The Influence of Soil Properties on Pesticide Toxicity to Soil Invertebrates: Towards a More Environmentally Relevant Risk Assessment

Pesticides can enhance crop production but may simultaneously harm soil invertebrates, including earthworms, enchytraeids, and springtails. These organisms support ecosystem functions such as organic matter (OM) breakdown, nutrient cycling, and soil structure maintenance, contributing to essential ecosystem services like food production and water management. To protect these animals, environmental risk assessments (ERA) for pesticides are performed in the EU. Part of the ERA consists of standardized laboratory toxicity tests in artificial soils (AS), which have different properties than agricultural soils. The differences in OM and clay content between AS and agricultural soil may potentially result in underestimating risks. To address this, a correction factor (CF) of 2 is currently applied for lipophilic pesticides, but its adequacy is debated. This thesis aimed to improve understanding of how soil properties influence pesticide toxicity to soil invertebrates, focusing on OM content and the validity of the CF of 2. Five research questions were addressed, focusing on toxicity-OM relationships across endpoints, species, and pesticide types, as well as the role of clay type in AS. A literature review (Chapter 2) indicated that toxicity-OM relationships vary by chemical, species, and endpoint. However, most studies used natural soils differing in multiple characteristics, limiting conclusions. Additionally, there was a lack of toxicity data across multiple soils and species. Chapters 3, 4, and 6 addressed these gaps by conducting toxicity tests with Eisenia andrei, Folsomia candida, and Enchytraeus crypticus. Five pesticides (chlorpyrifos, lindane, cyproconazole, carbendazim, imidacloprid) covering a range of lipophilicities (log Kow 0.6-4.7) were tested in AS with varying peat contents (10%, 5%, 2.5%) and in a natural reference soil (LUFA 2.2; ~4.5% OM). Toxicity decreased with increasing OM across all species, but relationships varied between pesticides, endpoints, and species. Moreover, pesticide toxicity in LUFA 2.2 often deviated from AS-based trends, suggesting additional soil properties should be considered in ERA. In many cases, the CF of 2 underestimated toxicity differences, showing it is not always conservative enough, even for non-lipophilic pesticides. Chapter 5 addressed a practical issue in soil ecotoxicology: quantifying test animals. For species like springtails and enchytraeids, test replicates can contain >1000 juveniles, making manual counting time-consuming and error-prone. The study evaluated RootPainter, an open-source image analysis tool using neural networks, to automate counting of E. crypticus juveniles across different soils and pesticide treatments. Automated counts closely matched manual counts, with comparable toxicity estimates, while reducing counting time by 97.5%, demonstrating its efficiency and accuracy. Chapter 7 examined the influence of clay type in AS on pesticide toxicity to E. andrei. Toxicity tests with kaolin and bentonite clays, using carbendazim and imidacloprid, showed that kaolin clay resulted in the highest toxicity. This confirms that clay type affects toxicity but also suggests that kaolin clay provides a more conservative risk assessment than bentonite. In Chapter 8, the research questions were revisited, reinforcing that soil OM content significantly influences pesticide toxicity, with lower toxicity in soils with higher OM. While ERA acknowledges this effect, the CF of 2 lacks scientific robustness and may over- or underestimate risks. A key recommendation is integrating natural soils into ERA as an intermediate tier between laboratory and field studies. This would reduce uncertainties by providing more realistic exposure conditions, though further research is needed to determine its implementation. Overall, this thesis highlights the need to refine ERA methodologies by incorporating soil-specific factors, ensuring better protection of soil invertebrates and their essential ecosystem functions.