Water used to wash fresh produce is often treated with chemical disinfectants to prevent cross-contamination and reduce microbial growth. The wash system is a dynamic process in which the microbial load of the incoming raw material, the concentration of sanitizer, and the organic matter change constantly along the production day. Regarding the organic matter of the wash water, the major factors that contribute to the organic load include the organic matter present in the dirt and soils, on the vegetable or fruit surface, and the organic materials released from the cut edges or damaged areas. For antimicrobial agents added to the wash, if the concentration is not sufficiently replenished, it usually declines rapidly as a result of its reaction with soluble organic materials present in the wash water. In the presence of an insufficient level of antimicrobial agent, microbial pathogens can remain viable in the wash water and transferred to the uncontaminated product. In the presence of a sufficient level of antimicrobial agent, microbial cells that are released from the contaminated product are immediately inactivated in the wash water, thus preventing cross-contamination.[1]
There are different commercially available agents for wash water disinfection, including chlorine derivatives such as sodium hypochlorite, calcium hypochlorite, electrolyzed water (EW), and chlorine gas. Chlorine dioxide (ClO2) is a neutral chlorine compound that is very different from elementary chlorine both in its chemical structure as in its behavior.
Understanding the Chemistry of ClO2
The chlorine in ClO2 exists in an oxidation state of +4, compared to an oxidation state of +1 for chlorine (in hypochlorous and hypochlorite ions). For this reason, chlorine and ClO2 have different pathways for disinfection and formation of by-products. The efficacy of ClO2 is less affected by pH and organic matter. The mechanism of action by which ClO2 inactivates microorganisms is not entirely understood. However, it is known that ClO2 kills microorganisms by either altering or disrupting transport of nutrients across the cell wall and also penetrating into the cell and disrupting protein synthesis.[2]
Most commercial generators use sodium chlorite as the common chemical precursor to generate ClO2 for drinking-water applications. The efficacy of ClO2 has a small dependence on pH in the range 5–9; thus, there is no need to carefully control the pH. Furthermore, the concentration of ClO2 necessary to reach a target level is lower than in the case of chlorine, resulting in a lower ClO2 dose to maintain an effective residual level.
Understanding the Presence of Chlorate Residues
Chlorine can be a solid, liquid, or gas and can accumulate in wash water from liquid chlorine (sodium hypochlorite) as chlorates are already formed during production/storage; as the solid chlorine (calcium hypochlorite), the amount of chlorates produced is lower. In the case of chlorine as a gas, chlorates are absent.[3]
Depending on the system used for ClO2 production into gas or liquid forms, chlorate can accumulate at high or low levels when added to the wash water. If ClO2 is generated on-site by a ClO2 generator, chlorate residues are produced in low amounts, but if ClO2 is manufactured as a stabilized aqueous solution, chlorate residues can be accumulated in high levels due to the chemical instability of ClO2.
In the case of chlorine, the maintenance of an appropriate range of pH in the water when using chlorine is crucial to ensure the maximum concentration of hypochlorous acid (HOCl), the form of chlorine with the highest antimicrobial activity. In a recent study, we evaluated the effect of two inorganic acids (phosphoric and sulfuric) and two organic acids (carbonic and citric) as pH regulators.[4] We observed that the different pH regulators tested did not influence the accumulation of chlorates in the wash water.
The Interview with Mabel Gil, Ph.D., Senior Researcher in the Food Science and Technology Department at the Spanish National Research Council
In your recent publication, “Use of Chlorine Dioxide to Treat Recirculated Process Water in a Commercial Tomato Packinghouse: Microbiological and Chemical Risks,” you mention that when using chlorine dioxide (ClO2) in the wash water, the transfer of chlorate to fruits is very low, with chlorate levels found in the washed product being lower than 0.05 mg/kg. This is highly important for our industry given the recent publication of the EC 2020/7249 where the implementation of Footnote A[5] has been published, but could you please explain our members what would be the most practical way for juice industry to measure and monitor these residues in fruits and vegetables when ClO2 is used during the different steps of the process of the manufacturing of juices, for example, for the washing of fruits and vegetables?
