Suitability of chlorine dioxide as a tertiary treatment for municipal wastewater and use of reclaimed water for overhead irrigation of baby lettuce

Accepted Manuscript Suitability of chlorine dioxide as a tertiary treatment for municipal wastewater and use of reclaimed water for overhead irrigation of baby lettuce Luana Tombini Decol, Francisco López-Gálvez, Pilar Truchado, Eduardo César Tondo, Maria I. Gil, Ana Allende PII:

S0956-7135(18)30445-6

DOI:

10.1016/j.foodcont.2018.08.036

Reference:

JFCO 6300

To appear in:

Food Control

Received Date: 1 March 2018 Revised Date:

11 July 2018

Accepted Date: 29 August 2018

Please cite this article as: Luana Tombini Decol, Francisco López-Gálvez, Pilar Truchado, Eduardo César Tondo, Maria I. Gil, Ana Allende, Suitability of chlorine dioxide as a tertiary treatment for municipal wastewater and use of reclaimed water for overhead irrigation of baby lettuce, Food Control (2018), doi: 10.1016/j.foodcont.2018.08.036 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Running Title: ClO2 disinfection of wastewater to be used as agricultural water.

2 3 Suitability of chlorine dioxide as a tertiary treatment for municipal wastewater

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and use of reclaimed water for overhead irrigation of baby lettuce

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Luana Tombini Decol1; Francisco López-Gálvez2; Pilar Truchado2; Eduardo César

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Tondo1; Maria I. Gil2, Ana Allende2*

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Tecnologia de Alimentos, Universidade Federal do Rio Grande do Sul (ICTA/UFRGS).

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Av. Bento Gonçalves 9.500, prédio 43212, Campos do Vale, Agronomia, CEP: 91501-

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970, Porto Alegre/RS, Brazil.

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Food Science and Technology, CEBAS-CSIC, Campus Universitario de Espinardo,

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30100, Murcia, Spain.

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Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of

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Laboratório de Microbiologia e Controle de Alimentos, Instituto de Ciência e

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Corresponding author: Ana Allende – E-mail: [email protected]

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ACCEPTED MANUSCRIPT ABSTRACT

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Reclaimed wastewater used for agricultural irrigation should meet specific

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microbiological standards in order to prevent microbial contamination of the irrigated

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produce. The objective of this study was to evaluate the suitability of chlorine dioxide

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(ClO2) for the disinfection of secondary-treated municipal wastewater and its

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subsequent use for overhead irrigation in greenhouse production of baby lettuce. The

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impact of reclaimed water tertiary-treated with ClO2 on E. coli concentration, the

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presence of pathogenic bacteria, and the occurrence of chlorates as disinfection by-

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products was evaluated in water and in baby lettuce. E. coli was quantified using both

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conventional plating methods and a quantitative real time PCR (qPCR) method with

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propidium monoazide (PMA) pre-treatment to differentiate between viable and non-

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viable bacteria. Population density of cultivable E. coli was significantly lower (p

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<0.05) in reclaimed water, tertiary-treated using ClO2 (ClO2W), when compared with

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secondary-treated municipal wastewater (SW). However, no significant differences in

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viable but non-cultivable (VBNC) E. coli loads were observed between treatments when

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quantifying using the PMA-qPCR method. These results could indicate that ClO2

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treatment of wastewater water did not kill the bacteria present in the water but it

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induced bacteria to enter a VBNC state. The proportion of samples positive for the

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presence of pathogenic bacteria was lower in ClO2W (1/8) compared with SW (7/8).

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Significantly lower E. coli counts (p <0.05) were detected in plants irrigated with

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ClO2W compared with those irrigated with SW. Relationship between higher E. coli

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counts and the presence of pathogens was observed when lettuce samples were analysed

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by PMA-qPCR (Mann-Whitney U Test, p<0.05). Baby lettuce irrigated with ClO2W

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showed a significantly higher concentration of chlorates than lettuce irrigated with SW.

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The quantification of viable bacteria using molecular methods suggests that the efficacy

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ACCEPTED MANUSCRIPT of ClO2 could be overestimated when conventional plating quantification methods are

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used. Additionally, the accumulation of chlorates in the tissue should be considered as it

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represents an adverse effect of this disinfection treatment.

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Keywords: Fresh produce; fruits and vegetables; agricultural water; water

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disinfection; foodborne pathogens; disinfection by-products; chemical risk.

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Introduction Water reclamation and reuse for irrigation in agriculture are priority innovation

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practices. Water reuse is indicated by the European Commission (EC) as an important

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topic for the circular sustainable economy, a regenerative system in which resource

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input and waste, emission, and energy leakage are minimized. Currently, reclaimed

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water is mostly used in agricultural irrigation, especially in semi-arid and arid regions to

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overcome water scarcity (Becerra-Castro et al., 2015). Limited awareness of potential

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benefits among targeted end-users, and lack of a supportive and coherent framework for

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water reuse are major reasons for reducing this practice in the EU (EC, 2017a).

