorfen provoked then structural changes but also functional changes in the capacity of biofilm CAT activity to respond to a sudden increase in concentration, suggesting a selection of species with higher antioxidant capacity. This study highlighted the difficulty of interpretation of AEA values due to their temporal variation and to the absence of absolute threshold value indicative of oxidative stress induced by contaminants. Nevertheless, the determination of AEA pattern throughout acute exposure test is of high interest to compare oxidative stress levels undergone by different biofilm communities and thus determine their antioxidant capacity.

Interactive effects of dissolved nitrogen, phosphorus and litter chemistry on stream fungal decomposers

The enrichment of ecosystems by nutrients such as nitrogen (N) and phosphorus (P) has important ecological consequences. These include effects on plant litter decomposition in forest soils and forested headwater streams, where fungi play a pivotal role. However, our understanding of nutrient relationships on fungal communities associated with decomposing litter remains surprisingly incomplete. We conducted a fully factorial microcosm experiment with known communities of fungal decomposers from streams to assess the importance of dissolved N and P supply, as well as the atomic nutrient ratio (N:P), on fungal community succession, diversity, biomass and reproduction on three leaf-litter species differing in nutrient and lignin concentrations. Fungal biomass accrual and spore production were strongly controlled by external N supply, whereas P supply was much less important. The magnitude of these effects was mediated by litter quality, with stronger effects of dissolved N and P on lignin-poor and high N:P litter. N supply also influenced fungal diversity and species composition, acting as a pacemaker of community succession. Collectively, our data indicate that N was in much greater demand than predicted by standard stoichiometric models. The most parsimonious explanation for this deviation relates to the need of litter fungi to invest large amounts of N into degradative exoenzymes.