In vitro assessment of the toxicity of pesticides on AOM

Pesticides are considered major environmental pollutants. The toxicity of these compounds is regulated by a European Union framework that is based on assays established for aquatic and terrestrial organisms, but not for soil microorganisms. Recent studies have indicated that ammonia-oxidizing microbes (AOM), which perform the first and typically rate-limiting step of nitrification, are ideal microbial indicators of agrochemicals’ toxicity because of their key functional role, their sensitivity to xenobiotic compounds, and the availability of established tools to measure activity and abundance both in vitro and in situ. In my thesis, I am working on the in vitro assessment of the toxicity of pesticides on ammonia oxidizing microorganisms and other microorganisms participating in the nitrification process in order to develop and standardize in vitro tests, as the first sensitive analysis, to assess pesticide toxicity on phylogenetically and ecophysiologically distinct soil nitrifying isolates at activity level. Also, for compounds that exhibit strong effects in vitro, the toxicity mechanism is explored via transcriptomics and proteomics.

First step of our study was the identification of the most sensitive stains among ammonia oxidizing bacteria (AOB), ammonia oxidizing archaea (AOA) and nitrite oxidizing bacteria (NOB). For this purpose, we monitored the activity of 12 representative soil-derived nitrifying isolates grown in liquid cultures via nitrite production or consumption, over a broad range of pesticides concentrations. Specifically, we used six AOB strains (Nitrosomonas europaea, Nitrosomonas communis, Nitrosomonas oligotropha, Nitrosomonas ureae, Nitrosospira multiformis and Nitrosospira briensis), three AOA strains (Candidatus Nitrosocosmicus franklandianus, Candidatus Nitrosotalea sinensis and Nitrosospaera viennensis), and three NOB strains, (Nitrobacter sp. NHB1, Nitrobacter winogradskyi and Nitrospira defluvii). According to our first results all pesticides affected at least one non-target nitrifier with the different strains exhibiting various levels of sensitivity. Our findings are expected to contribute to the development of novel ecotoxicity tools and a risk assessment scheme characterising impacts of pesticide on non-target soil microbes.

Experimental setup

Fig. 1: Experimental set up of the in vitro test for the identification of the most sensitive strains among the different nitrifying groups. The toxicity of selected pesticides of all major classes (herbicides, insecticides, fungicides) was studied in a range of phenotypically and ecologically distinct strains of ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA) and nitrite-oxidizing bacteria (NOB) representing globally distributed lineages found in soil. Toxicity was assessed at the functional level by measuring nitrite production or consumption in liquid cultures of AOB/AOA and NOB respectively, amended with a broad range of pesticides concentrations, and relevant toxicity endpoints (EC50s) were calculated.

In my thesis, I am studying if and how pesticides can affect ammonia-oxidizing microorganisms and other microorganisms participating in the nitrification process in order to develop bioassays using cultures of soil nitrifiers, as a novel ecotoxicity tool for characterizing the impacts of pesticides on non-target soil microbes.

Experiment in pictures

Fig. 2: Incubation of liquid cultures of ammonia oxidizing archaea.
Fig. 3: Liquid cultures of ammonia oxidizing bacteria ready to add the pesticides.

Fig. 4: Nitrite concentrations in liquid cultures are determined colorimetrically in a 96-well plate assay based on the analytical biochemistry Griess test.


Fig. 5: ES1 Eleftheria Bachtsevani presented her first results in the Soil Microbial Ecology poster session and discuss the ARISTO project and her work with other researchers in 18th International Symposium on Microbial Ecology (ISME), Lausanne, Switzerland, 14-19 August.

Fig. 6: ARISTO members, fellows, and PIs, participated in the 18th International Symposium on Microbial Ecology (ISME) in Lausanne, Switzerland, 14-19 August.