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National audience; L'usage des pesticides est une menace pour la biodiversité et l'activité des communautés microbiennes présentes dans les agrosystèmes. L'altération des communautés microbiennes peut avoir des conséquences sur des fonctions écosystémiques conduisant à la diminution de la fertilité et des capacités épuratrices des sols contribuant à diminuer les rendements des cultures et à augmenter la pollution des sols et des eaux, respectivement. Il reste toutefois difficile d'estimer l'impact des pesticides sur des fonctions écosystémiques et de suivre leur résilience suite à des changements de pratique agricole. Dans ce contexte, le projet IMPEC visait à tester et à développer de nouveaux indicateurs microbiens pour estimer l'impact des pesticides sur des fonctions écosystémiques liées au (i) cycle des nutriments et (ii) service épuration des compartiments terrestres et aquatiques. Ces nouveaux indicateurs ont été utilisés pour évaluer a priori et a posteriori l'impact écotoxicologique de pesticides sur la composition taxonomique et sur des traits fonctionnels microbiens des sols et des sédiments. Ils ont été pour partie transférés via le développement de normes ISO pouvant être utilisées pour évaluer l'impact écotoxicologique a priori et a posteriori des pesticides sur les microorgansimes du sol. Une synthèse des principaux résultats du programme IMPEC ainsi que les perspectives en termes de transfert et de recherche seront présentées.

Etudes après études, les pesticides sont identifiés comme une menace pour la diversité et l’activité des microorganismes des sols. Bien que cette menace soit identifiée, nous restons démunis pour estimer l’impact écotoxicologique des pesticides sur des fonctions écosystémiques des sols. Ainsi, l’évaluation a priori des effets des pesticides sur les microorganismes des sols repose sur l’estimation de leur impact sur la minéralisation du carbone et de l’azote. Ces tests trop globaux ne rendent pas compte de l’effet des pesticides sur les microorganismes. Cette étude a testé un bouquet d’indicateurs pour évaluer l’impact écotoxicologique de pesticides sur les communautés microbiennes supportant des fonctions écosystémiques des sols et des sédiments. Elle a permis le développement de nouveaux outils et de normes ISO pouvant être utilisés pour évaluer l’impact a priori et a posteriori des pesticides sur la diversité et l’activité des microorganismes des sols et des sédiments., What microbial indicators to assess the ecotoxicological impact of pesticides on terrestrial and aquatic ecosystem functions? Studies after studies, pesticides are identified as a major threat for the diversity and activity of soil microorganisms. Although identified as a threat, there is a lack of methods to estimate the ecotoxicological impact of pesticides on soil ecosystemic functions. Indeed a priori evaluation of pesticide effects on soil microorganisms relies solely on the estimation of their impact on carbon and nitrogen mineralisation. These global tests did not allow to properly measure the effects of pesticides on soil microorganisms. This study tested a panel of indicators to evaluate the ecotoxicological impact of pesticides on microbial communities supporting soil and sediment ecosystemic functions. It leads to the development of new tools and ISO standards ready to be used to evaluate the a priori and a posteriori impact of pesticides on the diversity and activity of soil and sediment microorganisms.

Ce numéro comprend les articles issus du colloque de restitution Ecophyto Recherche « Des outils et des dispositifs pour éclairer les décisions, explorer les possibles et accompagner la transition vers des systèmes économes et multi-performants », qui s’est tenu à Saint-Malo, du 22 au 24 mars 2017.; Quels indicateurs microbiens pour évaluer l’impact écotoxicologique des pesticides sur des fonctions écosystémiques terrestres et aquatiques ?

