The land application of wastewater residuals is vital for circular agriculture but introduces complex contaminant mixtures and antimicrobial resistance determinants (ARDs) into terrestrial ecosystems. Current frameworks rarely assess cumulative risks of chemical mixtures and ARD proliferation or how the delivery matrix (biosolids vs. effluent) influences their ecological fate. To address these gaps, we executed a tiered risk assessment utilizing data from a multi-trophic mesocosm study to evaluate human dietary, avian predator, and aquatic pathways, comparing effluent-irrigated and biosolids-amended treatments. To assess unattenuated gross ARD exposures, we propose two metrics—average daily genetic dose (ADGD) and estimated environmental genetic concentration (EECgenetic). While contaminant phytoaccumulation was restricted, stringent regulatory thresholds for PFAS yielded elevated human dietary risks across both delivery matrices. For chemicals, biosolids drove greater risks to predators (from PBDEs) and edge-of-field aquatic hazards (from carbamazepine) compared to effluent. Conversely, for ARDs, biological amplification limited delivery matrix’s influence. Soil-ingesting invertebrates accumulated and amplified ARDs across both matrices, delivering high unattenuated ADGDs (>1010 copies kgBW-1 d-1) to secondary predators. Because quantitatively assessing ARD proliferation is precluded by lack of established dissemination thresholds and parameters evaluating attenuated exposure, we propose a three-equation framework to operationalize these gross genetic metrics. This framework partitions unattenuated exposure into in vivo colonization and secondary environmental shedding. To evaluate these colonization risks, we conceptualize a site-specific genetic reference dose (RfDgenetic). Ultimately, this study highlights the need to incorporate complex mixture toxicity and ARD proliferation risks into sustainable wastewater reuse policies and provides a roadmap to operationalize this integration.