There is not an easy and practical way for the juice industry to measure and monitor chlorates. The methodology for the analysis of chlorate residues is by high-performance liquid chromatographic (HPLC) coupled to a triple quad mass spectrometer (MS/MS) using a method described for polar residue analysis in foods of plant origin.[6] (This method is the “EU Reference Method for Pesticides requiring Single Residue Methods (EURLSRM).” Quantification of chlorates can be performed employing isotope labeled analogue as internal standards added directly to the sample at the beginning of the procedure to compensate for any factors having an influence on the recovery rates such as volume deviations, analyte losses during sample preparation, as well as matrix effects during measurement. The chromatographic equipment is quite sophisticated for a plan factory, and generally the analytical tests are performed in accredited laboratories using validated methodologies. Differences in the testing results have made big differences whether or not to comply with the maximum residue limits (MRLs) permitted in the different products. Thus, this methodology has been modified recently by improving sample preparation, reducing particle sizes as well as using the isotopically labeled analogue chlorate 18O3 as internal standard.[7] As a result, this method is a standard analytical procedure for chlorate analysis in different matrices, such as liquid or solid samples of foods of plant origin such as fruits, vegetables, cereals, dry pulses, oily seeds and nuts, as well as honey. This methodology offers high selectivity and precise and reproducible measurements for chlorate quantification. However, as this equipment is not suitable for in-line applications in juice plants and requires highly skilled technicians, installation, and maintenance costs, the only possibility to prevent chlorate accumulation is by the control of wash water disinfection.
Chlorate accumulated in the wash water can be indirectly estimated knowing the amount of chlorate generated by the sanitizer as well as the amount of sanitizer added to maintain a residual level. As this procedure also relies on the analysis of chlorate produced by the sanitizer, prevention of chlorate residues must be done through the best practices for monitoring and control of water sanitation. Water quality parameters and sensors for calibration and validation of water sanitation are needed. The best location for sampling and frequency of precision measurements must be established. Practical considerations on wash water sanitation will allow the identification of the “fit for purpose” of the water, the need of sanitizer addition, the routine monitoring of the critical parameters, and how to adjust when the parameters exceed the critical limit. In our research group, we have been conducting several projects to provide to the industry practical data-supported results for the water sanitation to know how much sanitizer to use, how to measure, where, and when.
Accurate monitoring and recording of water sanitation is an essential component of a sound quality and safety program. Water must be treated with a sanitizer to minimize that waterborne microorganisms, whether postharvest plant pathogens or agents of human illness, be rapidly acquired and taken up from the water to produce. Water is used in numerous procedures in postharvest handling such as dumping, washing, cooling, transporting, cleaning and sanitation, and must be of suitable quality that the safety of fresh produce is not compromised. However, managing water sanitation accurately is a difficult task due to multiple chemical options, multiple product types, multiple equipment designs, historical limits for sanitizers different physicochemical water quality characteristics, and multiple sensors and test methods.
The parameters related to water quality and water sanitation must be identified. The window limits of the sanitizers should be high enough to be effective as antimicrobial, but at the same time, as low as possible to avoid chemical risks associated with disinfection by-products.
With operational limits, I guess you are referring to standardize the conditions of how to better apply the sanitizer?
Yes, under industrial scale conditions, sanitizers should be managed properly for the prevention of both microbial and chemical risks for the consumers. A suitable residual concentration should be maintained to avoid microbial hazards. For example, in the case of ClO2, the residual concentration should be high enough to be effective as antimicrobial, but at the same time, it should be as low as possible to avoid chlorate accumulation.
I ask this to clarify this point in the sense that it needs to be clearly understood as some don’t understand the relationship between the sanitizer and the microorganisms that can contaminate our raw materials and thus our final product.
Sanitizers are used to maintain the quality of the water and prevent cross-contamination of the product. In general, it could be assumed that the cleaning action of the washing process removes microorganisms from the product and the sanitizing agent eliminates them in suspension.
