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Antibiotic resistance genes (ARGs) are emerging contaminants found in the water and sediments surrounding animal feedlots. In this study, the fate of five tetracycline-resistance and 16S-rRNA genes released in swine waste were monitored for 21 days in the water column and biofilms in 12 mesocosms mimicking different natural receiving water bodies. Four treatments were employed in triplicate: two light exposures (light/dark) and two loading scenarios (single/periodic). As seen previously, light exposure had a significant effect on disappearance rates of tet genes in both the water column and biofilms, although absolute rates were significantly lower in the biofilms. Further, periodic versus single loading events resulted in >2 orders of magnitude higher tet gene levels in associated tanks. Regardless of treatment, ARGs migrated quickly to biofilms, with 3% and >85% of detected tet determinants found in biofilms on days 1 and 4, respectively. Overall, these are the first quantitative data on specific ARG disappearance rates in biofilms, and also the first evidence of progressively accumulating ARG levels in biofilms under loading conditions typical of natural receiving waters. In summary, ARGs migrate rapidly to biofilms where they persist longer than adjacent waters, which suggests biofilms likely act as reservoirs for ARGs in nature.