Background and objectivesThe fate and effects of human and veterinary antibiotics in the environment have been the subject of intense investigation for nearly two decades. While the occurrence and persistence of antibiotics and antimicrobial resistance in environment have become major human health and research issues, their impacts on microbial ecosystem processes (i.e. nutrient recycling) are not well-understood. Many gaps still persist regarding the ecotoxicological assessment of antibiotics as related to environmental risk assessment (ERA). ERA researches have been mainly focused on normalized single species bioassay, and current ecotoxicological studies should aim at bridging the gap existing in the understanding and assessment of antibiotic impact on microbial ecosystem processes. The ERA methodologies for antibiotics have been developed based on the current standard ecotoxicology tests existing guidelines for other chemicals, but such regulatory assessment has been questioned (Boxall et al., 2012). Actually, the modes of action of antibiotics are mainly specific for bacteria, but for bacterial toxicity tests play only a minor role during the ecotoxicological assessment of antibiotics (Kümmerer et al., 2004; Brandt et al., 2015). Consequently, this may overlook adverse effects of antibiotics on microbial ecosystem functions. This issue additionally requires the characterization of the community – function relationships, to identify sensitive, resistant and resilient populations, and the main edaphic factors controlling antibiotic availability and thus microbial exposure to antibiotic. The growing interest for organic wastes recycling as soil amendments, can lead to various diffuse antibiotic contaminations, requiring an ecotoxicological assessment of such agricultural practice. The scope of this work aims at identifying microbial community-based tests and endpoints to improve targeted protection of key microbial soil processes and also unravel the diversity – function relationships involving in the resilience of soil microbial functioning, following application of contaminated amendments. Methods and resultsHere, we investigated the effects of sulfamethoxazole (SMX) on microbial nitrogen transformations. A dose-effect approach was performed with different doses of SMX added to different organic wastes (compost of sewage sludge - green waste (SGW), farmyard manure (FYM)), before their mixing with soil, in microcosms. The final concentrations of SMX in amended soils ranged from 0.022 to 2.22 mg kg-1dw, with control soil microcosms amended with SGW or FYM without SMX. Nitrogen forms, potential nitrification and denitrification activities were determined in soils, after 8, 28 and 84 days following amendments. Abundances and composition of specific microbial guilds (ammonia oxidizing bacteria and archae and some denitrifiers) were determined with molecular tools. The total and available concentrations of SMX were extracted with organic and aqueous solutions, respectively, and then quantified by UHPLC-MS/MS.Discussion and conclusionsOnly the nitrification activity was adversely impaired by SMX, following a dose-response pattern, in SGW amended soils, but not in FYM amended soils. These effects had short-term outcomes for nitrogen nutrient dynamic, decreasing soil nitrate content, SGW amended soils, from 0.22 mg SMX kg-1dw. Organic matter is a key environmental factor influencing the bioavailability and the effects of antibiotics on soil microorganisms. In this experiment, the chemical assessment of SMX availability did not show strong differences between the SGW or FYM amended soils. Actually, the acute toxicity of SMX, following a bacteriostatic action, can be only detected on growing microbial populations, such as nitrifiers, in SGW amended soils with high NH4+ levels. Denitrification activity was only slightly affected by high SMX doses, in SGW amended soils, despite an obvious non limitation of nutrients (nitrate and organic carbon) improving population growth. Different level of taxonomic diversity can explain this different sensitivity between nitrification and denitrification. In addition, the magnitude of effects of low doses of SMX (from 0.22 to 0.66 mg kg-1dw) slightly decreased after 84 days of exposure, compared to days 8 and 28, in accordance with the decrease of SMX availability, measured in SGW amended soils. Analytical chemistry (See presentation Goulas et al.) improves our understanding of community responses, with the determination of the exposure to antibiotics. Furthermore, modification of community composition could contribute to the functional resilience against antibiotic stress, through the substitution of sensitive taxa by more tolerant taxa which can colonize vacant niche (Ollivier et al., 2013).This work reveals that, in high nutrient context, soil nitrification appeared as a sensitive indicator of SMX effects on soil microbial functioning, while denitrification was more resistant. Also, close to realistic environmental concentration, the results highlighted resilience of soil nitrification, following a short-term impairment. The dissipation of SMX, leading to a decrease of soil microorganism exposure, promoted this resilience, in addition to changes in community composition. The availability of antibiotic and ecological characteristics of microbial communities (resistance and resilience) must be integrated in ERA methodologies.Boxall A, Rudd M.A, Brooks B.W., …Topp E, Van Der Kraak G. 2012. Pharmaceuticals and personal care products in the environment: what are the big questions? Environ Health Perspect. 120, 1221–1229.Brandt KK, Topp E, et al., 2015. Ecotoxicological assessment of antibiotics: A call for improved consideration of microorganisms. Environ Inter. 85, 189–205.Kümmerer K, Alexy R, Hüttig J, Schöll A. 2004. Standardized tests fail to assess the effects of antibiotics on environmental bacteria. Water Res. 38, 2111–2116.Ollivier J, Schacht D, Kindler R, Groeneweg J, Engel M, Wilke B-M, Kleineidam K and Schloter M. 2013. Effects of repeated application of sulfadiazine-contaminated pig manure on the abundance and diversity of ammonia and nitrite oxidizers in the root-rhizosphere complex of pasture plants under field conditions. Front Microbio. 4, 22.