Usually, we need to do a validation of the sanitizers under laboratory conditions and then extrapolate this to facility conditions. As a general overview of the validation steps, could you please add your insights on the steps below to validate a sanitizer in a generalized way?
1. Test the sanitizer in question at different concentrations that are allowed according to the corresponding legislation that lies in the country where we are doing this experiment/validation, as this will have to take into account different contact times and surfaces we are sanitizing.
Correct
2. Use the microorganisms of interest that we want to challenge against the different concentrations of sanitizer that we are testing, using also different microbial loads that can mimic the contamination in facility environment.
Correct
3. Once we find the right concentrations of the sanitizer that can kill the microorganism in question, then we test those conditions in our facilities to validate the sanitizer.
Correct
4. The sanitizer used in question needs to be compatible with the type of surfaces that we are putting it into contact with.
It must also be compatible with the product, preserving the quality, for example, blanching carrots.
5. We need to pursue to use the sanitizer in question in terms of complying with the legislation, not overcoming the concentrations allowed of the sanitizer, using always the lowest concentration possible of the sanitizer to avoid future resistance of the microorganism in question in our facilities, using lower concentrations will leave less residuals of the sanitizer on the surface of raw materials and machinery, and thus also less rinsing and less water to remove residuals will be needed; however, this can only be achieved successfully when proper validation of the sanitizer is done.
Correct
Given that your results mention that washed products with ClO2 in solution leave less than 0.05 mg/kg, do you consider that the added presence of chlorates from other sources like drinking water for juice reconstitution can overcome the levels that you are highlighting if ClO2 is also used to disinfect the drinking water? I ask this because the EC 2020/749 highlights that the maximum MRLs of chlorates in fruit and vegetables must be of 0.05 mg/kg for citrus fruits, 0.05 mg/kg for pome fruits, 0.05 mg/kg for stone fruits, 0.05 mg/kg for berries and small fruits, 0.1 mg/kg for tomatoes, etc. If added water is used in juice reconstitution, how can we ensure that we don’t overpass the limits established in EC 2020/749 as well as the limits established in the EC Directive 98/83 (0.2 & 0.7 mg/L)?[8]
There is no practical and economical treatment available to remove chlorate once it has been formed in drinking water. If added water is used in juice reconstitution, we cannot ensure that we don’t exceed the limits established if the ClO2 treatment generates chlorates. In the case of the fruits and vegetables, they can be rinsed in clean water to remove residual sanitizer from produce surfaces. It is essential to make sure that the ClO2 system used to disinfect drinking water is not a source of chlorate, preferably use the on-site ClO2 generator. When water is used in juice reconstitution, we must ensure that the disinfection system used does not generate chlorate to overpass the limits established.
Going back to previous question, do you consider that in order to have levels less than 0.05 mg/kg would it be important to validate the use of the sanitizer in this case ClO2 on different fruit surfaces as well as in the source of water we have in our facilities? I ask this because as we explained above, the validation of sanitizers has to be done according to: a) the food matrix we are disinfecting, b) type of microorganism that we are dealing with, c) pH of water (as this will enhance or nor the efficacy of certain sanitizers), and d) the contact materials that will be in touch with the sanitizer, as this will avoid the damage of certain surfaces in our facilities, as some sanitizers are strong oxidants and can corrode materials or damage gaskets, etc.
Yes, the validation of each sanitizer must be done first under lab scale experimentation mimicking industrial conditions to demonstrate that the proposed variables are able to control cross-contamination and prevent chlorate residues, and after under commercial conditions. In the case of ClO2, it is recommended to maintain a constant concentration of ClO2 to confirm that the minimum operating concentration prevents the risk of cross-contamination and, if a high content of organic matter is present, the chlorate accumulation can be prevented. Verification activities should be performed to confirm that the validated critical limits are operating according to the parameters established via experimentation trials under industrial conditions. Gombas et al.[1] described different validation options for ensuring the effectiveness of antimicrobial washes, with an emphasis on developing practical guidelines for validation. However, the industry needs a collection of pathogen and spoilage surrogates that can be used for validation studies, which is very difficult for them. Validation of disinfectants/sanitizers must be determined in the context of a multivariate system with full knowledge of the variables that can impact the efficacy such as: type of microorganism, pH, temperature, salinity, dissolved organic materials, debris, soil, product demand, turbidity, agitation, contact time, product to water ratios, initial water source, etc.
