Mixtures effect of antibiotics on soil microbial nitrogen processes

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.