Demonstration of practical, effective and environmentally sustainable agricultural water treatments to achieve compliance with microbiological criteria

Growers should be assisted in determining the risk associated with agricultural water and the best mitigation option to remove pathogens if needed. Water disinfection is one of the most recommended intervention strategies for irrigation water. The main purpose of this project is to demonstrate a practical, effective, and environmentally sustainable water disinfection treatment. Within this regard, we propose the use of stabilized chlorine dioxide (ClO2) as a suitable disinfection treatment. We will try to establish if stabilized ClO2 could be a suitable disinfection treatment to assurance the compliance with the established microbial limits, particularly fecal indicator bacteria such as E. coli. First, agricultural waters from different water sources will be characterized by microbiological and physicochemical parameters. Optimal operational conditions for stabilized ClO2 as a suitable disinfection treatment will be established first at a pilot scale. After that, demonstration of practical, effective and environmentally sustainable agricultural water disinfection treatment will be carried out at commercial fields, where the impact of stabilized ClO2 on the environment will be also evaluated. We believe that the obtained conclusions will be very valuable for growers who will be able to integrate this technology in their water management practices.

Irrigation water has been recognized as a risk factor for fecal contamination of fresh produce. Growers should be assisted in determining the risk associated with agricultural water and the best mitigation option to remove pathogens if needed. Water disinfection is one of the most recommended intervention strategies for irrigation water. The main purpose of this project is to demonstrate a practical, effective, and environmentally sustainable disinfection treatment for agricultural water. Within this regard, we propose the use of stabilized chlorine dioxide (ClO2) as a suitable disinfection treatment for irrigation water. Stabilized ClO2 may be a suitable treatment to use in commercial agricultural fields to assurance the compliance with the established microbial criteria, particularly fecal indicator bacteria such as E. coli. In this proposal, three Industry Cooperators will collaborate. Two of the most important leafy green growers in EU, ‘Primaflor’ and ‘Florette’, located in the Southeast and Northeast of Spain, will provide agricultural water from different sources used in commercial growing fields. Additionally, the official distributor of stabilized ClO2 ‘STC’ will collaborate in the disinfection tests providing the needed reagent. First, agricultural waters from different sources will be characterized by microbiological and physicochemical parameters. Physicochemical characteristic such as chemical oxygen demand (COD), turbidity, total soluble solids (TSS), maximum filterable volume (MFV) and pH will be examined. Quantification of qPCR-based E. coli (molecular E. coli) and cultivable E. coli levels, as well as detection of foodborne pathogens (Salmonella spp. and VTEC) will be performed. The selected water sources will be: 1) streams bordering farmlands and urban areas, 2) water reservoirs and 3) water from rivers. The effectiveness of stabilized chlorine dioxide (ClO2), will be studied first at a pilot plant scale and secondly at commercial fields where different water sources are used. Doses thresholds of stabilized ClO2 in different water sources will be optimized based on reduction of E. coli levels and inactivation of foodborne pathogens based on the established microbiological criteria. Once minimum effective doses against foodborne pathogens are established, application of selected treatment will be carry out in three different commercial growing fields. Industry Cooperators will provide with all the facilities needed to test the water treatment in their commercial fields. The efficacy of these treatments will be based in reductions of E. coli levels. The impact of repeated ClO2 treatments on the environment will be carried out by evaluating the impact of the highest dose for pathogen removal on the soil microbial community. Cultivable phyllosphere-associated bacteria based on conventional methods and characterization of viable but non cultivable bacterial population of soils using Illumina next-generation sequencing will be evaluated. The gathered results will allow concluding if stabilized ClO2 can be defined as an effective and eco-friendly disinfection technology. We believe that the obtained conclusions will be very valuable for growers who will be able to integrate this technology in their water management practices.

1. Microbial and physicochemical characterization of different water sources commercially use to grow leafy greens in Spain 

2. Pilot plant studies to establish effective dose thresholds of stabilized ClO2 in different water sources a. Required doses for minimal compliance with microbiological criteria established in recommended guidelines of good agricultural practices as well as current legislation b. Required doses for removal of E. coli to drinking water standards c. Required dose for removal of foodborne pathogens such as Salmonella spp. and VTEC 

3. Practical demonstration of the stabilized ClO2 stability under field conditions 

4. Evaluation of the impact of repeated ClO2 treatments at the highest selected doses for pathogen removal on the soil microbial community

The results obtained confirm that E. coli load is very variable depending on the source of irrigation water. Surface water has been classified as the irrigation water type at “most risk” and this study confirms that statement. However, among the different types of surface water that growers might have available, water from drainage ditches should be avoided. Additionally, it was confirmed that levels of E. coli higher than 1.5–2.0 log cfu/100 mL were closely related to a higher probability of the occurrence of pathogens. It should be taken into account that samples positive for the presence of pathogenic bacteria were occasionally isolated from water samples with E. coli levels below 1.5 log cfu/100 mL. Plate count techniques seem to be a suitable quantification method, as no significant differences were observed between this classical method and the molecular PMA-qPCR technique. In cases where chemicals might be used for disinfection or to prevent algae formation, however, the plate count techniques might underestimate the E. coli population in irrigation water. The mathematical model based on initial E. coli load of the irrigation water and the physicochemical parameter UV254 can be a useful tool to predict the initial ClO2 concentration that growers can use to reduce the initial E. coli load below the established limits. In both studies (open field and commercial greenhouse) the selected ClO2 concentrations were efficient to reduce the E. coli load in irrigation water below the established microbial limits (FSMA). In the open field study, the microbiological quality of the irrigation water was very good, and the need for the disinfection treatment was very limited. However, when irrigation water of poor microbiological quality was used, as in the commercial greenhouse study, the selected treatment significantly reduced the E. coli load in the water. The comparison of two quantification methods (PMA-qPCR and plate count techniques) revealed that the slight differences observed between the E. coli loads in untreated and treated irrigation water disappeared. Therefore the applied disinfection treatment may not to be efficient enough to kill the bacterial cells but it may inactivate them. The abundance and diversity of the water microbiota were impacted by disinfection treatment with ClO2. However, disinfection with ClO2 in irrigation water slightly affected the bacterial community of baby spinach and soil, as only small changes were detected at lower taxonomic levels. Abundance of Pseudomonadaceae and Enterobacteriaceae decreased in baby spinach treated with ClO2 compared with the control. This finding is remarkable because a large proportion of foodborne and pathogenic bacteria associated with fresh produce belong to these families. Therefore, stabilized ClO2 could be defined as an eco-friendly disinfection technology.