Your results indicate that using 3 mg/L ClO2 is a suitable option for the reuse of wash water as not only reduces microbial populations but also it doesn’t lead to the presence of disinfection by-products, such as chlorite and chlorate ions, etc. in the final product. However, knowing the versatility of ClO2, which is not only used for water disinfection for human consumption but also as a surface sanitizer (e.g., industry equipment) and fruit & vegetable disinfectant, we would like you to explain more about the non-presence of residuals of chlorates in final product when ClO2 is used?
Any sanitizer that can generate chlorate residues needs to be used in a designed system able to improve the disinfection of the wash water through cost-effective strategies directly focused on reducing the organic matter. For example, washing produce by means of showers to remove any bulk contamination and excess exudates prior to disinfection will facilitate the use of solutions with lower concentrations of sanitizers and shorter exposures times, reducing chlorate accumulation in the wash water and in the washed product. Another step to reduce chlorate is the rinse of the product with tap water to remove sanitizer residues from produce and equipment surfaces. These recommendations can be used when using ClO2 for surface disinfection and a post-rinse to remove residuals in the final product.
Going back to the previous question, your results show that using 3 mg/L ClO2 are enough to disinfect “re-use water” that previously had a microbial load, but what if we are using water that has not been used before? In this case, the concentration used could be even less, which would give the juice industry a wider margin of use of ClO2 especially when drinking water is used in the reconstitution of juices. Could you please comment?
Yes, if you are using water that has not been used before, then you do not need to maintain the same residual/concentration of ClO2. However, in the case of process wash water, this is different. Most processors recirculate process wash water to save water and energy. By doing that, organic matter from the dirt and soil present on the vegetable surface and the organic matter released from the damaged areas of the produce accumulates in the water. As mentioned before, water of inadequate microbiological quality has the potential to be a direct source of contamination and a vehicle for spreading pathogens. Therefore, wash water must be treated with chemical antimicrobials to prevent cross-contamination. For the antimicrobial agent, if the concentration is not maintained correctly, it usually declines rapidly in the washing operation as a result of its reaction with soluble organic materials present in the wash water. In the case of drinking water, there is no need to maintain 3 mg/L of ClO2 due to the low organic matter content and disinfection demand.
As you know, chlorine dioxide is mentioned in the Recast of Drinking Water Directive 98/83 on the quality of water intended for human consumption only when levels of chlorate reach up to 0.7 mg/L. Knowing that ClO2 could leave less residuals of chlorates when generated on site, one could argue that if high chlorate residuals are found in the final product, it could be due to a misuse of the sanitizer, for example, as it was not validated previous to its use. ClO2 is more stable chemically talking than chlorine for instance, but up to what extent we can say that if residuals of chlorates are found, they don’t come from ClO2 in case this has been used in the facility? Could you please clarify more about this referring to the chemistry of chlorine dioxide versus chlorine and then clarify about residuals forms of chlorate of each one?
Depending on the ClO2 system used, chlorate residues can be generated. As it is an exceptionally reactive gas, it cannot be stored due to its instability but rather must only be manufactured to meet requirements at its place of use, reducing chlorate residues. Liquid ClO2 has a high solubility in water as a true gas at low temperatures. Its chemical stability is higher at low concentrations (<10 g/L). Chlorine instead hydrolyzes quickly and reversibly in water at any pH. ClO2 at high pH (>8) gets decomposed quickly to ClO3– and ClO2– ions; however, it is quite stable in weak acid conditions.[9]
The presence of chlorate residues in food is probably due to their occurrence as disinfection by-products from disinfection agents used in the food chain, from production to processing, including irrigation water and water used in postharvest unit operations such as hydrocooling and washing. It is not possible to distinguish the different sources that cause the residuals of chlorate.