Background and objective :During the last two decades, the environmental concern regarding the antibiotics has increased considerably. The persistence of both antibiotics and antimicrobial resistances in the environment has become a major human health and research issue. However their ecotoxicological impacts on microbial ecosystem functions involving in biogeochemical processes, are still not well-understood (Roose-Amsaleg & Laverman, 2016). Furthermore, the environmental risk assessment, based on existing guidelines for other chemicals, may overlook adverse effects of antibiotics on environmental microorganisms and related functions (Boxall et al., 2012), due to the lack of representativeness of ecotoxicological tests for bacterial toxicity in guidelines for environmental risk assessment of antibiotics. In addition, while ecosystems are contaminated by mixtures of chemicals rather than individual substances, toxicity mixture effects of antibiotics on key microbial processes have been not investigated (Brandt et al., 2015). The scope of this study was to unravel the mixture toxicity of antibiotics on soil microbial nitrification and denitrification processes, to improve knowledge of suitable ecotoxicological endpoints, for risk assessment.Methods and results : The tested antibiotics were belonged to several classes (tetracycline, sulfonamide, macrolide, fluoroquinolone) with various modes of action. The effects of each antibiotic individually and as mixtures were assessed across dose-response approaches (performed with R software, DRC package) on potential nitrification in slurry bioassay (NF EN ISO 14238) and denitrification (Roose-Amsaleg et al., 2013) of soil microbial communities. Antibiotic mixtures were built-up in order to test additivity, based on Toxic Unit (TU) ratio, in addition to other mixtures close to environmental surveys (literature data). The antibiotic exposure was verified by measuring the concentrations of antibiotics at the beginning and the end of the experience. Complementary investigations were realized on community structure parameters of specific N-cycling microbial guilds with molecular approaches, to help us in identifying key actors involving in structure – function relationships (species interaction). The first results demonstrated strong differences of magnitude effect among each antibiotic individually, in relation with the mode of action of AB and different levels of sorption on soil components, influencing their bioavailability in the nitrification bioassay. The results of the experimental approaches were analyzed regarding mathematical modeling interpretation based on the concepts of concentration addition or independent action. In many cases, independent action concept better predicted mixture toxicity of different classes of antibiotics, harboring different mode of action. The responses of structural parameters of microbial guilds were observed at higher concentration than the functional endpoints. The decreases of abundances of several microbial groups were correlated with those of the respective activities.Discussion and conclusion : Ecotoxicological effects were recorded at very low concentrations, in our bioassays, based on model microbial communities, in optimal growth conditions. These conditions are not representative of the bulk soil conditions but can correspond to some microbial hot-spots in functional domains, in soils (i.e. rhizosphere). Whilst many previous studies have mainly focused on normalized single species bioassay, significant well-designed studies, improving the integration of ecotoxicological endpoints at the community level, should grab the existing gap on understanding and environmental assessment of mixture impact on microbial ecosystem processes. The archeal nitryfiers / bacterial nitrifyers ratio seems to be related to the magnitude of nitrification responses, only for some antibiotic and the mixture (Konopka et al., 2015). These results can be explained by the lower sensitivity of Archeae to certain antibiotics, notably those targeting the bacterial cell-wall components.

The environmental concern regarding the antibioticshas increased considerably, due to their intensive uses in human and veterinary medicines and the growing interest in organic wastes recycling as soil amendments. While the occurrence and persistence of antibiotics and antimicrobial resistance in environment have become major human health issues, their ecotoxicological impacts on microbial ecosystem processes (organic matter degradation and nutrient recycling) are not well understood. The exposure (fate and bioavailability of antibiotics) and ecological characteristics of microbial communities (resistance and resilience) influence the impacts. Organic matter is a key environmental factor influencing the dissipation and the bioavailability of antibiotics, in soils. Also, organic matter and nutrient inputs influence microbial communities in amended soil. Our objectives were to investigate the effects of the sulfamethoxazole (SMX) on microbial nitrogen transformations. A dose-effect approach was performed with different doses of SMX added to different organic wastes (compost of sewage sludge -green waste (SGW), farmyard manure (FYM)), before their mixing on soil microcosms. The final concentrations of SMX in amended soils ranged from 0.022 to 2.22 mg kg-1dw, with control soil microcosms amended with SGW or FYM without SMX. Nitrogen forms, potential nitrification and denitrification activities were determined in soils, after 8, 28 and 84 days following amendments. The total and available concentrations of SMX were extracted with organic and aqueous solutions, respectively, and then quantified by UPLC-MS/MS. Only the nitrification activity was adversely impaired by SMX, following a dose-response pattern, in SGW amended soils, but not in FYM amended soils. These effects were related to the soil nitrate contents in soil-SGW, as functional output. The chemical assessment of SMX availability did not show strong differences between the SGW or FYM amended soils, in opposite to nitrification response. Actually, the acute toxicity of SMX, following a bacteriostatic action,can be only detected on growing microbial populations, such as nitrifiers, in SGW amended soils with high NH4+levels. Denitrification activity was not consistently affected by SMX, despite high nutrient levels (nitrate and organic carbon). Nitrification appeared as a sensitive indicator of SMX effects on soil microbial functioning, while denitrification seemed to be more resistant. Different level of taxonomic diversity can explain these differences. In addition, the magnitude of effects of low doses of SMX (< 0.5 mg kg-1dw) slightly decrease after 84 days of exposure, compared to days 8 and 28, in accordance with the decrease of SMX availability, measured in SGW amended soils. This highlighted resilience capabilities of soil nitrification, following short-term impairments, which could be promoted by dissipation of SMX, leading to a decrease of its bioavailability. Analytical chemistry plays an important role in understanding ecotoxicity through the assessment of exposure scenario.

