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Bees play a crucial role as natural pollinators, ensuring the maintenance and stability of the world's biodiversity and agricultural crops. Native bees in neotropical regions belong to the Meliponini tribe, a larger group that differs significantly in behavior and biology from honeybees (e.g., Apis mellifera) and solitary bees (e.g., Osmia spp.). Hence, the exposure and effects of pesticides is also likely to vary among these different species. The aim of this study was to develop an analytical method to determine the presence of the neonicotinoid clothianidin in the Brazilian native stingless bee Tetragonisca angustula (local common name: Jataí). The method used for the chemical analysis involved a QuEChERS technique combined with UHPLC-MS/MS analysis. The developed method was subsequently used to analyze collected field samples. In addition, the acute toxicity of the pesticide to T. angustula was evaluated in a laboratory bioassay evaluating both lethal and sublethal endpoints. The analytical method was successfully developed with detection and quantification limits of 1.55 and 5 μg L−1, respectively, along with a linear range of 1–5 ng mL−1. Clothianidin was detected in environmental samples (9.2–32.9 ng g−1), and the exposure experiments demonstrated acute oral toxicity to adults of T. angustula , (24 h-LD 50 of 0.16 ng a.i./bee), as well as no significative interference in acetylcholinesterase activity. Considering the obtained toxicity endpoints for T. angustula and those reported in the literature for other bee species, this study revealed that T. angustula is more (lethally) sensitive to clothianidin than other bee species, including those commonly used in environmental risk assessment studies. This thus also supports the call for using native test species in (regional) risk assessment evaluations. [Display omitted] • Optimized and validated method for the determination of clothianidin in native bees. • Determination of the oral susceptibility of Tetragonisca angustula to clothianidin. • Quantification of the lethal concentration doses of clothianidin in native bees. [ABSTRACT FROM AUTHOR]