California’s Salinas Valley is a high-production region for leafy greens (Gorski et al., 2022; Lacombe et al., 2022). Unfortunately, Salinas Valley has been associated with multiple foodborne outbreaks, making it a focal point for investigating produce-related pathogen systems. The Food and Drug Administration (FDA) and the Center for Disease Control (CDC) have stated that agricultural water is a key factor in pathogen conveyance (Gelting et al., 2011; Lacombe et al., 2022). Pathogenic bacteria held commonly in cattle feces are carried by runoff, flooding, and wildlife directly into produce fields or into open-source water, which may be further used in growing operations (FDA, 2021; Gorski et al., 2022; Lacombe et al., 2022). Water treatment is then a critical point to intervene in leafy green production to prevent contamination of food products. Currently, most agricultural water in the United States is treated with chlorine-based sanitizers; however, these sanitizers are labeled to treat fungal pathogens, and no label yet exists for standardizing the treatment of pathogenic bacteria such as Shiga toxin-producing Escherichia coli. Federal government agencies, including the FDA and the Environmental Protection Agency (EPA), have more recently developed testing protocols to define standard effective procedures for treating low-quality water (EPA, 2024). This is important because the efficacy of some chemical sanitizers can be heavily impacted by pH and turbidity, while other methods may be more flexible.

Chlorine dioxide (ClO₂) is an alternative chlorine-based sanitizer used in agriculture water treatment. Aqueous ClO₂ is a strong oxidizing agent that disrupts bacterial membrane permeability, metabolism, and structural proteins via electrophilic abstraction (Bridges et al., 2020; Jefri et al., 2022; Nadupalli et al., 2011; Ofori et al., 2018, 2017; Zhang et al., 2023). Irrigation infrastructure is vulnerable to the build-up of algal and bacterial growth, and ClO₂ is useful in biofilm inactivation, making it a diverse tool for growers to sanitize and unclog irrigation lines and other water transport systems (Krüger et al., 2023). Generator-based ClO₂ requires large infrastructure investment and trained personnel to handle equipment; however, the development of manufacturer kits has made ClO₂ a simpler and viable alternative for water treatment. This method allows for concentrated batches (200–500 ppm) of aqueous ClO₂ to be produced with dry precursors. This gives the operator the flexibility to choose the appropriate dose for application based on the manufacturer’s instructions. However, there is a knowledge gap as to the appropriate dose for human pathogen disinfection based on water quality.

Recent Shiga toxin-producing Escherichia coli (STEC) outbreaks are a reminder of the importance of reducing contamination in food production operations. The Leafy Greens STEC Action Plan (LGAP) is a document created by the FDA to address repeated leafy-green-related foodborne outbreaks (FDA, 2023). LGAP addresses recurring STEC outbreaks by facilitating collaboration between public and private agency efforts to answer questions regarding produce safety. These efforts serve to reduce the frequency and seriousness of outbreaks (EPA, 2024; FDA, 2021). LGAP included methodology to determine the concentration of chemical sanitizer required to achieve a target of 3-Log (99.9%) pathogen reduction (FDA, 2021). FDA and EPA designed a protocol for testing “worst case scenario” water to test sanitizers’ disinfection efficacy in water with high turbidity, conductivity, total dissolved solids, and varied pH. Due to location and water rights, these water parameters are designed to reflect poor conditions faced by farmers pulling from open-source water systems, including canals and rivers. It is important to define disinfection parameters for these sources so that growers can efficiently inactivate human pathogens before water use.

Another human pathogen, Listeria monocytogenes, has been reported across the Salinas Valley in surface waters (Gorski et al., 2022). Long-term sampling and genotyping indicated that the majority of L. monocytogenes strains that were isolated contained virulence genes and pathogenicity islands, making them a potential threat to public health and produce safety (FDA, 2023; Gorski et al., 2022). Although L. monocytogenes outbreaks are most frequently associated with post-harvest production environments and gaps in good manufacturing practice, the prevalence and pathogenicity of L. monocytogenes in Salinas Valley makes it a key organism to monitor in pre-harvest water alongside STEC (Gorski et al., 2022). Therefore, STEC and Listeria monocytogenes were chosen for the present study as useful pathogens for testing sanitizing treatments in water samples from California’s central coast growing region.

