Clarisse Mallet

Isolation and characterization of mesotrione-degrading Bacillus sp. from soil

Dissipation kinetics of mesotrione, a new triketone herbicide, sprayed on soil from Limagne (Puy-de-Dôme, France) showed that the soil microflora were able to biotransform it. Bacteria from this soil were cultured in mineral salt solution supplemented with mesotrione as sole source of carbon for the isolation of mesotrione-degrading bacteria. The bacterial community structure of the enrichment cultures was analyzed by temporal temperature gradient gel electrophoresis (TTGE). The TTGE fingerprints revealed that mesotrione had an impact on bacterial community structure only at its highest concentrations and showed mesotrione-sensitive and mesotrione-adapted strains. Two adapted strains, identified as Bacillus sp. and Arthrobacter sp., were isolated by colony hybridization methods. Biodegradation assays showed that only the Bacillus sp. strain was able to completely and rapidly biotransform mesotrione. Among several metabolites formed, 2-amino-4-methylsulfonylbenzoic acid (AMBA) accumulated in the medium. Although sulcotrione has a chemical structure closely resembling that of mesotrione, the isolates were unable to degrade it.

Toxicity assessment of the maize herbicides S-metolachlor, benoxacor, mesotrione and nicosulfuron, and their corresponding commercial formulations, alone and in mixtures, using the Microtox® test

The Microtox(®) test, using the prokaryote Vibrio fischeri, was employed to assess the toxicity of the maize herbicides S-metolachlor, benoxacor, mesotrione and nicosulfuron, and their formulated compounds: Dual Gold Safeneur(®), Callisto(®) and Milagro(®); alone and in mixtures. For each compound we obtained original IC50 values, with consistent higher toxicities for formulated compounds compared to active ingredients alone. Mixtures of the four herbicides, prepared according to application doses encountered in agriculture, were found to be toxic at a lower concentration than single molecules. Mesotrione and nicosulfuron mixture appeared to be highly toxic to V. fischeri, however, this recommended post-emergence combination for maize crops got its toxicity decreased in formulated compound mixtures, suggesting that chemical interactions could potentially reduce the toxicity. Data comparisons to theoretical models showed a good prediction of mixture toxicity by Concentration Addition concept. Results seemed to exclude any synergistic effects on V. fischeri for the tested herbicide mixtures. Additional work coupling these bioassay data to ecosystemic level studies (aquatic and soil compartments) and data on additives and degradation products toxicity, will help to fill the gap in our knowledge of the environmental impact of these xenobiotics and in the choice of a more sustainable use of pesticides.

Dynamics of ammonia-oxidizing Archaea and Bacteria in contrasted freshwater ecosystems.

Thaumarchaeota have been recognized as the main drivers of aerobic ammonia oxidation in many ecosystems. However, little is known about the role of ammonia-oxidizing Archaea (AOA) and Bacteria (AOB) in lacustrine ecosystems. In this study, the photic zone of three contrasted freshwater ecosystems located in France was sampled during two periods: winter homothermy (H) and summer thermal stratification (TS), to investigate the distribution of planktonic AOA and AOB. We showed that AOB were predominant in nutrient-rich ecosystems, whereas AOA dominated when ammonia concentrations were the lowest and during winter, which could provide a favorable environment for their growth. Moreover, analyses of archaeal libraries revealed the ubiquity of the thaumarchaeal I.1a clade associated with higher diversity of AOA in the most nutrient-poor lake. More generally, this work assesses the presence of AOA in lakes, but also highlights the existence of clades typically associated with lacustrine and hot spring ecosystems and specific ecological niches occupied by these microorganisms.

Responses of Limagne “Clay/Organic Matter-Rich” Soil Microbial Communities to Realistic Formulated Herbicide Mixtures, Including S-Metolachlor, Mesotrione, and Nicosulfuron

Soil is a primary resource used by mankind to ensure its needs mainly through agriculture. Its sustainability is regulated by the indigenous organisms it contains such as microorganisms. Current agricultural practices employ mixtures of pesticides to ensure the crops yield and can potentially impair these non-target organisms. However despite this environmental reality, studies dealing the susceptibility of microorganisms to pesticide mixtures are scarce. In this context, we designed a 3-month microcosm study to assess the ecotoxicity of realistic herbicide mixtures of formulated S-metolachlor (Dual Gold Safeneur®), mesotrione (Callisto®), and nicosulfuron (Milagro®) on the abundance, the diversity, and the activities of microorganisms from a “clay/organic matter-rich” soil, with a particular attention given to N-cycle communities. These communities appeared to be quite resistant to realistic mixtures even if transient effects occurred on the N-cycle-related communities with an increase of ammonification and an inhibition of nitrification as a short-term effect, followed by an increase of denitrification and an accumulation of nitrates. As nitrates are known to be highly leachable with a strong pollution potential, intensive studies should be carried out at field level to conclude on this potential accumulation and its consequences. Moreover, these data now need to be compared with other agricultural soils receiving these herbicide mixtures in order to bring general conclusion on such practices.

