Microsoil - A comprehensive assessment of the effects of chemicals on soil microorganisms

Soil health is an important factor for sustainable agriculture and must be maintained and promoted. Soil microorganisms are integral components of the soil and play an essential role in biogeochemical cycles of nutrients such as carbon, nitrogen or phosphorus. They promote the decomposition of organic matter and thus humus formation and are crucial for maintaining soil health. Agricultural soils are exposed to a wide range of stresses from chemicals such as pesticides and veterinary pharmaceuticals. As a result, the resilience and thus the ability of soil microorganisms to respond to stressors such as climate change can decrease significantly. Accordingly, the European Commission is committed to update the terrestrial risk assessment guidelines for chemicals.

In order to achieve the fundamental protection goal of "maintaining soil biodiversity" for microorganisms, Fraunhofer IME, in cooperation with the German Environment Agency, has carried out the MICROSOIL project.

The aim of the project was to identify meaningful test systems and endpoints to enable the assessment of the effects of pesticides and veterinary pharmaceuticals on microorganisms in agricultural soils.

Three research objectives were formulated:

• Identification of suitable methods for assessing changes in soil function and the structure of the soil microbiome, i.e. the community of bacteria, fungi and archaea
• Investigation of the effects of antibiotics and veterinary pharmaceuticals on the development of resistance genes in soil microorganisms
• Investigation of the influence of multiple applications of plant protection products and background contamination on microbial pollutant degradation in soils.

Effects on the function and structure of soil microbial communities

In the current environmental risk assessment for plant protection products, it is assumed that the microbial communities in the soil and their ecosystem services are not at risk if the predicted environmental concentration after 100 days does not affect the nitrogen transformation (OECD guideline 216) by more than 25% compared to a control. On the basis of a literature review, five methods were identified that appeared suitable to replace or supplement the current regulatory relevant test, the nitrogen transformation, in an environmental risk assessment. The sensitivity of the individual methods was examined with six test substances in three soils. Based on our results, it is recommended to extend the first-tier risk assessment and include an additional test method for enzyme activity (ISO 20130: Measurement of enzyme activity patterns in soil samples with colorimetric substrates in microtiter plates, 2021), which observes effects on exoenzymes released by bacteria, and a test method with arbuscular mycorrhizal fungi (symbiontic fungi) (ISO 10832: Effects of pollutants on mycorrhizal fungi – Spore germination test, 2011). A molecular biological approach (automated rRNA intergenic spacer analysis – ARISA) to assess effects on the structure of the soil microbiome is also recommended, but requires further research before it can be included in an environmental assessment scheme.

Effects of antibiotics and veterinary pharmaceuticals on the development of resistance genes in soil microorganisms

Experiments on the toxicity of antibiotics to individual microorganism strains and the development of antibiotic resistance play an important role in the risk assessment of antibiotics. To this end, the minimum inhibitory concentrations of antibiotics are determined for clinically relevant bacterial strains. Such data are available from the EUCAST database for various antibiotics. However, the data is not available for soil-relevant bacterial strains. Therefore, as part of the project minimum inhibitory concentrations (MIC) for four soil-relevant bacterial strains were determined. For three of the four antibiotics/veterinary pharmaceuticals tested, no systematic difference was found between the MICs for the tested soil-relevant and clinical strains (from the EUCAST database). However, in one case, a soil bacterium showed a lower MIC compared to clinical strains. The results suggest that standard ecotoxicological tests may underestimate the risk of certain veterinary pharmaceuticals in terms of the development of antibiotic resistance in soil bacteria. However, the data is currently too limited to verify the suspected development of antibiotic resistance in soil microorganisms.

Influence of multiple applications of plant protection products on microbial pollutant degradation in soils

The degradability of plant protection products and veterinary pharmaceuticals is routinely determined in laboratory studies (OECD guideline 307) based on the maximum application rate of the plant protection product and using reference soils. However, several plant protection products may be applied during a single vegetation period, and these may contain several active substances, which is not considered in the current environmental risk assessment. Experiments on the degradation capacity of soil microorganisms when two reference soils were exposed to the fungicide pyraclostrobin and the herbicide ethofumesate were designed to show whether multiple applications of one of the two active substances, of a binary mixture, or potential background contamination affect the degradability of plant protection products in soil.

Multiple applications of the same plant protection product, as well as the presence of another plant protection product in the soil, had both positive and negative effects on the degradation performance of soil microorganisms. For example, a slight decrease in the degradation of ethofumesate was observed when the soil was previously contaminated with pyraclostrobin. The results suggest that further research into multiple exposure is needed to consider more realistic application patterns and agricultural practices.

Conclusion

The MICROSOIL project highlights the need for the European Commission's mandate to update the terrestrial ecotoxicology guidance document for plant protection products and provides specific recommendations on how this guidance should be updated in relation to soil microorganisms.