Beyond the chemical load: Wastewater residuals as drivers of antibiotic resistance proliferation and dissemination into soil, water, and food webs

Land application of wastewater residuals (treated biosolids and effluent) introduces antibiotic resistance determinants (ARDs), including resistance genes and mobile genetic elements, into the environment. However, the environmental fate-particularly the influence of the delivery matrix (biosolids vs. effluent) on ARD mobility and trophic transfer-remains poorly understood. We investigated the proliferation and dissemination of selected ARDs (6 antibiotic resistance genes and the Class 1 integron-integrase gene, intI1) in a 60-day greenhouse mesocosm study. Each mesocosm comprised soil, leachate, earthworms, and 3 plant species, exposed to complex contaminant mixtures delivered via biosolids or effluent. We utilized a high-organic matter (OM; 50%) soil to evaluate ARD fate in an environment that promotes contaminant sorption and microbial growth. We observed significant gene proliferation and mobility, suggesting ARD proliferation is not strictly limited by bulk soil OM content. Fate pathways diverged based on matrix-organism combination. Leaching was gene-specific rather than matrix-dependent; for instance, effluent facilitated qnrS leaching while sul1 and sul2 leached disproportionately from biosolids. Occurrence of the genes in biota revealed a physiological divergence. Plants, limited to passive absorption of porewater-associated targets and surface colonization by rhizosphere microorganisms, exhibited low gene bioaccumulation factors (BAF < 1), though both matrices facilitated stochastic, gene-specific uptake in pea pods. Conversely, earthworms actively accumulated and amplified targets-likely by selectively ingesting nutrient-rich, ARD- and contaminant-laden biosolids aggregates-reaching concentrations exceeding 107-108 copies g-1 dry weight and BAF > 3 (up to 89). These distinct pathways of proliferation of these resistance determinants are not captured in generic risk assessment methodologies. Therefore, risk frameworks should adopt gene- and matrix-specific monitoring-prioritizing mobility for effluent-irrigated lands and trophic transfer potential for biosolids-amended soils-to minimize ARD spread while promoting sustainable wastewater residual reuse.