Following FDA and EPA guidelines, this study aims to define the disinfection efficacy of chlorine dioxide against bacterial pathogens across a wide range of water samples associated with the Salinas Valley, a key production region for leafy greens. Initially, we identified variations in agricultural water samples’ pH, turbidity, conductivity, and total dissolved solids (TDS). Pathogenic bacteria were exposed to a gradient of ClO₂ dosages to estimate the required concentration of disinfectant to achieve a 3-Log (99.9%) reduction in each water sample. Further, we monitored water metrics at three sites in the Salinas Valley to observe changes in chemistry and microbial content and understand if seasonal changes in water quality exist.

ClO₂ is a strong oxidizer and a useful alternative to conventional chlorine-based methods for disinfecting pathogens in various water sources (Barbeau et al., 2005; Bridges et al., 2022; Goodburn and Wallace, 2013). Dry precursor batch treatments of ClO₂ are efficacious in both ground and open-source waters, suggesting its suitability for agricultural water treatment. The present study demonstrates the inactivation of STEC and Listeria monocytogenes using dry precursors to create ClO₂ in variable agricultural water qualities. It suggests concentrations for a 3-log reduction of human pathogens.

Irrigation water metrics may fall outside optimal NaClO water quality ranges, meriting the need for alternative treatments. Field samples from Salinas Valley had water metrics outside the optimal NaClO treatment range (Table 2). Standard agricultural water treatment utilizes NaClO (bleach) due to low cost and existing infrastructure. NaClO has limitations, including reduced efficacy in water with pH > 8 and reactivity with organic matter, such as nitrogen fertilizers that produce hazardous disinfection byproducts (DBPs) (Chang et al., 2000; Goodburn and Wallace, 2013). In addition, extensive use of NaClO has raised concerns regarding persistent populations of pathogens remaining in the food supply (Hu et al., 2020; Praeger et al., 2018; Xu et al., 2022; Zhang et al., 2023). In this study of ClO₂ against STEC and L. monocytogenes, variation in reduction between water samples appeared at lower dosages while reduction was similar at 5 and 10 mg/L. Across treatments, there was no correlation between sample pH, turbidity, conductivity, and log reduction in either STEC or L. monocytogenes (Table 4). While the source of water played a role in water quality and ClO₂ efficacy, no single variable significantly impacted the inactivation of pathogen. This is in alignment with previous research that demonstrate ClO₂ broad spectrum ability to inactivate potential pathogens in irrigation water (Truchado et al., 2018).

Groundwater aquifers are a good source of irrigation water due to consistent water metrics and infrequent bacterial contamination. Pathogenic bacteria treated in groundwater required the lowest concentration to achieve 3-Log reduction (Table 3). In addition, Ag Well and Domestic Well samples showed a more significant reduction than ddH₂O lab water. Recently, Krüger et al. (2023) reported increased ClO₂ inactivation in phosphate buffered-saline solution compared to tap water when exposing Pseudomonas aeruginosa, E. coli, and Staphylococcus aureus to 0.4 mg/L ClO₂ for 5 min. Although it is unclear why conductive samples increase ClO₂ disinfection, a positive correlation between ClO₂ and factors related to conductivity was demonstrated. Therefore, alkaline groundwater samples possessing moderate conductivity and low turbidity are potential candidates for ClO₂ treatment.

In contrast to groundwater, open-source water in agricultural areas is subject to frequent contamination by runoff that contributes to fluctuating water metrics and pathogen load (Gorski et al., 2022; Jefri et al., 2022; Lacombe et al., 2022). Open-source field samples ENV₁ and ENV₂ had the lowest reduction across water samples despite having more moderate water quality compared to EPA_6.5/EPA_8.4 laboratory standards. The complexity and inconsistency in open-source water make it difficult to treat compared to groundwater sources, where high dosage and multiple disinfection methods are introduced to accommodate diversity within the sample. Regional water quality and contamination may vary throughout the year due to seasonal changes in precipitation, temperature, and animal activity. During the 2023–2024 season, extreme variation in water quality throughout seasons was observed at the open-source sites, likely due to sporadic rain events in the 2023–2024 summer-fall-winter. The pH decreased, and turbidity increased significantly at one site (ENV₂) in the winter (Figure 5). E. coli MPN counts were also higher in the winter, coinciding with the elevated turbidity, posing potential issues for water treatment. ClO₂ is less reactive to organic material and will not break down into chloramines as readily as NaOCl (Jefri et al., 2022; Nadupalli et al., 2011). Several articles report no major influence on ClO₂ disinfection in pH ranging from 3.0 to 9.0 (Chang et al., 2000; Huang et al., 1997; Jefri et al., 2022; Xu et al., 2022). Therefore, ClO₂ is well suited for open-source water decontamination because its broader spectrum activities accommodate unpredictable season changes.