Sensitivity of laccase activity to the fungicide tebuconazole in decomposing litter

The present study investigates the sensitivity of laccase activity to the fungicide tebuconazole (TBZ) in order to seek for new functional toxicity descriptors in aquatic microbial communities associated to decomposing litter. With this aim, we analyzed the sensitivity of laccase from the different microbial components (fungi and bacteria growing separately and in co-existence), as well as that of their corresponding enzyme fractions (cell bound and diffusible), forming microbial communities in Alnus glutinosa leaves. Results show that fungi are pivotal for laccase activity in leaves and that their activity is repressed when they co-exist with bacteria. The sensitivity of laccase activity to the TBZ was only detectable in leaves colonized by fungi separately (Alatospora acuminata populations), but absent in those colonized by bacteria separately and/or mixed fungi plus bacteria. Specifically, the increase of TBZ concentration enhances laccase activity in Alatospora acuminata populations but decreases ergosterol concentration as well as the amount of 18S RNA gene copies. This activity response suggests a detoxification mechanism employed by the fungus in order to reduce TBZ toxicity. Besides, enzyme fractioning showed that laccase activity in the cell bound fraction (76% of the total activity) was sensitive to the fungicide, but not that in the diffusible fraction (24% of total activity). Hence, TBZ would influence laccase activity in the presence of fungal cells but not in enzymes already synthesized in the extracellular space. The present study highlights the importance of the biological complexity level (i. e. population, community, ecosystem) when seeking for appropriate functional ecotoxicity descriptors in aquatic microbial communities.

Stimulation or inhibition: Leaf microbial decomposition in streams subjected to complex chemical contamination

Leaf litter decomposition is a key mechanism in headwater streams, allowing the transfer of nutrients and energy into the entire food web. However, chemical contamination resulting from human activity may exert a high pressure on the process, possibly threatening the structure of heterotrophic microbial communities and their decomposition abilities. In this study, the rates of microbial Alnus glutinosa (Alnus) leaf decay were assessed in six French watersheds displaying different land use (agricultural, urbanized, forested) and over four seasons (spring, summer, autumn, winter). In addition, for each watershed at each sampling time, both upstream (less-contaminated) and downstream (more-contaminated) sections were monitored. Toxicities (estimated as toxic units) predicted separately for pesticides and pharmaceuticals as well as environmental parameters (including nutrient levels) were related to microbial decay rates corrected for temperature and a range of fungal and bacterial community endpoints, including biomass, structure, and activity (extracellular ligninolytic and cellulolytic enzymatic activities). Results showed that agricultural and urbanized watersheds were more contaminated for nutrients and xenobiotics (higher pesticides and pharmaceuticals predicted toxicity) than forested watersheds. However, Alnus decay rates were higher in agricultural and urbanized watersheds, suggesting compensatory effects of nutrients over xenobiotics. Conversely, fungal biomass in leaves was 2-fold and 1.4-fold smaller in urbanized and agricultural watersheds than in the forested watersheds, respectively, which was mostly related to pesticide toxicity. However, no clear pattern was observed for extracellular enzymatic activities except that β-glucosidase activity positively correlated with Alnus decay rates. Together, these results highlight microbial communities being more efficient for leaf decomposition in polluted watersheds than in less contaminated ones, which is probably explained by changes in microbial community structure. Overall, our study showed that realistic chemical contamination in stream ecosystems may affect the biomass of Alnus-associated microbial communities but that these communities can adapt themselves to xenobiotics and maintain ecosystem functions.

Interactive Effects of Pesticides and Nutrients on Microbial Communities Responsible of Litter Decomposition in Streams

Global contamination of streams by a large variety of compounds, such as nutrients and pesticides, may exert a high pressure on aquatic organisms, including microbial communities and their activity of organic matter decomposition. In this study, we assessed the potential interaction between nutrients and a fungicide and herbicide [tebuconazole (TBZ) and S-metolachlor (S-Met), respectively] at realistic environmental concentrations on the structure (biomass, diversity) and decomposition activity of fungal and bacterial communities (leaf decay rates, extracellular enzymatic activities) associated with Alnus glutinosa (Alnus) leaves. A 40-day microcosm experiment was used to combine two nutrient conditions (mesotrophic and eutrophic) with four pesticide treatments at a nominal concentrations of 15 μg L-1 (control, TBZ and S-Met, alone or mixed) following a 2 × 4 full factorial design. We also investigated resulting indirect effects on Gammarus fossarum feeding rates using leaves previously exposed to each of the treatments described above. Results showed interactive effects between nutrients and pesticides, only when nutrient (i.e., nitrogen and phosphorus) concentrations were the highest (eutrophic condition). Specifically, slight decreases in Alnus leaf decomposition rates were observed in channels exposed to TBZ (0.01119 days-1) and S-Met (0.01139 days-1) than in control ones (0.01334 days-1) that can partially be explained by changes in the structure of leaf-associated microbial communities. However, exposition to both TBZ and S-Met in mixture (MIX) led to comparable decay rates to those exposed to the pesticides alone (0.01048 days-1), suggesting no interaction between these two compounds on microbial decomposition. Moreover, stimulation in ligninolytic activities (laccase and phenol oxidase) was observed in presence of the fungicide, possibly highlighting detoxification mechanisms employed by microbes. Such stimulation was not observed for laccase activity exposed to the MIX, suggesting antagonistic interaction of these two compounds on the ability of microbial communities to cope with stress by xenobiotics. Besides, no effects of the treatments were observed on leaf palatability for macroinvertebrates. Overall, the present study highlights that complex interactions between nutrients and xenobiotics in streams and resulting from global change can negatively affect microbial communities associated with leaf litter, although effects on higher trophic-level organisms remains unclear.