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Wastewater usually contains pathogenic microorganisms, many of which are able to

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survive in the environment and be transmitted to humans through fresh produce (EPA,

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2004; Steele & Odumeru, 2004; Uyttendaele et al., 2015; López-Gálvez et al., 2016a).

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Although reclamation treatments can improve the microbiological quality of water, the

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effluents of wastewater treatment plants can be vehicle of microbiological and chemical

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hazards that can affect the safety of irrigated vegetables (Pérez-Sautu et al., 2012). In

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fact, the microbiological safety of irrigation water is one of the most important factors

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to be considered in the production of leafy greens (Allende & Monaghan, 2015;

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Uyttendaele et al., 2015; Decol et al., 2017).

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Chemical disinfection treatments are often used as tertiary treatments for the

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reclamation of municipal wastewater. Among the chemical disinfectants, chlorine is one

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of the most commonly used biocides for wastewater disinfection and irrigation water

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treatment. However, chlorine is highly reactive with organic matter and causes the

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generation of organo-halogenated disinfection by-products (e.g. trihalomethanes and

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haloacetic acids) (Ayyildiz, et al., 2009; Nikolaou & Lekkas, 2001; Rodriguez &

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Serodes, 2001). Chlorine dioxide (ClO2) has been defined as a potential alternative to

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ACCEPTED MANUSCRIPT chlorine for disinfection of agricultural water. The main advantage of ClO2 over

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chlorine is the formation of fewer types and lesser amounts of organo-halogenated by-

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products (Veschetti et al., 2003; López-Gálvez et al., 2010; Van Haute et al., 2015; Van

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Haute et al., 2017). However, the use of ClO2 can lead to the presence of other DBPs

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such as chlorites (ClO2-) and chlorates (ClO3-) in the treated water via

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disproportionation reactions (AHDB, 2016). ClO2 shows a higher oxidation capacity

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and bactericidal capability compared to chlorine (Hassenberg et al., 2017). Bactericidal

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effectiveness of ClO2 depends on several factors, including disinfectant dose, contact

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time, water temperature, pH, and organic load (Junli et al., 1997; Ayyildiz, et al., 2009).

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Sprinkler irrigation is the most utilized irrigation system for the commercial

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growth of baby leaves (Oron, 2002; Pachepsky et al., 2011). Current Spanish and US

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legislation classify reclaimed water based on specific microbiological standards in

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different categories, each one for specific crops and irrigation system (Real Decreto

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1620/2007, 2007). For example, only reclaimed water with less than 2 log CFU E. coli

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/100 mL can be applied in direct contact with the edible part of the crop using overhead

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irrigation.

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Based on these factors, the aim of present study was to evaluate the suitability of

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ClO2 for the reduction of the microbiological contamination present in secondary-

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treated wastewater used for overhead irrigation of commercially grown baby lettuce.

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The effect of ClO2 on the concentration of the fecal indicator E. coli, and on the

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presence of the bacterial pathogens Shiga-toxigenic Escherichia coli (STEC) and

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Salmonella spp. were assessed. These pathogenic microorganisms were selected as the

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most relevant foodborne pathogens on leafy greens (Ahmed et al., 2012; Ferguson et al.,

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2012; EFSA and ECDC, 2015; Decol et al., 2017). Additionally, the potential

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occurrence of chlorates in water and in the irrigated plants was evaluated.

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Materials and methods

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2.1. Experimental setup Ten day-old Red oak lettuce plants obtained from a local nursery (Semilleros

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Jimenado S.A., Torre Pacheco, Spain) were grown in a greenhouse located next to the

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wastewater treatment plant (WWTP) (Murcia, Spain) (37°47′48″ N, 0°57′33″ W). Data

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acquisition including climatological data and the irrigation system’s layout have been

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described in previous studies (López-Gálvez et al., 2014). In the present study, two

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types of water were used for overhead irrigation system. The first water type was

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secondary effluent from the WWTP (SW) obtained by the treatment of municipal

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wastewater. Secondary treatment consisted in activated sludge systems followed by

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coagulation-flocculation for the removal of suspended solids, colloids and organic

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matter present in wastewater (Renault et al., 2009; López-Gálvez et al., 2016b). The

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second irrigation water type consisted of secondary effluent from the WWTP treated

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with chlorine dioxide (ClO2W). The plants were grown on trays with peat as substrate.

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A total of eight lettuce trays with 294 plants each were used for each irrigation water

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type.

