Periodic Reporting for period 2 - ARISTO (The European Industry - Academia Network for RevIsing and Advancing the Assessment of the Soil Microbial TOxicity of Pesticides)
Periodo di rendicontazione: 2022-12-01 al 2024-11-30
Pesticides are major environmental pollutants whose placement in the market is under stringent regulatory control. Still certain pieces of this regulatory framework like the assessment of the toxicity of pesticides on the soil microbiota have been identified as problematic by EFSA and the scientific community and should be revisited in light of methodological advances, new scientific knowledge and deficiencies of the obsolete methods used (OECD 216 N transformation test). ARISTO addressed these scientific and regulatory gaps through a unique doctoral program, aiming to train the next generation of Microbial Ecotoxicologists, a crew of 9 PhD students that received intersectoral training by 7 academic and 9 industrial beneficiaries. The ARISTO project has revolutionized the way we assess the toxicity of pesticides on soil microbiota. It proposes a tiered risk assessment approach built around two key functional microbial groups, ammonia-oxidizing microorganisms (AOM) and arbuscular mycorrhizal fungi (AMF), to determine the potential effect of pesticides on the soil microbiota (Figure 1). The implementation of this tiered approach is supported by relevant tools (e.g. Single Species Tests – Tier I; Protocols for defining indirect pesticide effects; In silico tools for predicting pesticide toxicity on the soil microbiota) developed in the frame of the project. The main research challenge of ARISTO was addressed through 5 objectives structured in corresponding research WPs:
1. to develop and standardize pioneering in vitro tests, to assess pesticides toxicity on phylogenetically and ecophysiologically distinct soil ammonia oxidizing microorganisms (AOM) (fellow 1) and arbuscular mycorrhizal fungi (fellow 2)
2. to develop and implement advanced experimental lab and field tests to assess pesticides toxicity on natural soil assemblages of AOM (fellow 3) and AMF (fellow 4), as a more realistic assessment step
3. to go beyond the "single organism (in vitro)" and "single group assays (natural soil assemblages)" and offer an ecosystem-level assessment of the toxicity of pesticides on soil microbial networks (fellow 5) and on microorganisms from different trophic-levels within the soil food-web (predator - prey) (fellow 6), and the consequences of these on ecosystem functioning
4. to develop novel tools and procedures to determine in a comprehensive way the soil microbial toxicity of pesticide mixtures (fellow 7) and bio-pesticides (fellow 8) which remains unexplored
5. to develop and validate advanced in silico tools for prioritizing transformation products (TPs) of pesticides with potential toxicity to soil microorganisms (fellow 9)
1. to develop and standardize pioneering in vitro tests, to assess pesticides toxicity on phylogenetically and ecophysiologically distinct soil ammonia oxidizing microorganisms (AOM) (fellow 1) and arbuscular mycorrhizal fungi (fellow 2)
2. to develop and implement advanced experimental lab and field tests to assess pesticides toxicity on natural soil assemblages of AOM (fellow 3) and AMF (fellow 4), as a more realistic assessment step
3. to go beyond the "single organism (in vitro)" and "single group assays (natural soil assemblages)" and offer an ecosystem-level assessment of the toxicity of pesticides on soil microbial networks (fellow 5) and on microorganisms from different trophic-levels within the soil food-web (predator - prey) (fellow 6), and the consequences of these on ecosystem functioning
4. to develop novel tools and procedures to determine in a comprehensive way the soil microbial toxicity of pesticide mixtures (fellow 7) and bio-pesticides (fellow 8) which remains unexplored
5. to develop and validate advanced in silico tools for prioritizing transformation products (TPs) of pesticides with potential toxicity to soil microorganisms (fellow 9)
Building around the tiered risk assessment approach, the PhD students in WP4 have developed and standardized assays to determine the toxicity of pesticides on AOM and AMF strains in vitro (DL. 4.1. and 4.2.). These constitute ready to implement Tier I – Single Species Tests that will be going through further ISO standardization as a main outcome of ARISTO. These results are described in two articles submitted in Environ. Sci. Technol (AOM) and Ecological Indicators (AMF). A booklet with detailed protocols for the cultivation of the tested AOM and AMF strains (DL 4.3.) and a database with EC50 values for a range of pesticides on AOM and AMF is provided (DL 4.4.). In WP5, the toxicity of pesticides on natural soil microbial assemblages was determined in lab and field trials (Tier II and III). Mesocosm experiments determined pesticide effects in the frame of climate change pressure with results indicating that AOM were affected mostly by drought/rewetting but