Impacts of antibiotic mixtures on microbial ecosystem functions need to be unraveled to improve the environmental risk assessment. This study aims to characterize and predict the toxicity of antibiotic mixtures on soil nitrification and denitrification processes. Different antibiotics (tetracycline, sulfonamide, macrolide, fluoroquinolone) with distinct mechanisms of action, were tested alone and in mixtures by dose-response approaches on soil substrate-induced nitrification (SIN) and minimum inhibitory concentration of soil den- itrifier enrichment (MIC-E). Abundance of ammonia-oxidizers (archeal and bacterial) and denitrifiers (clade I and II of N2O-reducers) were investigated by molecular approach. For nitrification, the experimental results for different antibiotic mixture ratio were further ana- lyzed regarding mathematical modeling interpretation based on the concepts of concentration addition (CA) or independent action (IA). The SIN bioassay highlighted bacteriostatic effects at sulfonamide or macrolides concen- trations lower than observed environmental concentrations. The differences in toxicities of antibiotics individually tested were explained by the sorption coefficient of the various an- tibiotics. Surprisingly, despite different cellular targets of the tested antibiotics, modelling mixture analyses revealed few differences in toxicity prediction between IA or CA concepts. Denitrification showed a higher tolerance than nitrification to the antibiotics and the lowest MIC-E for tetracycline. For nitrification and denitrification, the mixture toxicity was mainly driven by the more toxic compound. The archeal/bacterial nitrifiers ratio was positively related to the magnitude of nitrification inhibition; archaea showing invariant abundances while the antibiotic doses increased. The abundances of the denitrifier nosZ clade II greatly decreased under any antibiotic exposures unlike the nosZ clade I guilds.

The environmental concern regarding the antibiotics has increased considerably, due to their intensive uses in human and veterinary medicines and the growing interest in organic wastes recycling as soil amendments. While the occurrence and persistence of antibiotics and antimicrobial resistance in environm ent have become major human health issues, their ecotoxicological impacts on microbial ecosystem processes (organic matter degradation and nutrient recycling) are not well understood. The exposure (fate and bioavailability of antibiotics) and ecological ch aracteristics of microbial communities (resistance and resilience) influence the impacts. Organic matter is a key environmental factor influencing the dissipation and the bioavailability of antibiotics, in soils. Also, organic matter and nutrient inputs in fluence microbial communities in amended soil. Our objectives were to investigate the effects of the sulfamethoxazole (SMX) on microbial nitrogen transformations. A dose - effect approach was performed with different doses of SMX added to different organic w astes (compost of sewage sludge - green waste (SGW), farmyard manure (FYM)), before their mixing on soil microcosms. The final concentrations of SMX in amended soils ranged from 0.022 to 2.22 mg kg - 1 dw , with control soil microcosms amended with SGW or FYM without SMX. Nitrogen forms, potential nitrification and denitrification activities were determined in soils, after 8, 28 and 84 days following amendments. The total and available concentrations of SMX were extracted with organic and aqueous solutions, res pectively, and then quantified by UPLC - MS/MS. Only the nitrification activity was adversely impaired by SMX, following a dose - response pattern, in SGW amended soils, but not in FYM amended soils. These effects were related to the soil nitrate contents in soil - SGW, as functional output. The chemical assessment of SMX availability did not show strong differences between the SGW or FYM amended soils, in opposite to nitrification response. Actually, the acute toxicity of SMX, following a bacteriostatic action, can be only detected on growing microbial populations, such as nitrifiers, in SGW amended soils with high NH 4 + levels. Denitrification activity was not consistently affected by SMX, despite high nutrient levels (nitrate and organic carbon). Nitrification appeared as a sensitive indicator of SMX effects on soil microbial functioning, while denitrification seemed to be more resistant. Different level of taxonomic diversity can explain these differences. In addition, the magnitude of effects of low doses of S MX (< 0.5 mg kg - 1 dw ) slightly decrease after 84 days of exposure, compared to days 8 and 28, in accordance with the decrease of SMX availability, measured in SGW amended soils. This highlighted resilience capabilities of soil nitrification, following short - term impairments, which could be promoted by dissipation of SMX, leading to a decrease of its bioavailability. Analytical chemistry plays an important role in understanding ecotoxicity through the assessment of exposure scenario.