Introduction
Aquaponic production is recognized as a sustainable agriculture solution, effectively utilizing the nitrogen by-products from aquaculture as nutrients for fresh produce (Verma A.K., et al., 2023). AWARE (Aquaponics from wastewater reclamation GA N.101084245) aims to further this concept by establishing the first European Recirculation Aquaponics System (RAS) for using reclaimed water to grow fish (tilapia) and plants (lettuce) for human consumption. This study intends to assess the food safety of foods produced in a small-scale experimental recirculating aquaponics system that utilizes reclaimed water while ensuring animal welfare.
Materials and Methods
Aquaponics experimental system: experiments were conducted in a small-scale, Recirculatory Aquaculture System (RAS) installed in Castellana Grotte coupled with a municipal wastewater plant. The experimental system comprised three different RAS modules, RAS1, 2 and 3. RAS1 operated using dechlorinated tap water (CONTROL), while RAS2 and RAS3 were supplied with reclaimed water. RAS were stocked with Nile tilapia (Oreochromis niloticus) and a 5 m2 of crop planting surface with capacity for 90 lettuce plants (Lactuca sativa Batavian Red). Microbiological and chemical hazards, along with animal welfare, were monitored by collecting samples at the beginning (M0) and every three weeks at M1 and M2 between September and December 2024.
Microbiological analysis
Microbiological hazard analyses. Listeria monocytogenes, Salmonella spp., Escherichia coli and intestinal enterocci were determined in tilapias and lettuces produced in the new aquaponics system accordingly with EU regulation (EC 2073/2005; EU 2020/2184; 2020/741) by following the standardized ISO methods. Aeromonas hydrophila and Pseudomonas aeruginosa were also included as non-regulated pathogenic bacteria associated to aquaponics foods (Dorick J. et al., 2024). The presence of several human enteroviruses, including Sapovirus (SaV), Norovirus (NoV GI and NoV GII), Hepatitis A (HAV), Hepatitis E (HEV) was determined by real time PCR following the ISO 15216-1; 2017. CrAssphage, and Pepper Mild Mottle Virus (PPMoV) was also assessed as indicators of fecal contamination in aquatic environments that can reflect the effectiveness of treatment systems (Gyawali, P. et al. 2019).
Microbiological characterization. Total DNA, extracted from tilapia’s gut and gills and lettuce’s roots, in parallel with water and biofilter samples, was used for 16S rRNA gene metabarcoding aiming the characterization of the bacterial community and assessment of potential cross-contamination. In addition, the genotypes of bacterial isolates of tilapia and lettuce were compared.
Chemical hazards analysis: Regulated contaminants and maximum levels defined in the EU Regulation No 1881/2006 (e.g. nitrate, mycotoxins, certain metals (Pb, Cd, Hg, Sn), dioxins and PCBs, PAHs) and unregulated contaminants (CECs) were determined by means of ICP-MS, GC-HRMS and LC_HRMS.
Animal welfare: Animal welfare assessment was determined by combining growth performance, somatic indices, differences in inflammatory gene markers.
Results
Microbiological analyses: Regarding microbiological hazards, no significant differences in food-borne pathogenic bacteria were observed between tilapias and lettuces produced with potable (RUN1) and reclaimed water (RUN2, RUN3). L. monocytogenes was only detected in the soil at M0. Salmonella spp. was detected by qPCR but its presence was not confirmed by the ISO method. E. coli and Enterococcus were quantified in tilapia, plants, and soil at the beginning of the experiment (M0). Aeromonas hydrophila and Pseudomonas aeruginosa were not detected. Regarding virus analysis, only NoV GI was detected in muscle al levels lower of the LoQ. Other enteric viruses including SaV, HEV or NoVGII were detected in internal organs mainly in M2. NoV GII and SaV, as well as PMMoV, were identified in the leaves of the lettuces in M2. Additionally, higher levels of PMMoV in Tilapias were observed in M2 compared to M0 and M1. Regarding microbial characterization, the bacterial community analysis evidenced distinct bacterial communities in tilapia and in lettuce, which were relatively stable during the experimental cycle. The hypothesis of possible cross-contamination between water, tilapia and lettuce was not confirmed based on the community analysis. However, the inspection of the genotypes of the bacterial isolates, which is more sensitive, may provide further information.
Chemical hazards: Initial results indicate that the levels of contaminants are well below the maximum levels defined in the EU Regulation No 1881/2006.
Animal welfare: No significant differences in fish mortality were observed among the different treatments. The evaluation of growth performance revealed significant differences between RAS2 and RAS3 (ANOVA F(2,6)=7.62, p=0.02); however, these did not differ significantly from RAS 1. Similar patterns were observed for fish size. Fish in RAS 2 showed increased length, although the differences were not statistically significant (ANOVA F(2,6) = 4.65, p = 0.06). The hepato-somatic index indicated reduced liver weight in RAS1, but this difference was not significant (F(2,6)=2.92, p = 0.13). In contrast, the spleen-somatic index showed significant differences among treatments (F(2,6)=12.5, p=0.007). Specifically, RAS1 differed significantly from both the control (p < 0.01) and RAS 2 (p = 0.01).
Conclusions: the first conclusion is that microbiological risk analysis of tilapia and lettuce produced in the aquaponic system designed at AWARE using reclaimed water showed that these foods are safe. Additionally, there was no evidence of microbiological cross-contamination between water, tilapia and lettuce. Regarding chemical hazards, analysis indicate that the levels of contaminants are below the maximum levels defined in the EU-regulation. Finally, fish mortality remained unaffected across treatments, although physiological responses suggest differing resource allocation strategies.
REFERENCES
1.Verma A. K., Chandrakant M.H., John V. C., Peter R. M., John I. E. 2023. https://www.sciencedirect.com/science/article/pii/S0040162523003943
2.Dorick J., Kumar G. D., Macarisin D., Widmer A., Stivers T., Dunn L. L. 2024. https://www.sciencedirect.com/science/article/pii/S0362028X24000140
3.Gyawali, P., Croucher, D., Ahmed, W., Devane, M., Hewitt, J., 2019. https://doi.org/10.1016/j. watres.2019.02.003