Assessing the chronic effect of the bioavailable fractions of radionuclides and heavy metals on stream microbial communities: A case study at the Rophin mining site.

This study aimed to assess the potential impact of long-term chronic exposure (69 years) to naturally-occurring radionuclides (RNs) and heavy metals on microbial communities in sediment from a stream flowing through a watershed impacted by an ancient mining site (Rophin, France). Four sediment samples were collected along a radioactivity gradient (for 238U368 to 1710 Bq.Kg-1) characterized for the presence of the bioavailable fractions of radionuclides (226Ra, 210Po), and trace metal elements (Th, U, As, Pb, Cu, Zn, Fe). Results revealed that the available fraction of contaminants was significant although it varied considerably from one element to another (0 % for As and Th, 5-59 % for U). Nonetheless, microbial communities appeared significantly affected by such chronic exposure to (radio)toxicities. Several microbial functions carried by bacteria and related with carbon and nitrogen cycling have been impaired. The high values of fungal diversity and richness observed with increasing downstream contamination (H' = 4.4 and Chao1 = 863) suggest that the community had likely shifted toward a more adapted/tolerant one as evidenced, for example, by the presence of the species Thelephora sp. and Tomentella sp. The bacterial composition was also affected by the contaminants with enrichment in Myxococcales, Acidovorax or Nostocales at the most contaminated points. Changes in microbial composition and functional structure were directly related to radionuclide and heavy metal contaminations, but also to organic matter which also significantly affected, directly or indirectly, bacterial and fungal compositions. Although it was not possible to distinguish the specific effects of RNs from heavy metals on microbial communities, it is essential to continue studies considering the available fraction of elements, which is the only one able to interact with microorganisms.

Cyanosphere Dynamic During Dolichospermum Bloom: Potential Roles in Cyanobacterial Proliferation.

Under the effect of global change, management of cyanobacterial proliferation becomes increasingly pressing. Given the importance of interactions within microbial communities in aquatic ecosystems, a handful of studies explored the potential relations between cyanobacteria and their associated bacterial community (i.e., cyanosphere). Yet, most of them specifically focused on the ubiquitous cyanobacteria Microcystis, overlooking other genera. Here, based on 16s rDNA metabarcoding analysis, we confirmed the presence of cyanosphere representing up to 30% of the total bacterial community diversity, during bloom episode of another preponderant cyanobacterial genus, Dolichospermum. Moreover, we highlighted a temporal dynamic of this cyanosphere. A sPLS-DA model permits to discriminate three important dates and 220 OTUs. With their affiliations, we were able to show how these variations potentially imply a turnover in ecological functions depending on bloom phases. Although more studies are necessary to quantify the impacts of these variations, we argue that cyanosphere can have an important, yet underestimated, role in the modulation of cyanobacterial blooms.

Impact of Leptospermone, a Natural ß-Triketone Herbicide, on the Fungal Composition and Diversity of Two Arable Soils.

Impact of leptospermone, a ß-triketone bioherbicide, was investigated on the fungal community which supports important soil ecological functions such as decomposition of organic matter and nutrients recycling. This study was done in a microcosm experiment using two French soils, Perpignan (P) and Saint-Jean-de-Fos (SJF), differing in their physicochemical properties and history treatment with synthetic ß-triketones. Soil microcosms were treated with leptospermone at recommended dose and incubated under controlled conditions for 45 days. Untreated microcosms were used as control. Illumina MiSeq sequencing of the internal transcribed spacer region of the fungal rRNA revealed significant changes in fungal community structure and diversity in both soils. Xylariales, Hypocreales, Pleosporales and Capnodiales (Ascomycota phyla) fungi and those belonging to Sebacinales, Cantharellales, Agaricales, Polyporales, Filobasidiales and Tremellales orders (Basidiomycota phyla) were well represented in treated soil microcosms compared to control. Nevertheless, while for the treated SJF a complete recovery of the fungal community was observed at the end of the experiment, this was not the case for the P treated soil, although no more bioherbicide remained. Indeed, the relative abundance of most of the saprophytic fungi were lower in treated soil compared to control microcosms whereas fungi from parasitic fungi included in Spizellomycetales and Pezizales orders increased. To the best of our knowledge, this is the only study assessing the effect of the bioherbicide leptospermone on the composition and diversity of the fungal community in soil. This study showed that leptospermone has an impact on α- and ß-diversity of the fungal community. It underlines the possible interest of microbial endpoints for environmental risk assessment of biopesticide.

