Microbiological risk assessment using QMRA in preharvest agriculture water treatment systems for leafy greens

While much research has been done on efficacy of commonly used agricultural water treatment sanitizers including Peroxyacetic Acid (PAA), Calcium/Sodium Hypochlorite, and Chlorine Dioxide to reduce pathogens/indicators in water, little research has focused on the potential added benefit of these sanitizers on pathogens that are already established on crop surface/plant tissue or in soil. Additionally, variability in water treatment or “breakthrough” has also not been properly characterized in an agricultural setting. This proposal aims to directly address these knowledge gaps by using laboratory and field evaluation coupled with Quantitative Microbial Risk Assessment (QMRA). Ultimately, the study will provide growers and regulators with an improved understanding of the impact of water treatment on risk reduction for consumers.

To date, agricultural water treatment research has heavily focused on the efficacy of available chemistries/devises at reducing microbiological indicators including generic Escherichia coli (E. coli) and Total Coliform bacteria in water. While key learnings have led to a strong understanding of the conditions needed to achieve reductions of pathogens/indicators in agricultural water itself, there remains a significant knowledge gap of the impact that treated agricultural water has on pathogens already established on plant surface(s) or in soil. Additionally, there is a lack in understanding of the risk to human health that agricultural water treatment variability or “breakthrough” has when associated with the consumption of fresh leafy greens. This proposal aims to directly address these knowledge gaps by using laboratory and field evaluation coupled with Quantitative Microbial Risk Assessment (QMRA). The specific objectives of this project are: (1) Determine the die-off or log-reduction of E. coli O157:H7 and generic E. coli surrogates pre-established on leaf surface and in soil following agricultural water treatment using commonly used water treatment sanitizers (PAA and Calcium Hypochlorite); (2) Conduct in-field evaluations with grower collaborators of water treatment variability or “breakthrough” using traditional grab sampling techniques for microbiological indicators (generic E. coli and Total Coliform bacteria) coupled with real-time in-line monitoring for physical/chemical parameters (PAA, free chlorine, pH, temperature, ORP, flow rate in gpm); and (3) Use real world collected data from Objectives 1 and 2 to conduct a QMRA for STECs in leafy greens (romaine and spinach). Success of the project will include a comprehensive understanding of the impact of residual agricultural water treatment chemistries on pathogen persistence in water, on plant tissue and in soil and how it relates to risk. It is anticipated that scientific data will be generated which growers can use to justify enhanced benefits of their antimicrobial water treatment beyond die-off of organisms found in agricultural water alone. The study will also evaluate treatment systems at a commercial scale production to document critical time periods and durations of treatment variability that may result in a loss of effectiveness due to decreased residual, and all taken together will result in improved estimates of risk to human health. Ultimately, the study aims to provide growers and regulators with an improved understanding of the impact of water treatment on risk reduction for consumers.

1. Determine the die-off or log-reduction of Shiga-toxigenic Escherichia coli (STEC) and generic E. coli surrogates pre-established on leaf surfaces and in soil following agricultural water treatment with commonly used water treatment sanitizers (PAA and calcium hypochlorite). 

2. Conduct in-field evaluations of water treatment variability or “breakthrough” using traditional grab sampling techniques for microbiological indicators (generic E. coli and Total Coliform bacteria) coupled with real-time in-line monitoring for physical/chemical parameters (PAA, free chlorine, pH, temperature, ORP, flow rate in gpm). 

3. Use real-world collected data from research Objectives 1 and 2 to conduct a QMRA for STEC in leafy greens (romaine and spinach).

Findings

• Agricultural Water Treatment Effectiveness: Treatments using peracetic acid (PAA) and calcium hypochlorite reduced microbial contamination in all evaluated scenarios, demonstrating a beneficial impact on bacteria present in water, soil, and plant surfaces.

• Animal Intrusion Poses the Highest Risk: The fecal slurry (FC) scenario had the highest risk and the least reduction in contamination due to the presence of organic matter and elevated bacterial concentrations, making it the most challenging to mitigate.

• Calcium Hypochlorite Demonstrated Greater Efficacy: In field trials, calcium hypochlorite resulted in higher microbial risk reductions compared to PAA, particularly in controlling E. coli on plant surfaces and in soil.

• Variability in Treatment Success: Risk reduction varied across contamination types, emphasizing the need for targeted treatment strategies to address different contamination scenarios effectively.

• QMRA Findings: The Quantitative Microbial Risk Assessment (QMRA) model demonstrated that E. coli concentration was the most critical factor influencing microbial risk. Sensitivity analysis indicated that microbial load in irrigation water must be minimized to effectively reduce contamination risks.

• Persistence and Regrowth Observations: Some bacteria exhibited regrowth post treatment, with E. coli surviving in soil for up to 72 hours post exposure to treated water. Further study is needed to understand persistence in different environmental conditions.

• Partial Reduction on Romaine Lettuce: While treated irrigation water significantly reduced E. coli levels on romaine lettuce, neither PAA nor chlorine treatments completely eliminated the bacteria. The level of reduction varied depending on sanitizer type and concentration, with PAA typically having a greater impact on immediate reductions but chlorine showing more sustained effectiveness.

• Risk Per Exposure Analysis: The highest individual exposure risk was observed in the animal intrusion scenario (2.28 × 10⁻⁸), equating to a 1 in 43.9 million chance of infection per exposure. However, risk compounds over repeated exposures, emphasizing the need for ongoing mitigation.

Recommendations

• Enhance Water Treatment Strategies: Optimize treatment approaches based on contamination sources, ensuring appropriate sanitizer residuals to maximize microbial reduction and limit treatment variability.

• Focus on High-Risk Contamination Scenarios: Develop enhanced mitigation strategies specifically targeting animal intrusion scenarios, where bacterial loads are highest and most persistent.

• Implement Real-Time Monitoring & Response Systems: Continuous monitoring of water quality and sanitizer effectiveness is needed to promptly identify and address treatment inconsistencies and breakthrough events.

• Improve Understanding of Bacterial Persistence: Further research is required on bacterial regrowth in soil and plant surfaces post treatment to enhance QMRA accuracy and optimize treatment application strategies.

• Evaluate Long-Term Treatment Efficacy: Investigate the prolonged impact of sanitizer application on microbial loads in soil and plant surfaces beyond the initial 72-hour period.

• Enhance Grower Adoption of Best Practices: Provide industry stakeholders with data-driven guidance on effective water treatment protocols to improve compliance with Leafy Greens Marketing Agreement (LGMA) metrics and reduce contamination risks.