Ecotoxicological impact of sulfamethoxazole on nitrogen cycling microbial communities, in agricultural amended soil

Background and objectives The 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 (Kummerer 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 results Here, 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 conclusions 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 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. 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