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Two preliminary tests were carried out in order to adjust the ClO2 doses and the

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experimental conditions for lettuce growth. For the selection of the optimum

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disinfectant doses, the reduction of E. coli in the water and the phytotoxic effects in the

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plants were taken into account. During November and December 2016, a final

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experiment that lasted 21 days was performed using the optimum conditions previously

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established. In the final experiment, minimum and maximum temperatures inside the

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greenhouse were 14.4 °C and 28.3 °C, respectively, with an average of 17.2 °C. The

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relative humidity (RH) in the greenhouse ranged from 52.6 % to 91.6 % with an average

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of 76.8 %. The day length during this period was approximately 10 hours. The

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approximate total amount of irrigation water applied throughout the experiment was 1.6

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m3 per treatment. During the growing cycle, the lettuce plants were irrigated once a day

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for 5-15 minutes. During the experiments, the concentration of culturable and presumptive viable E.

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coli, as well as the presence of pathogenic bacteria were assessed in water and lettuce

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samples. Additionally, residual ClO2 concentration and other physicochemical

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parameters of the irrigation water such as pH, temperature, oxidation reduction potential

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(ORP), absorbance at 254 nm (UV254), and the concentration of chlorates (ClO3-) were

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analyzed.

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2.2. Preparation, measurement, and application of chlorine dioxide The company Servicios Técnicos de Canarias (STC S.L.U., Las Palmas de Gran

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Canaria, Spain) provided reagents and instructions for the preparation of the stable

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chlorine dioxide solution AGRI DIS® (ClO2). A concentrated ClO2 solution (7000

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mg/L) was prepared weekly and kept in an opaque plastic jerry can at ambient

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temperature. Chronoamperometric measurement of ClO2 concentration was performed

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using the equipment ChlordioXense® (Palintest, Gateshead, United Kingdom). A

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diluted ClO2 solution (100- 300 mg/L ClO2) was prepared daily just before starting the

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irrigation using the concentrated ClO2 solution and tap water. The diluted ClO2 solution

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was pumped directly to the pipes using a peristaltic pump. Pipe length and contact time

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from the ClO2 application point to the sprinklers were ≈50 m and ≈6 min, respectively.

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The ClO2 doses applied were selected based on the results of the preliminary trials.

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2.3. Sample collection

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ACCEPTED MANUSCRIPT Water sampling was performed every 2 to 5 days over the lettuce growing cycle.

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Irrigation water was sampled during the irrigation of lettuce from the sprinkler emitters

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located at the end of the irrigation lines. Each sampling day, three to five 2-L water

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samples were collected using sterile plastic jars. For microbiological analysis, in order

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to quench residual ClO2, 5 mL of sodium thiosulfate (17.5 g/L) were added to the

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ClO2W samples. A total of n=74 water samples (38 SW, and 36 ClO2W samples) were

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collected in eight different sampling days along the lettuce growing cycle. For

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pathogenic bacteria analysis, one 10-L sample per treatment was collected each

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sampling day (n=16). Additionally, for the measurement of chlorates, three samples (45

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mL) per treatment (n=74) were taken each sampling day.

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Lettuce sampling was performed 5 times during the last two weeks of the lettuce

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growing cycle. Samples were taken every 2 to 5 days, and the last sampling was done at

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the end of the cycle. Each sampling day, three samples (60 g each) per treatment were

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aseptically taken and were transported to the lab in refrigerated conditions. A total of

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n=30 lettuce samples were collected (15 samples per treatment) in five different

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sampling days. Lettuce samples were always taken before irrigation.

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2.4. E. coli analysis

Culturable E. coli quantification was carried out on water and lettuce samples. For

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water samples, depending on the expected E. coli concentration, pour plating (1 mL)

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and/or membrane filtration (10 and 100 mL) were used. Samples were filtered through

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0.45 µm membrane filters (Sartorius, Madrid, Spain) using a filter holder manifold

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(Millipore, Madrid, Spain). Chromocult coliform agar (Merck, Darmstadt, Germany)

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was used for membrane incubation and pour plating. Plates were incubated for 24 h at

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37 °C before interpretation of the results. Dark blue-violet colonies were considered

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ACCEPTED MANUSCRIPT positives for E. coli. For lettuce samples, 25 g were homogenized in 100 mL of sterile

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0.1% buffered peptone water (BPW, Scharlab, Barcelona, Spain) for the quantification

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of culturable E. coli. The homogenate was serially diluted and 1 mL aliquots were pour

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plated using Chromocult coliform agar. Incubation of the plates and interpretation of

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results were performed as explained before for water samples.