not by pesticides. In parallel, a series of pot experiments identified potential off-target effects of a range of pesticides on the soil and intraradical AMF community. Finally, two field experiments were undertaken in France and Germany to define the impact of three selected pesticides on AOM and AMF natural soil assemblages. All these efforts are summarized in DL 5.1. and DL5.2. In WP6, ARISTO looked at the effects of pesticides across trophic levels and on microbial networks. Soil microcosm studies manipulated with different fractions of the soil microbiota showed for the first time the contribution of indirect effects on the soil microbiota. These results were published in the J. Hazard. Mat. (https://doi.org/10.1016/j.jhazmat.2024.134231(si apre in una nuova finestra)). Parallel soil microcosms showed that the main driver of the bacterial community composition was the diversity of protists, and, if this is affected by the pesticides, then cascading effects on the bacterial community should be expected (DL 6.3). In WP7, fellows addressed open issues in ERA like the potential toxicity of pesticide mixtures and biopesticides on soil microbiota. Lab tests showed that abiotic stressors (e.g. heat or high moisture) combined with applications of pesticide mixtures have a compounded effect on the soil microbiota. Parallel soil microcosms showed that the OECD 216 test failed to identify effects of biopesticides on AOM which were clearly highlighted by q-PCR. This work was published in Pest Manag. Sci. (https://doi.org/10.1002/ps.7961(si apre in una nuova finestra)). Furthermore, the use of Species Sensitivity Distributions (SSDs) using amplicon sequencing data was explored to obtain threshold values for risk assessment (HC5). Finally, in WP8, an in silico workflow for predicting and prioritizing pesticide transformation products according to their potential toxicity to AOM was developed (Zhang, Fenner, Bioinformatics, 39(7), 2023). Secondly, a model for predicting the toxicity of pesticides and their TPs on AOB was developed and validated with data from WP4. Finally, a graph neural network (GNN) model that had been pre-trained with lipophilicity data (logP) identified structural alerts linked to logP and toxicity towards nitrification (manuscript submitted to Env. Sci. Technol.).
ARISTO has produced data that will benchmark assessment of the soil microbial toxicity of pesticides in the following ten years:
- Proposed a tiered risk assessment approach with AOM and AMF as microbial ecotoxicity indicators
- Developed and validated, ready to implement in this tiered approach, Single Species Tier I tests for AOM and AMF
- Established protocols to assess pesticides toxicity on natural soil microbial assemblages of AMF and AOM
- Revealed the strong contribution of indirect effects of pesticides on the soil microbiota and provided experimental protocols on how to determine these effects
- Developed methods to determine the effects of pesticides along the soil-food web across different trophic levels that have been overlooked so far
- Demonstrated the low resolution of the OECD 216 test over ISO standardized q-PCR methods for assessing the toxicity of pesticides on the soil microbiota
- Highlighted the potential use of SSDs for derivation of quantitative toxicity thresholds by amplicon sequencing microbiome data
- Developed in silico tools for(a) predicting and prioritizing pesticide transformation products according to their potential toxicity to AOM (b) identifying structural alerts linked with toxicity on AOM
The results have been presented in regulatory bodies and industrial actors which provided feedback that was considered for a final fine tuning of the approach and tools developed.
- Proposed a tiered risk assessment approach with AOM and AMF as microbial ecotoxicity indicators
- Developed and validated, ready to implement in this tiered approach, Single Species Tier I tests for AOM and AMF
- Established protocols to assess pesticides toxicity on natural soil microbial assemblages of AMF and AOM
- Revealed the strong contribution of indirect effects of pesticides on the soil microbiota and provided experimental protocols on how to determine these effects
- Developed methods to determine the effects of pesticides along the soil-food web across different trophic levels that have been overlooked so far
- Demonstrated the low resolution of the OECD 216 test over ISO standardized q-PCR methods for assessing the toxicity of pesticides on the soil microbiota
- Highlighted the potential use of SSDs for derivation of quantitative toxicity thresholds by amplicon sequencing microbiome data
- Developed in silico tools for(a) predicting and prioritizing pesticide transformation products according to their potential toxicity to AOM (b) identifying structural alerts linked with toxicity on AOM
The results have been presented in regulatory bodies and industrial actors which provided feedback that was considered for a final fine tuning of the approach and tools developed.