Food Safety of Aquaponic Products

<p>Aquaponics SystemThe aquaponics system used in this project integrates fish production, biofiltration, and plant production. The microorganisms in the biofilter and settling tank clean the water of harmful nitrogenous compounds, and the plants uptake the soluble nutrients and reduce the amounts of harmful bacteria in a manner similar to that of a constructed wetland. Systems in temperature-controlled areas may have tilapia as the major fish component, while systems in areas where temperature cannot be controlled will have hybrid bluegill (Lepomis macrochirus x cyanellus), largemouth bass (Micropterus salmoides), channel catfish (Ictalurus punctatus), or a combination of the three fishes. The tanks for the fish and the biofilter will consist of food-grade plastic. The plant production elements will consist of aquaponics trays, which will have floating rafts of plants. The trays will be periodically filled, allowed to sit full, and then drained and refilled at intervals as required for plant health.Water will be tested for chlorine, chloramine, and ammonia using a LaMotte Smart3 colorimeter before introduction to fish. If necessary, water will be purified of chlorine using degassing columns and/or filtering through zeolite and activated charcoal. Water in the settling tanks, aquaponics trays, and discharge holding tanks will be tested for plant nutrients using a LaMotte Smart3 colorimeter. Water quality parameters will be measured in the fish tanks, settling tanks, and aquaponics trays for conductivity (mS/cm1), and temperature (oC) using a Hanna conductivity meter. Dissolved oxygen (mg/L) and pH will be measured using a Hach SensION multiparameter meter. Turbidity (FAU) will be measured using a LaMotte Smart3 colorimeter. Insect, bacterial, fungal, and other pests will be controlled through integrated pest management (IPM) with emphasis in organic, non-pesticide treatments, because of the high susceptibility of fish to chemicals. Plant matter infected by pests that are not susceptible to the aforementioned tests will be removed and burned, and involved trays, pots will be sterilized.Fish will be fed commercially prepared feed or feed developed in-house. Feed will be weighed as it is given to fish. At least a 10% subsample of fish will be weighed and measured at intervals depending on season and ambient temperature. Fish diseases will be managed with methods that do not render fish unsafe for human consumption. Fish that are wild-caught for comparison will be obtained through angling, electrofishing, or seining. Ages of wild caught fish will be estimated using scales, otoliths, or fin ray cross sections.Food Safety EvaluationSimultaneous Detection of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella enteritidis in aquaponic nutrient solutionThe objectives of the study are to use conventional methods such as with the IDEXX system to identify E. coli and fecal coliforms to check for the presence and molecular tools to confirm the identity of E. coli O157:H7, Listeria monocytogenes and Salmonella enteritidis in the nutrient solution to determine the safety and accuracy of this aquaponic system.Initial evaluation will seek to quantify the amount of fecal indicator bacteria fecal coliforms and E. coli in water samples using Most Probable Number (MPN) Analysis. Solution will be collected in sterile polypropylene bottles immediately placed on ice and kept at 4°C until microbiological analyses were completed. The IDEXX (IDEXX laboratories, Maine) system for water quality analysis will be used for enumeration procedures of E. coli (Colilert®).</p><p>The samples will be analyzed with enzymes specific for each using a Quanti-Tray and IDEXX® Quanti-Tray Sealer. The sample trays will be incubated in trays at 35°C (Colilert®) for 24 hours. Results will be compared to a Most Probable Number (MPN) chart.Sampled nutrient solutions will be extracted for DNA using the MO BIO Ultra Clean Soil DNA Isolation Kit (MO BIO Laboratories Inc., Carlsbad, CA). The DNA concentration will then be determined using the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE). Multiplex PCR will be performed in a total volume of 50 μl containing 2 μl of template DNA and 48 μl of PCR master mix composed of 1x PCR buffer, 5.0 mM MgCl2, 80 nM of each of the detection primers, 200 μM dATP, dCTP, and dGTP, 400 μM dUTP, 0.25 U of AmpliTaq Gold DNA polymerase (Applied Biosystems Foster City, USA), and 0.5 U of AmpErase UNG (uracil-N-glycosidase; Applied Biosystems Foster City, USA) as described by Yasmin et al. (2007). Optimization of the multiplex PCR system for the simultaneous detection of E. coli O157:H7, Listeria monocytogenes and Salmonella enteritidis will be done using DNA from pure cultures of each organism. Each organism will be used as templates for DNA extraction analysis and used as positive controls for the PCR reactions. PCR amplification will be done using a Peltier Thermal Cycler PTC-200 (GMI, Inc., Ramsey, Minnesota. The expected size for E. coli O157:H7, L. monocytogenes and Salmonella are 120, 234 and 375 bp, respectively. This study will aid us in determining if the presence of indicator bacteria signifies the presence of pathogenic bacteria. Additionally, the potential detection of the target pathogen (s) will aid us in identifying the safety of this aquaponic system.Mercury Analysis with DMA-80Cultured fishes, their food, and water from the fish tanks, settling tanks, and aquaponics trays will be analyzed for mercury. Fish samples will be wrapped in aluminum foil, immediately frozen and transported to the laboratory and kept frozen at -80oC. Before analysis, fish samples will be gently thawed, sorted into similar sizes, and carefully dissected with a clean scalpel to select desirable tissues (muscle, gills, liver, skin, and kidney) for analysis. Pooled samples from each tissue will be homogenized and freeze dried before extraction of methyl Hg according to the procedure of Carbonell et al. (2009). At the end of the extraction, 10ul of the extract will be loaded in quartz boats and introduced into DMA-80 system with thermal combustion (drying 120 s at 250oC, thermal decomposition 120 s at 650oC). Fish feed samples (10-200mcg), being already dried, will not require the drying step, and will otherwise be treated similarly. Water samples will be dried for 300 s at 300oC (Rüdel, et al. 2011). The amalgamator in the DMA-80 selectively traps Hg after the system is flushed with oxygen for 60s to remove any remaining gases or decomposition products. At the end of the elapsed flush time, the amalgamator is rapidly heated (12s), releasing Hg vapor. Absorbance is measured at 253.7 nm as a function of Hg concentration. Total mercury in the fish samples will be quantified by thermal combustion of 0.05g wet weight at 750oC for 120s using DMA-80. The difference between total Hg and MeHg will be classified as inorganic Hg. A-two range calibration (0-40 and 40-500 ng of Hg) of the instrument will be performed before Hg analysis because the instrument operates on two measuring cells. After initial calibration, a blank run will be performed to free the system from Hg residual and contamination. All sample boats to be used for analysis will be run through the system to get rid of any residual Hg on the boats. Certified reference material will be analyzed at the beginning and end of each set of samples (typically 10) to verify that the instrument remains calibrated during the course of the study. A blank (i.e., an empty sample boat or boat with purified water) will also be analyzed at the end of each sample set to prevent Hg from being carried over between samples.</p>