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Molecular quantification of E. coli in irrigation water and baby lettuce was

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performed following the combined use of propidium monoazide (PMA, Biotium Inc,

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Hayward, CA, USA) and quantitative polymerase chain reaction (q-PCR) (PMA-qPCR)

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as previously described in Truchado et al. (2016) with some modifications. Three water

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samples (200 mL) per treatment (SW and ClO2W) and sampling day were centrifuged at

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3000 g for 20 min. In the case of lettuce samples, three samples (25 g each) were

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homogenized in 100 mL of sterile 0.1% BPW using a Stomacher at low speed for 1

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min. The homogenate of each sample was centrifuged at 3000 g for 10 min. Then, each

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obtained pellet was activated with PMA (20 µM) and kept at -20 ºC until the DNA

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extraction was performed. Master Pure TM Complete DNA and RNA purification kit

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(Epicenter, Madison, USA) following the manufacturer’s instructions was used. For

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molecular quantification, primers and probes for detecting genes of E. coli 23S rRNA as

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well as the PMA-qPCR procedure were identical to those previously described

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(Truchado et al., 2016). Standard curves were made using known concentrations of

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genomic DNA isolated from E. coli CECT 515T. The E. coli concentration in the stock

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solution was verified by plating on PCA.

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2.5. Detection of pathogenic microorganisms

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For the detection of Shiga-toxigenic E. coli (STEC), E. coli O157:H7 and

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Salmonella spp. in water samples, 10-L samples were filtered at the greenhouse through

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ACCEPTED MANUSCRIPT Modified Moore Swabs (MMS) as previously described (Sbodio et al., 2013). The

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MMS were transferred aseptically into sterile stomacher plastic bags and transported to

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the lab in refrigerated conditions. Once in the lab, 200 mL of 20 g/L buffered peptone

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water (Scharlab, Barcelona, Spain) was added to the bags that were incubated for 24 h

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at 37 °C for enrichment. After incubation, a volume of 7 mL was transferred into 15 mL

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centrifuge tubes and stored at −20 °C with 30% glycerol until the analyses were

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performed.

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For the lettuce samples analyses, the homogenate described in paragraph 2.3.1

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was supplemented with 125 mL of BPW (40 g/L) and homogenized by massaging by

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hand the stomacher bags. Afterwards, bags were incubated for 24 h at 37 °C for

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enrichment, and then a volume of 7 mL was transferred into 15 mL centrifuge tubes and

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stored at −20 °C with 30% glycerol until the analyses were performed.

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Aliquots of 1 mL from each frozen sample were added to 9 mL of selective

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enrichment broth specific for the target pathogens. Modified Buffered Peptone Water

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(20 g/L) supplemented with sodium pyruvate (Scharlau, Barcelona, Spain; mBPWp)

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incubated for 24 h at 42 °C was used for STEC and E. coli O157:H7. Tetrathionate

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Broth (TT; Scharlau) incubated for 24 h at 37 °C was used for Salmonella. Prevalence

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of pathogenic microorganisms in water (n= 16) and lettuce samples (n=30) were

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performed using the Salmonella-STEC GeneDisc Pack in a Genedisc Cycler multiplex

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PCR (Pall® Corporation, WA, USA) following manufacturer instructions. Confirmation

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of presumptive positive samples was performed by isolation in selective culture media.

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For STEC, CHROMagar STEC (CHROMagar, Paris, France) incubated for 24 h at 37

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°C was used. For E. coli O157:H7, two culture media were used: CT-SMAC (Scharlab,

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Barcelona, Spain) and CHROMagar O157 (CHROMagar, Paris, France), followed by

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further confirmation using a latex test (Oxoid, Basingstoke, UK). For Salmonella, the

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IBISA method (AES Chemunex, Bruz, France) was used, followed by further

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confirmation using a latex test (Oxoid, Basingstoke, UK).

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2.6. Physicochemical analysis of irrigation water Temperature, pH and ORP were measured using a multimeter (pH and redox 26,

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Crison, Barcelona, Spain). For measuring UV254, water was filtered through 0.45 µm

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syringe nylon filters (Fisherbrand-Fisher Scientific, Waltham, USA), and a UV-VIS

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spectrophotometer (Jasco V-630, Tokyo, Japan) and quartz cuvettes with a path length

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of 1 cm (Hellma, Müllheim, Germany) were used.

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2.7. Presence of chlorates in irrigation water and in lettuce

Chlorates (ClO3-) content in water and lettuce was analysed by LC-MS as

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described in Gil et al. (2016), using an analytical standard of chlorate (RTC, ICS-004-

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100, Fluka, Sigma-Aldrich, Spain) for quantification. Areas of the peaks detected by

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MS were used for the quantification of chlorates. Results were expressed in mg/L and in

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mg/kg for water and lettuce samples, respectively.

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2.8. Statistical analysis

Counts derived from microbiological analyses were log transformed and entered

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in an Excel spreadsheet (Microsoft Excel, 2016). Results were compiled and graphs

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were made using Sigma Plot 11.0 Systat Software, Inc. (Addilink Software Scientific,

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S.L. Barcelona). SPSS statistics 21 (IBM, Armonk, NY, USA) was used for statistical

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analysis at a significance level of 5% (p = 0.05). The Kolmogorov–Smirnov test and

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Levene's test were used to assess normality and equality of variance, respectively. When

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data was not following a normal distribution, nonparametric tests were applied. Mann–

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Whitney U and Kruskal–Wallis tests were used to determine the difference between the

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raw data of the indicators and the presence of pathogens. The Pearson’s correlation

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coefficient was calculated (p<0.01) to assess links between physicochemical

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characteristics of wastewater (SW and ClO2W).

