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Aquaponic (AP) food production systems integrate aquaculture and hydroponics in soilless controlled environments while conserving space and natural resources (soil, water, air). Increasing global demand for high-quality quality nutritious, safe food continues to increase exponentially. Information specific to AP is needed to validate operational and handling practices for AP food safety. The first study is to assess the persistence of E. coli TVS 354 in bench-scale aquaponic systems (180 days after primary plant harvest). Methods: follow-up on the previous study and evaluate growth and persistence of E. coli TVS 354 at 180 days after primary plant harvest. Samples were collected from 10 independent aquaponic systems and bacterial growth was evaluated including aerobic plate count (APC), coliform bacteria (CB), heterotrophic plate count (HPC), and cultural enrichment for E. coli TVS 354 levels. Results: E. coli TVS 354 was not detected in samples from hydrotons (25 g), filters, fish feces, core and roots. The aerobic bacteria count of fish and plant tank samples ranged between 2.5 to 3 log CFU/mL for all treatments, while aerobic bacteria count on the core of the lettuce plant was significantly lower, ranging from 1.44 to 2.08 log CFU/mL. The second study is to evaluate the survival of Listeria innocua 2066 in effluent from fish tanks and plant tanks of aquaponic research units. Methods: four different treatments: fish tank water, fish tank water-replenished, plant tank water, and plant tank water-replenished. Each bottle was inoculated with Listeria innocua 2066, and bacterial growth and water chemistry conditions were evaluated over 7 days to identify physiochemical parameters associated with Listeria innocua 2066 survival and growth. Results: In 24 hours a significant reduction (p<0.05) in Listeria inccoua 2066 populations was observed for all treatments with a total reduction 3.9-3.7 log CFU/mL. Listeria innocua 2066 counts were lowest on day 7 for treatment groups, ranging from 1.1 log CFU/mL to 1.0 log CFU/mL. Treatments that were replenished daily from the aquaponic systems had higher counts of Listeria innocua 2066 compared to treatments that were non-replenished. The lowest APC on day 0, immediately after inoculation, ranged from 4.16 log CFU/mL to 4.32 log CFU/mL. A significant increase (P<0.05) in APC count was observed for all treatments in 24 hours while there is no significant difference in APC values between the treatments (p=0.35). The final and third study is to evaluate survival of inoculated Listeria innocua 2066 and nonpathogenic Escherichia coli and changes in AP physicochemical parameters. Methods: A four-week bench-scale AP experiment (n=12) with four goldfish (Carassius auratus)/aerated 37L tank, a 3-step biofilter, and four lettuce plants (Lactuca sativa var. Truchas)/37L hydroponic tank was conducted. Treatments were high and low-dose (6-log and 2-log CFU/mL, respectively) of Listeria innocua 2066-Er or E. coli-Rifr TVS 354, and uninoculated controls. Listeria innocua-2066-Er and E. coli-Rifr populations, mesophilic counts (APC), and physicochemical parameters (pH, temperature, dissolved oxygen, turbidity, ammonia, nitrite, and nitrate) were analyzed in plant tank water, and biofilters until plant harvest. Listeria innocua 2066-Er and E.coli-Rifr enumeration and APC (Petrifilm®) from lettuce shoots, roots and rockwool were determined at harvest. Results: Listeria innocua 2066 and E. coli TVS 354 populations declined significantly within 24 hours post-inoculation and were undetectable at day 14 and day 12 respectively (P <0.05). This decline was observed for both high and low-dose treatments. Listeria innocua 2066 and E. coli TVS 354 were detected in biofilters until week 4. At harvest, Listeria innocua 2066 and E. coli TVS 354 were recovered from lettuce roots, and rockwool, but not from plant leaves, and 100mL plant tank water grab samples. Lower leaves preharvest had a significantly higher APC (5.1- 6.4 log CFU/mL) relative to harvested lettuce upper leaves (2.8 - 4.2 log CFU/mL). Plant tank water pH had a significant effect (P<0.05) on Listeria innocua 2066 and E. coli TVS 354 survival. Significance: These results provide insights into the survival dynamics and sites of E. coli TVS 354 and Listeria innocua 2066 in AP and associated physicochemical conditions. These findings contribute to our understanding of potential food safety risks and associated risk factors such as inputs, physicochemical factors, and other environmental conditions in aquaponics systems.