Multi-trophic biodiversity responses to pesticide residues in Mollisol croplands

Mollisols are among the world’s most fertile agricultural soils, but long-term intensive management, including widespread pesticide use, has introduced chronic pressures to belowground communities. However, the occurrence of multi-class pesticide residues and their impacts on multi-trophic biodiversity in black-soil croplands remain poorly understood. Here, we surveyed 111 topsoil samples from Northeast China’s Mollisol croplands to quantify 22 pesticides, assess ecological risks, and characterize multi-trophic biodiversity (bacteria, fungi, protists, and metazoa) using DNA metabarcoding. Pesticide residues were widely detected, with high frequencies observed for herbicides such as nicosulfuron (79.3%), atrazine (52.3%), and acetochlor (49.5%); insecticides dominated by neonicotinoids (e.g., imidacloprid 70.3%, clothianidin 64.9%) and pymetrozine (49.5%); and fungicides including chloroneb (53.2%). Risk quotient screening identified atrazine, clothianidin, and difenoconazole as compounds of elevated potential concern. At the mixture level, 85.6% of samples exceeded the cumulative risk threshold, although cumulative risk was generally dominated by one or a few compounds, with multi-compound contributions observed in only 20.7% of samples. Biodiversity associations were strongly trophic-level specific. Pesticide residues showed the strongest negative association with metazoan α-diversity, accounting for approximately 29% of the independently explained model variance, whereas bacterial diversity was more closely associated with soil properties and fungal diversity with cropping systems. However, protistan diversity showed weak explanatory patterns within the current framework. Overall, pesticide residues were widespread in Northeast China’s Mollisol croplands, with potential ecological concern largely attributable to a few high-risk compounds. The pronounced association with metazoan diversity supports incorporating higher-trophic soil fauna and compound-specific mixture risk into future soil-health assessments.