High-Throughput Miniaturized Biotransformation Testing Using Activated Sludge Enables Rapid Chemical Persistence Assessment

The unprecedented scale and pace of chemical development challenges human and ecosystem health unless new chemicals are developed using safe-by-design approaches. Therefore, tools for efficient environmental persistence assessmentamong other critical assessment capabilitiesare urgently needed, as outlined in the European Commission's Safe and Sustainable by Design (SSbD) framework and the European Chemical Agency (ECHA)'s 2025 report on key regulatory challenges. Current persistence tests require large sample amounts and extended timelines making them unsuitable for early stage chemical development. We developed and validated a miniaturized, higher-throughput biotransformation assay using municipal activated sludge as the source of microbial inoculum. For 33 pesticides and pharmaceuticals, biotransformation rate constants showed strong correlation with large volume controls (R(2) > 0.84) and consistent relative biotransformation rankings across time and different sources of activated sludge (Spearman correlations > 0.8). Our 24-well plate test requires 2 mL per test (vs hundreds of mL in standard tests) and provides biotransformation data within 48 h (vs weeks or months) due to the dense biomass and high bioavailability of substrates in our targeted substance space (i.e., log K (oc) ≲ 4). This miniaturized test lends itself to further automation and enables persistence assessment during chemical design, directly supporting SSbD principles.