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3.

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3.1. ClO2 treatment

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In our study, the initial ClO2 concentration applied for the treatment of the

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secondary wastewater ranged between 3.3 and 9.2 mg/L. These initial ClO2 doses were

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necessary to reduce the levels of E. coli of reclaimed water below 2 log CFU/100 mL,

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which is the threshold recommended for irrigation water in the guidance addressing

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microbiological risks of fresh fruits and vegetables at primary production (EC, 2017b).

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Fig. 1 shows the initial and residual ClO2 levels in the ClO2W samples taken the days in

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which water and lettuce samples were taken for microbiological and physicochemical

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analysis. The contact time between ClO2 and reclaimed water in the irrigation

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distribution system was approximately 6 minutes. In all the cases, the ClO2 residual in

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wastewater as measured in the irrigation emitter was less than 1 mg/L (<0.02 to 0.33

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mg/L) to avoid any damage of phytotoxicity to the plants (WEAH, 2016).

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Absorbance at 254 nm (UV254) was measured as an indicator of the organic

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matter content of the irrigation water distribution system. Values of UV254 for SW

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ranged between 0.05 and 1.36 cm-1. Based on previous studies the increasing order of

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reactivity of some disinfectants commonly used in the treatment of wastewater with

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organic matter is: peracetic acid
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Haute et al., 2017; Veschetti et al., 2003). Although ClO2 is less reactive with organic

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matter than chlorine (Van Haute et al., 2015; Rodriguez & Serodes, 2001), the

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ACCEPTED MANUSCRIPT concentration of ClO2 is affected by the presence of organic matter. In the present study,

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the concentration of organic matter in the water influenced the residual ClO2

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concentration. For example, when lower concentrations of organic matter were observed

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(UV254=0.05 cm-1 at day 4 and 0.08 cm-1 at day 6), higher ClO2 residuals were detected

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(0.26 and 0.33 mg/L) (Fig. 1). A significant (p<0.01) negative correlation of -0,508 was

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found between residual ClO2 concentration and UV254. Similar results have been

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described in other studies (Tomás-Callejas et al., 2012; Praeger et al., 2016; Hassenberg

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et al., 2017; Van Haute et al., 2017).

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3.2. Changes in microbiological quality of irrigation water by chlorine dioxide

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In our study, when SW and ClO2W samples were analyzed by conventional plate

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count methods, significant differences (p <0.05) in E. coli counts were observed (Fig.

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2A). E. coli counts of SW and ClO2W samples ranged between 2.00 and 4.76 log

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CFU/100 mL (IQR=3.32 – 3.82) and between 0.70 and 3.49 log CFU/100 mL

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(IQR=1.59 – 2.18), respectively. Considering the mean counts of SW and ClO2W, it

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was possible to calculate a mean reduction of 2.21 log CFU/100 mL. However, when E.

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coli levels were quantified using the PMA-qPCR method, the average difference in E.

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coli enumerations between SW and ClO2W was 1.07 logarithmic units, and no

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significant differences were detected (p>0.05) (Fig. 2B). E. coli levels in SW and

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ClO2W samples as determined by PMA-qPCR were 3.17 to 6.27 log cells/100 mL (IQR

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4.19 – 5.10) and 2.71 to 5.46 log cells/100 mL (IQR 3.66 – 4.54), respectively (Fig.

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2B). In agreement with our results, some studies have reported that the levels of E. coli

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cells quantified by PMA-qPCR assay in different water samples such as drinking water,

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wastewater, irrigation water and seawater were higher than those obtained by cultivation

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based techniques (Van Frankenhuyzen et al., 2013; Gensberger et al., 2014; Li et al.,

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ACCEPTED MANUSCRIPT 2014; Truchado et al., 2016; López-Gálvez et al., 2018a). PMA is a DNA photoreactive

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binding dye, which allows the differentiation between viable and dead cells, avoiding

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an overestimation of results by qPCR (Gensberger et al., 2014; Truchado et al., 2016).

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Therefore, the differences observed between PMA-qPCR and plate counts may be due

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to the presence of viable but not cultivable (VBNC) bacteria. The results obtained could

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indicate a bacteriostatic action of ClO2, which can induce the entrance of E. coli cells

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into a VBNC state. Oliver et al., (2005) reported that the VBNC stage can be induced

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when microorganisms are exposed to chemical disinfectants. The presence of VBNC

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bacteria in the reclaimed wastewater due to water reclamation processes has been

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demonstrated previously (Zhang et al., 2015; Lin et at., 2016; Kibbee and Örmeci,

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2017). Recently, Kibbee and Örmeci (2017) have evaluated the levels of E. coli present

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in secondary wastewater effluent after chlorine disinfection, showing that high numbers

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of VBNC E. coli survive chlorination. Therefore, the use of conventional plate counting

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methods might lead to an overestimation of the efficacy of the water disinfection

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treatments (Zhang et al., 2015). It should be taken into account that the only culture

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method used in the present study to detect injured E. coli cells after treatment was based

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in the use of a selective culture medium and incubation for 24 h at 37 °C.