Alkalinity is an important factor in determining water quality and the appropriate sanitizer. In the present study, the pH of each sample did not correlate with the reduction in either STEC or L. monocytogenes trials, but some patterns were observed (Table 4). Previously, Ofori et al. (2018) demonstrated that ClO₂ disinfection kinetics increased in alkaline lab waters compared to acidic conditions. Nadupalli et al. (2011) showed that ClO₂ reactivity increased in hydroxide-rich solutions and stunted reactivity in acidic conditions. While alkaline waters may improve the reactive rates of ClO₂ oxidation, higher levels of turbidity and organic matter may quickly react ClO₂ to the less reactive ClO₂^– ion, drastically reducing disinfection (Gagnon et al., 2005; Ayyildiz et al., 2009). In this case, a more significant reduction against STEC in groundwater samples with higher pH (8.1 and 7.4) is expected compared to pure ddH₂O with a pH of 6.7 (Figures 1, 2 and Table 2). Although pH appears not to be the driving force for variation in reduction, it may play a complex role in influencing ClO₂.

Bacterial cell envelope structure may influence pathogen tolerance to ClO₂. In this report, L. monocytogenes and STEC responded differently to chlorine dioxide treatment across water samples. Overall, ClO₂ was less effective in treating L. monocytogenes than STEC (Table 3). Previous reports demonstrated that L. monocytogenes responded less to ClO₂ and may be best treated using peroxyacetic acid (PAA) (Hua et al., 2019). Differences in the cell wall and membrane structure between gram-positive and gram-negative bacteria may play a role, as ClO₂ acts on these features of the cell (Bridges et al., 2020; Hua et al., 2019; Krüger et al., 2023; Ofori et al., 2018). Pathogens also displayed different responses across water samples. For example, L. monocytogenes treated in ddH₂O required the highest dosage to achieve 3-Log reduction compared to any other water sample (Table 3). Little is known about the biotic and abiotic factors that allow L. monocytogenes to persist in the environment and whether the lack of environmental stressors may render L. monocytogenes harder to inactivate (Gartley et al., 2022; Arcari et al., 2020; Labidi et al., 2023). The EPA updated protocol excluded L. monocytogenes from the group of pathogens tested (EPA, 2024). This adjustment reflects the different responses to the environment and disinfectant displayed by L. monocytogenes compared to STEC, and thus, they should not be treated the same way. Facility contamination remains a pressing concern in food safety, and the abundance of pathogenic L. monocytogenes in Salinas Valley waterways underlines the importance of defining effective treatments (Gorski et al., 2022).

Pathogens in this experiment were tested at the stationary phase; however, cells in the long-term survival (LTS) phase exhibit greater tolerance to physical and chemical stressors, including chlorine-based disinfectants (Bhullar et al., 2021; Wen et al., 2009). The long-term survival phase is reached following the death phase and is common for cells persisting in water and soil (Finkel, 2006; Fremaux et al., 2008). While testing disinfectants against cells in the stationary phase is a common practice, understanding how cells in the LTS phase tolerate ClO₂ treatment in environmental samples will be important for developing treatment recommendations and guidelines.

The primary goal of this report is to inform the development of water treatment guidelines designed to treat human pathogens in agricultural water. In this study, dosages of 1.5–3.5 mg/L ClO₂ were effective in achieving ≥3 log reduction against stationary phase STEC across water qualities (Table 3). Listeria monocytogenes should be treated differently, and higher dosages are required due to the resiliency displayed and the uncertain factors influencing survivability (Table 3). Further mechanism studies may be useful in investigating the process by which gram-positive pathogens react to ClO₂ and how effective other alternative disinfectants are against L. monocytogenes in agricultural water samples.