Eleftheria BACHTSEVANI defended her PhD on Dec. 16th, 2024.
Place : Amphithéatre 3, École Centrale de Lyon (36 Av. Guy de Collongue, 69134 Écully)
Jury :
Rapporteurs :
Kalliope PAPADOPOULOU, Professeur, University of Thessaly
Graeme PATON, Professeur, University of Aberdeen
Examinateurs :
Feth el Zahar HAICHAR, Professeure, INSA Lyon
Christopher VAN DER GAST, Professeur, Northumbria University
Encadrement :
Graeme W. NICOL, directeur de recherche, Ecole Centrale de Lyon, Directeur de thèse
Christina HAZARD, ingénieur de recherche , Ecole Centrale de Lyon, Co-directrice de thèse
Abstract :
The use of pesticides is essential for boosting agricultural yields, but much of these chemicals miss their targets, accumulating in soil and harming ecosystems and microorganisms. Despite the importance of soil microbes in ecosystem health, traditional pesticide risk assessments have not fully addressed the toxicity of pesticides on them. To address this, recent studies proposed a tiered system to assess pesticide effects on the key microbial group of ammonia-oxidizing microorganisms (AOM). The system progresses from in vitro toxicity tests (Tier I) to soil microcosms or pot studies (Tier II), and finally to field-scale evaluations (Tier III). The main objectives of this thesis were to i) develop and validate an in vitro bioassay using AOM and functionally related NOB as a standard Tier I assay, related to the single-species tests commonly used in aquatic ecotoxicology, ii) compare the toxicity of pesticides on AOM with an existing toxicity assessment assay, the Microbial Assay for Risk Assessment (MARA) kit from NCIMB (Scotland), and iii) characterize the physiology of Ca. Nitrobacter laanbroekii NHB1, one of the most sensitive strains identified in this thesis. The toxicity of representative pesticides—covering insecticides, fungicides, and herbicides— was assessed on several ammonia-oxidizing microorganisms (AOM), including ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA), and nitrite-oxidizing bacteria (NOB). Toxicity endpoints (EC50) were determined for each strain-pesticide combination, with Nitrosotalea sinensis Nd2 (AOA), Nitrosospira briensis (AOB), and Ca. Nitrobacter laanbroekii NHB1 (NOB) identified as the most sensitive strains and suitable bioindicators for the proposed single species bioassay. Fungicides and insecticides were generally more toxic to AOM than NOB, while herbicides showed variable toxicity across all nitrifier groups. This testing approach proved valuable for Tier I ecotoxicity assessments of pesticides on soil microbial communities. Building on these findings, toxicity of additional pesticides from different categories on the three most sensitive strains was assessed. Using the proposed single-species in vitro assay, EC50 values confirmed these strains as effective bioindicators for Tier I testing framework. N. sinensis was the most sensitive strain, followed by N. briensis and Ca. N. laanbroekii NHB1, indicating these strains are promising bioindicators for pesticide toxicity in soil. Fungicides were most toxic to N. sinensis, causing significant inhibition at agronomical concentration levels. The differential sensitivities of these strains to various pesticide classes highlight the need for a comprehensive approach that captures a broad spectrum of microbial responses. The evaluation of pesticides toxicity using the MARA kit, which uses 11 heterotrophic microbial strains to detect toxicity, demonstrated relatively low sensitivity, with pesticides affecting these strains only at high concentrations, in contrast to AOM which showed much higher sensitivity to pesticides. The physiology of Ca. Nitrobacter laanbroekii NHB1, one of the identified bioindicator was also characterised. The strain demonstrated an acidotolerant physiology with optimal growth at pH 6.0 and detectable growth down to pH 3.5. In co-culture, Ca. N. laanbroekii NHB1 enhanced the growth of acidophilic ammonia-oxidizing archaea (AOA), such as Nitrosotalea devaniterrae Nd1 and N. sinensis, by removing inhibitory nitrite. This cross-feeding mechanism underscores the importance of substrate concentrations in microbial interactions in acidic soils.
Keywords: ammonia oxidizing bacteria, ammonia oxidizing archaea, nitrite oxidizing bacteria, ecotoxicity, pesticides