There is a clear, critical need to describe offsite antimicrobial resistance (AMR) transport from agricultural operations in flowing waters to inform AMR monitoring and control efforts. Significant unknowns are the persistence and transport of AMR in flowing waters, including the role of environmental drivers such as variation in water quality in receiving waters, seasonality, and the suitability of specific AMR detection methods (e.g., culture versus molecular) and targets (e.g., different antibiotic resistance genes) to represent the wide variety of AMR determinants. Our technical approach will be designed to understand how AMR from multiple agricultural manures is transported and simultaneously degrades in flowing water systems, once in the water column. This will be done using a set of complementary experiments in (1) experimental mesocosms, (2) an outdoor experimental system with linked streams, ponds and wetlands, and (3) field verification in a real-world scenario in the agriculturally-dominated Pine Creek Watershed. Concurrently, we will integrate models with data across a gradient of environmental complexity to understand both culturable and molecular AMR marker degradation and movement in flowing waters. Ultimately, we will build tools and methods for efficient detection and quantification of AMR and construct models to help understand detection in the context of flowing water ecosystems. In addition to technical project tasks, we propose to develop and implement both educational and extension modules relevant to AMR management. Educational and extension modules will be implemented at experimental sites through multiple partners, and based on the best available science to date.Specific project objectives are:Objective 1. Quantify degradation, transport, and sorption of AMR in flowing waters using recirculating mesocosms and replicated experimental streams at the ND-LEEF facility. We will conduct controlled experiments that will test the role of environmental conditions on AMR fate and transport. Both culturable and molecular AMR markers will be quantified to determine agreement between differing AMR quantification approaches.Objective 2. Field validate AMR techniques and detection capabilities in natural stream and ditch ecosystems using the Pine Creek Watershed (PCW) in southwestern Michigan. We will measure parameters derived from experiments from Objective 1 (mesocosms and experimental streams at ND-LEEF) in a variety of natural systems located at PCW to validate our understanding of the interacting role of substrate, stream flow, water quality, biofilms, and seasonality play on AMR dynamics in flowing waters.Objective 3. Integrate empirical data from experiments across a gradient of environmental complexity into a dynamic model to understand and predict AMR transport, degradation, sorption, and movement in flowing waters. This iterative work will continue through all experiments (mesocosms, experimental streams, and field validation in streams and ditches of the PCW). We will use this iterative approach so that empirical results from early experiments inform models and the results of models inform later experiments (Figure 1).Objective 4. Develop education and extension modules about AMR, water quality, and conservation practices. Digital education modules will be developed with ND-Learning and implemented at ND-LEEF (site of Objective 1) and through electronic media allowing widespread dissemination, with target audiences of K-12 through to the general public. Extension modules will be developed in conjunction with our partners at the VBCD (location of Pine Creek, site of Objective 2) and will include extension to local producers/landowners (e.g., conservation meetings, farm field days).