The residues of chlorate found whether in the raw materials or final product could come then from sodium hypochlorite that has been approved for use as a biocide for human hygiene, disinfection, veterinary hygiene, food and animals feeds, drinking water[10] or ClO2 that, although banned as a pesticide, is being reviewed for use as a biocide[11] in the European Economic Area (EEA) and/or Switzerland[12,13] and that as mentioned above, it is also highlighted in the Drinking Water Directive when the residuals of chlorates reach a level of up to 0.7 mg/L. The same happens with chlorine that is also being reviewed for use as a biocide in the EEA and/or Switzerland in drinking water, disinfection, etc.[14] Please note we are not talking about sodium chlorate as this herbicide has been banned in Europe since 2009.
As we know, ClO2 is presumably stable in pure water in the dark, but it is photoreactive in sunlight, producing chlorate, chlorite, and chloride ions, especially in alkaline solution, at an industry level. Would you have any advice/protocol that can help better keep the chemical stability of the compound to avoid its degradation and thus have the less residuals possible in final product and/or raw materials?
The three main factors affecting ClO2 degradation to produce disinfection by-products are storage time, temperature, and light. The on-site production is the best way to avoid degradation of ClO2 and accumulation of disinfection by-products. Other advantages of the ClO2 on-site production is that transportation and storage is not necessary, no extra chemicals for pH correction are required and the lowest chloride and chlorate levels are produced.
Do you consider possible to differentiate that if chlorate residuals are found in juices are attributed from the raw materials (fruits or vegetables) or from processing aids (water) or is it definitively not possible to differentiate the source in the final product?
It is not possible to identify the source of chlorate in the final product. Chlorate residues can even be present in the raw materials uptaken during cultivation. The occurrence of disinfection by-products in irrigation water has been highlighted as a health risk of emerging concern since chlorate residues can be uptaken and accumulated in the edible portions during crop production. For instance, in a recent study we did, irrigation with chlorinated reclaimed/recycled water resulted in the accumulation of chlorate in lettuce (0.34–0.56 mg/kg), despite that the chlorate content in irrigation water was below the MRLs allowed for potable water (0.25–0.49 vs. 0.70 mg/L, respectively).[15] The chlorate content gradually increased from the inner leaves (younger) (0.21 mg/kg) to the outer leaves (oldest) (0.55 mg/kg), and the roots (0.56 mg/kg). The study shows that there was chlorate bioconcentration observed in lettuce heads, although it did not exceed the current MRLs for chlorate on leafy greens (0.7 mg/kg). In another study, we evaluated the potential accumulation of chlorate in baby lettuce irrigated with water treated with EW in a commercial greenhouse over three consecutive harvests and re-growths.[16] The use of EW caused the accumulation of chlorates in irrigation water (0.02–0.14 mg/L), and in the fresh produce (0.05–0.10 mg/kg), but in this case the levels reached were not above the MRL for leafy greens.
For people who are not familiar with the use of ClO2, this sanitizer is used in gaseous form or in solution, but from the practical point of view, which form you think is more practical to be used in facilities?
The most practical form of using ClO2 is in solution, but one drawback in the production is the accumulation of undesired by-products (chlorite and chlorate ions).
Do you consider comparable to measure chlorates residues in “peel samples” versus “water samples,” for example, the last wash cycle of the fruits and/or vegetables before they are further processed? Are the concentrations correlated in both peel and last wash cycle water?