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Several studies have shown that different factors may influence the bactericidal

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effect of ClO2 (Ayyildiz, et al., 2009; Junli et al., 1997). Some of the most important are

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temperature, pH, and presence of organic matter. The temperature of water can strongly

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influence the microbial inactivation capacity of ClO2. Barbeau et al. (2005) observed

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that 0.25 mg/L of free ClO2 were sufficient to inactivate 99% of E. coli in water after 16

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seconds at 30 °C. However, when the temperature of water was 5 °C, the same

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reduction was reached only after 110 seconds. Junli et al. (1997) reported that 10 °C

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increase of the water temperature doubles the inactivation power of ClO2 against

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microbiological inactivation capacity of ClO2, this influence was not observed in the

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present study. The reductions of E. coli observed in reclaimed wastewater demonstrated

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no correlation with water temperature (p>0.01) and this may be due to the narrow range

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of temperature variation in the irrigation water (15.2 to 19.3 °C).

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Globally, water demand is predicted to increase significantly over the coming

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decades. According to WWAP (2017), more than 70% of the water that is consumed all

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over the world is used for agricultural irrigation. Therefore, there is a big potential for

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the application of reclaimed water for irrigation (WHO, 2006). To evaluate the

359

microbiological suitability of reclaimed municipal wastewater for agricultural irrigation,

360

the recommendations and microbiological limits described in guidelines and regulations

361

should be used. For example, World Health Organization (WHO, 2006) recommends

362

that wastewater used for irrigation of agricultural crops likely to be eaten raw should

363

have a level of fecal coliforms ≤103 CFU/100 mL. In the United States, the Food and

364

Drug Administration (FDA) established a limit of 23 CFU/100 mL for E. coli

365

concentration in agricultural irrigation water when there is a direct contact with the

366

produce (Sugano et al., 2016). In Italy, the limit for E. coli concentration in treated

367

wastewater used for agricultural irrigation is 10 CFU/100 mL (Decreto Ministeriale,

368

2003). Spanish legislation establishes in reclaimed water in direct contact with produce

369

consumed raw a maximum of 102-103 CFU of E. coli per 100 mL in irrigation water

370

(number of sample units (n) = 10, threshold value for the number of E. coli (m) = 100

371

CFU/100 mL, maximum value (M) = 1000 CFU/100 mL, number of sample units where

372

the E. coli count may be between m and M (c) = 3) (Real Decreto 1620/2007, 2007).

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In the present study, E. coli concentration was above the 2 log/100 mL threshold

374

in all SW samples analyzed. On the other hand, due to the treatment with ClO2, 69.4%

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ACCEPTED MANUSCRIPT of ClO2W samples (25 out of 36) were in accordance with the Spanish legislation for E.

376

coli (Real Decreto 1620/2007, 2007) based on conventional plate counting techniques.

377

Therefore, even after ClO2 treatment, 30.6% of the ClO2W samples were not acceptable

378

for being used in overhead irrigation of raw consumed leafy green vegetables according

379

to the Spanish legislation.

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3.3. Microbiological characteristics baby lettuce irrigated with reclaimed water disinfected with chlorine dioxide

383

Culturable E. coli counts in baby lettuce irrigated with SW and ClO2W ranged

384

between <0.70 and 2.90 log CFU/g (IQR 0.69 – 1.30) and between <0.70 and 1.40 log

385

CFU/g (IQR 0.69 – 0.69), respectively, throughout the sampling period (Fig. 3A).

386

Significant differences (p <0.05) in culturable E. coli counts were observed between

387

baby lettuce irrigated with SW and ClO2W. These differences could be explained by the

388

higher microbial contamination of SW that caused a higher contamination over the

389

entire growing period. Similar results were demonstrated by Makkaew et al. (2016) and

390

Amahmid et al. (1999) investigating the influence of different levels of E. coli

391

contamination present in wastewater stabilization ponds in South Australia and

392

retention of E. coli contamination when compared with the other types of lettuces. In

393

our study, we used the variety red Oak leaf, whose morphology/topography may have

394

contributed to the retention of E. coli.

