Pesticides override other stressors to drive stream macroinvertebrate diversity loss: unraveling interactive effects and ecological thresholds

Freshwater ecosystems worldwide face increasing pressures from chemical pollutants, particularly pesticides, across both agricultural and urban settings. The individual and interactive impacts of pesticide contamination and other environmental stressors on freshwater macroinvertebrates remain insufficiently quantified under real-world, multi-stressor conditions. Here, we conducted seasonal monitoring at 39 sites in the Wei River Basin-one of China's most intensively managed agricultural and urban regions. A multiple-stressor analytical framework was then constructed to examine how pesticide contamination interacts with other environmental stressors to shape macroinvertebrate diversity. Results revealed that pesticides contributed more than 40% to both α-diversity loss (species richness) and β-diversity nestedness, exceeding the combined effect of the other factors that were considered. Interactive effects between pesticides and land use were stronger in the dry season, evidenced by a 2.1-fold increase in functional sensitivity and a 56% rise in interaction contributions. Structural equation modeling revealed that pesticides and land use differentially regulated community structure and function via direct and indirect pathways, respectively, with pronounced seasonal variation and coupled interactive effects. Community-level Threshold Indicator Taxa Analysis (TITAN) was conducted for the four high-priority pesticides. The ecological harm thresholds were estimated at 13.81 ng/L for Imidacloprid (5-95% quantile range: 4.02-104.47 ng/L), 1.78 ng/L for Hexazinone (0.65-4.20 ng/L), 10.70 ng/L for Atrazine (2.87-31.33 ng/L), and 0.34 ng/L for Simazine (0.01-2.29 ng/L). These compound specific thresholds, derived under real-world multiple stressor conditions, provide ecologically grounded benchmarks for regional watershed management and highlight the importance of compound level threshold derivation for pesticide risk assessment and the protection of macroinvertebrate communities. Building on established multiple-stressor frameworks, this study provides quantitative evidence that pesticide contamination is primarily associated with macroinvertebrate diversity loss in a major agricultural watershed, and demonstrates how hydrological seasonality modulates pesticide effects on taxonomic and functional diversity, offering evidence-based support for watershed pesticide regulation and biodiversity conservation.