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 ß-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.

Functional and structural responses of soil N-cycling microbial communities to the herbicide mesotrione: a dose-effect microcosm approach.

Microbial communities driving the nitrogen cycle contribute to ecosystem services such as crop production and air, soil, and water quality. The responses to herbicide stress of ammonia-oxidizing and ammonia-denitrifying microbial communities were investigated by an analysis of changes in structure-function relationships. Their potential activities, abundances (quantitative PCR), and genetic structure (denaturing gradient gel electrophoresis) were assessed in a microcosm experiment. The application rate (1 × FR, 0.45 µg g(-1) soil) of the mesotrione herbicide did not strongly affect soil N-nutrient dynamics or microbial community structure and abundances. Doses of the commercial product Callisto® (10 × FR and 100 × FR) or pure mesotrione (100 × FR) exceeding field rates induced short-term inhibition of nitrification and a lasting stimulation of denitrification. These effects could play a part in the increase in soil ammonium content and decrease in nitrate contents observed in treated soils. These functional impacts were mainly correlated with abundance shifts of ammonia-oxidizing Bacteria (AOB) and Archaea (AOA) or denitrifying bacteria. The sustained restoration of nitrification activity, from day 42 in the 100 × FR-treated soils, was likely promoted by changes in the community size and composition of AOB, which suggests a leading role, rather than AOA, for soil nitrification restoration after herbicide stress. This ecotoxicological community approach provides a nonesuch multiparameter assessment of responses of N-cycling microbial guilds to pesticide stress.

Soil surface colonization by phototrophic indigenous organisms, in two contrasted soils treated by formulated maize herbicide mixtures.

Soil phototrophic microorganisms, contributors to soil health and food webs, share their particular metabolism with plants. Current agricultural practices employ mixtures of pesticides to ensure the crops yields and can potentially impair these non-target organisms. However despite this environmental reality, studies dealing the susceptibility of phototrophic microorganisms to pesticide mixtures are scarce. We designed a 3 months microcosm study to assess the ecotoxicity of realistic herbicide mixtures of formulated S-metolachlor (Dual Gold Safeneur(®)), mesotrione (Callisto(®)) and nicosulfuron (Milagro(®)) on phototrophic communities of two soils (Limagne vertisol and Versailles luvisol). The soils presented different colonizing communities, with diatoms and chlorophyceae dominating communities in Limagne soil and cyanobacteria and bryophyta communities in Versailles soil. The results highlighted the strong impairment of Dual Gold Safeneur(®) treated microcosms on the biomass and the composition of both soil phototrophic communities, with no resilience after a delay of 3 months. This study also excluded any significant mixture effect on these organisms for Callisto(®) and Milagro(®) herbicides. We strongly recommend carrying on extensive soil studies on S-metolachlor and its commercial formulations, in order to reconsider its use from an ecotoxicological point of view.

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.

Dose-dependent effects of the herbicide mesotrione on soil cyanobacterial communities.

This study aimed to investigate the dose-response effects of an herbicide on soil photosynthetic microbial communities, particularly cyanobacteria, using a microcosm approach. Pure mesotrione (active ingredient), and Callisto (a commercial formulation of this triketone herbicide), were spread at different rates on soil microcosm surfaces. Soil Chlorophyll concentrations were quantified to assess the photosynthetic biomass, and the genetic structure and diversity of the cyanobacterial community were investigated by a group-specific polymerase chain reaction followed by denaturing gradient gel electrophoresis. Dose-dependent responses were evidenced for both functional and structural parameters. No effect was detected in soils treated with 1 × AR (1-fold recommended application rate) irrespective of the herbicide formulation. At 10 × AR (10-fold recommended application rate), only Callisto treatment induced significant decreases of photosynthetic biomass, whereas structural parameters were less affected. At the 100 × AR (100-fold recommended application rate), both pure mesotrione and Callisto had strong negative impacts on soil chlorophyll concentrations and cyanobacterial genetic structure and diversity. At both the 10 × AR and 100 × AR treatments, Callisto induced significant stronger effects than pure mesotrione. In addition, indicators of photosynthetic biomass, compared with structural parameters of cyanobacterial communities (within 14 days), responded (within 7 days) more quickly to herbicide stress. The results of this study underscore the relevance of soil photosynthetic microbial communities to develop indicators for herbicide risk assessment.

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.