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E. coli enumerations using PMA-qPCR rendered counts in SW and ClO2W

396

irrigated lettuce of 2.17 to 3.83 log cells/g (IQR 2.83 – 3.25) and 2.18 to 3.31 log cells/g

397

(IQR 2.55 – 2.98), respectively (Fig. 3B). PMA-qPCR analysis resulted in log counts

398

around 2 logarithmic units higher than those from the same samples analyzed by the

399

plating method in agreement with previous findings (Truchado et al., 2016). As

16

ACCEPTED MANUSCRIPT mentioned before for water samples, the differences observed between plate count and

401

PMA-qPCR methods could be due to the presence of VBNC E. coli by the biocide and

402

the stress induced by the environmental conditions. The phyllosphere is a hostile habitat

403

for microorganisms due to nutrient limitation, shift in temperature and solar radiation

404

exposure, which can induce by VBNC state of the bacteria (Wilson and Lindow, 2000).

405

This phenomenon should be considered with caution because bacteria can persist for

406

long periods of time in this state and they could retain their virulent potential (Dinu and

407

Bach, 2011).

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3.4. Correlation between E. coli levels and presence/absence of pathogens

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The prevalence of pathogens was detected by a multiplex PCR in 9 out of 16

411

samples of all irrigation water samples (56.2 %). Among the positive samples, 8

412

samples were confirmed using selective media and latex agglutination tests. Seven out

413

of eight corresponded to SW (1 Salmonella and 6 STEC) while only one sample of

414

ClO2W was positive for STEC. For the lettuce samples, the presence of pathogens was

415

detected in 4 out of 33 samples (13.33 %), by a multiplex PCR. Only 1 lettuce sample

416

irrigated with SW was confirmed for the presence of STEC using selective media and

417

latex agglutination tests. E. coli O157:H7 was not confirmed in any water or lettuce

418

sample (Table 2).

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Fig. 4 shows the relationship between the E. coli levels enumerated using plate

420

count and PMA-qPCR methods and the presence/absence of pathogenic bacteria in

421

irrigation water samples. For both quantification methods (plate count and PMA-qPCR)

422

positive samples for pathogenic bacteria showed significantly higher E. coli levels than

423

samples negative for the presence of pathogenic bacteria (Mann-Whitney U Test,

424

p<0.05) (Fig. 4B). The only exception was for SW when E. coli was enumerated using

17

ACCEPTED MANUSCRIPT conventional plating methods, which could be due to the high levels of E. coli found in

426

samples both positive and negative for pathogenic bacteria. Corroborating these

427

findings, Ferguson et al. (2012) and Truchado et al. (2016) reported that molecular

428

techniques are suitable techniques to predict the potential presence of pathogenic

429

bacteria in groundwater, and secondary reclaimed water, respectively.

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Other studies also found correlation between E. coli levels and the presence of

431

STEC and Salmonella in agricultural settings (Ceuppens et al., 2014; Holvoet et al.,

432

2014; López-Gálvez et al., 2014; Park et al., 2014; Castro-Ibáñez et al., 2015). These

433

findings support the hypothesis that considers E. coli as a good microbial indicator of

434

fecal contamination and the association with enteric pathogens.

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3.5. Occurrence of chlorates in chlorine dioxide treated reclaimed water and irrigated baby lettuce

438

ClO2 has recently been used as an alternative to chlorine as it does not lead to the

439

formation of chlorinated DBPs after reaction with organic matter. However, chlorite and

440

chlorate ions (ClO2-, ClO3-) can be formed due to disproportionation reactions, and they

441

are the main DBPs from ClO2 that may have a significant human health risk.

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When irrigation water samples were analyzed for the presence of ClO3-, SW

443

showed very low levels (0.00-0.01 mg/L), while the concentration in ClO2W ranged

444

between 1.44 and 5.94 mg/L (Fig. 5). In other studies, lower chlorates concentrations

445

were detected in irrigation water treated with disinfectants (Nitsopoulos et al., 2014;

446

López-Gálvez et al., 2018a; López-Gálvez et al., 2018b). However, in those studies,

447

water with a much better microbiological and physicochemical quality was used and,

448

therefore, lower disinfectant concentration was needed for the treatment. In our study,

449

due to the characteristics of the treated water, a high initial concentration of ClO2 was

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ACCEPTED MANUSCRIPT 450

needed to achieve the desired microbiological reduction (Fig. 1). The concentration of

451

ClO3- showed a significant positive correlation (p<0.01) of 0.65 with the initial ClO2

452

concentration. According to Korn et al. (2002), lower concentration of ClO2 and lower levels of

454

organic matter can reduce the presence of chlorates in treated water. In the present

455

study, the high amount of organic matter present in the water influenced the increase of

456

chlorate concentration due to the higher disinfection capacity needed.