We have data comparing the chlorate content in “peel samples” vs” water samples” but express the results per fruit. We observed that despite the high chlorate content in ClO2-treated wash water, the residues of chlorate in the washed tomatoes were lower than the MRLs for tomatoes (0.1 mg/kg). Some of the reasons for the low transfer of chlorate from water to the product would be the absence of irregularities in the surface of the fruit, the reduced number of stomata, and the hydrophobic nature of the cuticle.[17] We also have data comparing the chlorate residues in several fresh-cut products and in water. We observed that when the concentration of chlorate in the wash water increased, the residues in the product increased.[18] The chlorate accumulation in the product showed a linear response with the chlorate present in the wash water. However, for the same content of chlorate in wash water, the residues in the product varied depending on the type of product and the cut size. In shredded carrots, there was at least 5-fold greater content than in chopped lettuce for the same chlorate content in wash water. These results showed that the chlorate uptake by the product was 1.7% for chopped lettuce and 9.8% for shredded carrots at different contents of chlorate in the wash water. One reason could be the cut edges of shredded carrots exposed directly to the wash water and the small piece size that influenced the high uptake.
Are there industrial applications to measure chlorates in peel of fruits and vegetables? In your article you mentioned to have measured chlorate residues in the peel of tomatoes.
As far as I know, there is not a simple, accurate, rapid, and inexpensive test method except the analysis by HPLC-MS/MS to measure the chlorate residues accurately in fruits and vegetables. This is the analytical equipment we used to carry out the analysis of chlorate residues in the peel of tomatoes.
References
1. Gombas, D., et al. 2017. “Guidelines to Validate Control of Cross-Contamination during Washing of Fresh-Cut Leafy Vegetables.” J Food Prot 80: 312–330.
2. World Health Organization. 2008. “Benefits and Risks of the Use of Chlorine-Containing Disinfectants in Food Production and Food Processing.” Joint FAO/WHO expert meeting, Ann Arbor, MI, U.S., May.
3. Tudela, J.A., et al. 2019. “Chlorination Management in Commercial Fresh Produce Processing Lines.” Food Control 106.
4. Marin, A., et al. 2020. “Chlorinated Wash Water and pH Regulators Affect Chlorine Gas Emission and Disinfection By-Products.” Innov Food Sci Emerg Technol 66: 102533.
5. https://eur-lex.europa.eu/legal-content/GA/TXT/?uri=CELEX:32020R0749.
6. Anastassiades, M., et al. 2013. “Quick Method for the Analysis of Residues of Numerous Highly Polar Pesticides in Foods of Plant Origin Involving Simultaneous Extraction with Methanol and LC-MS/MS Determination (QuPPe-Method).” Stuttgart. Germany: Laboratory for Chemical and Veterinary Analysis of Food CVUA. Version 7.1. November.
7. https://www.eurl-pesticides.eu/userfiles/file/EurlSRM/meth_QuPPe-PO_EurlSRM.pdf.
9. Tran, T.V., L.M. Dobosz, and J.K. Argasinski. 2008. “The Effect of Highly Concentrated Chlorine Dioxide on Physical Properties of Fluoropolymers.” NACE: International Corrosion Conference Series.
10. https://echa.europa.eu/substance-information/-/substanceinfo/100.028.790.
12. https://echa.europa.eu/substance-information/-/substanceinfo/100.030.135.
13. https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=OJ:L:2019:031:FULL&from=EN.
14. https://echa.europa.eu/substance-information/-/substanceinfo/100.029.053.
15. Garrido, Y., et al. 2020. “Chlorate Accumulation in Commercial Lettuce Cultivated in Open Field and Irrigated with Reclaimed Water. Food Control 114: 107283.
16. López-Gálvez, F., et al. 2018. “Disinfection By-Products in Baby Lettuce Irrigated with Electrolysed Water. J Sci Food Agric 98: 2981–2988.
17. López-Gálvez, F., et al. 2020. “Use of Chlorine Dioxide to Treat Recirculated Process Water in a Commercial Tomato Packinghouse: Microbiological and Chemical Risks.” Frontiers Sustain Food Syst.
18. Garrido, Y., et al. 2019. “Chlorate Uptake During Washing Is Influenced by Product Type and Cut Piece Size, as well as Washing Time and Wash Water Content.” Postharvest Biol Technol 151: 45–52.
Alejandra Aguilar Solis, Ph.D., is the technical and scientific affairs manager at the European Fruit Juice Association.