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In baby lettuce irrigated with ClO2W, concentration of ClO3- ranged between 1.13

458

and 8.49 mg/kg (Fig. 5). These levels are much higher than the maximum residue limit

459

for chlorate allowed in the European Union in food (0.01 mg/kg; EC, 2005), although

460

these levels are currently being revised (EC, 2014). In a previous study from our group

461

(López-Gálvez et al., 2018a), lower chlorate concentrations were detected in baby

462

spinach cultivated in open field and irrigated by overhead irrigation with ClO2-treated

463

surface water. However, much lower disinfectant concentrations were used compared

464

with the present study, leading to lower chlorate concentration in irrigation water and in

465

the crop. Other studies performed using irrigation water treated with chlorine-based

466

disinfectants reported lower chlorate concentration in the crop compared with our study,

467

probably due to the lower concentrations of disinfectants applied (Nitsopoulos et al.,

468

2014; López-Gálvez et al., 2018b).

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4.

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Conclusions

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ClO2 treatment reduced culturable E. coli concentration and the prevalence of

472

pathogenic bacteria in the water used for irrigation. However, when viable bacteria was

473

enumerated by molecular techniques combined with the use of PMA, no significant

474

differences were observed between untreated and ClO2 treated water, indicating a

19

ACCEPTED MANUSCRIPT potential bacteriostatic action of ClO2, which can induce the entrance of E. coli cells

476

into a VBNC state. Baby lettuce irrigated with ClO2 treated water beared lower

477

concentrations of culturable E. coli than the plants irrigated with the untreated

478

secondary effluent of the WWTP. However, as in the case of the water samples, the

479

differences between treatments were not significant when the enumeration of E. coli

480

was performed by PMA-qPCR. Detection of pathogens in lettuce was almost null, and,

481

as a consequence, the potential effect of ClO2 on the occurrence of pathogens in the

482

lettuce plants could not be detected. The results obtained suggest that the use of plate

483

count methods to estimate the efficacy of disinfection methods could lead to an

484

overestimation of the microbial reductions. In any case, the accumulation of chlorates in

485

the crop as a consequence of the ClO2 treatment exceeded the maximum recommended

486

limits of chlorates (0.01 mg/kg), making this treatment unsuitable to be applied under

487

the conditions evaluated in the present study.

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Acknowledgments

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Authors are thankful for the financial support from the Center for Produce Safety Grant

Agreement

(Projects

492

(ProjectsAGL2013-48529-R and AGL2016-75878-R). Support provided by the

493

Fundación Séneca (19900/GERM/15) and the CNPq/MCTI with the PVE Project

494

313835/2013-6 is highly appreciated. P. Truchado is holder of a Juan de la Cierva

495

incorporation contract from the MINECO (IJCI-2014-20932).

2015-374

and

2017-01)

and

the

MINECO

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Uyttendaele, M., Jaykus, L-A., Amoah, P., Chiodini, A., Cunliffe, D., Jacxsens, L.,

681

Holvoet, K., Korsten, L., Lau, M., McClure, P., Medema, G., Sampers, I., & Jasti,

682

P.R. (2015). Microbial hazards in irrigation water: standards, norms, and testing to

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manage use of water in fresh produce primary production. Compr. Rev. Food Sci.

684

F., 14, 336–356.

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Van Frankenhuyzen, J.K., Trevors, J.T., Flemming, C.A., Lee, H., & Habash, M.B.

686

(2013). Optimization, validation, and application of a real-time PCR protocol for

687

quantification of viable bacterial cells in municipal sewage sludge and biosolids

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using reporter genes and Escherichia coli. J. Ind. Microbiol. Biot., 40, 1251–126.

689

Van Haute, S., López-Gálvez, F., Gómez-López, V. M., Eriksson, M., Devlieghere, F.,

690

& Allende, A. (2015). Methodology for modeling the disinfection efficiency of

691

fresh-cut leafy vegetables wash water applied on peracetic acid combined with

692

lactic acid. Int. J. Food Microbiol., 208, 102–113.

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Van Haute, S., Tryland, I., Escudero, C., Vanneste, M., & Sampers, I. (2017). Chlorine

694

dioxide as water disinfectant during fresh-cut iceberg lettuce washing:

695

Disinfectant demand, disinfection efficiency, and chlorite formation. LWT - Food

696

Sci. Technol., 75, 301–304. Veschetti, E., Cutilli, D., Bonadonna, L., Briancesco, R., Martini, C., & Cecchini, G.

698

(2003). Pilot-plant comparative study of peracetic acid and sodium hypochlorite

699

wastewater disinfection. Water Res., 37, 78–94.

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WEAH, Water Education Alliance for Horticulture, Treatment Technologies: Chlorine

701

dioxide. (2016). Available at: http://watereducationalliance.org/keyinfo.asp.

702

Accessed January 2018.

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Wilson, M., & Lindow, S.E. (2000). Viable but nonculturable cells in plant-associated

704

bacterial populations. In Colwell, R.R. & Grimes D.J. (Eds.) Non-culturable

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Microorganisms in the Environment (pp. 229–241). ASM Press, Washington,

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D.C.

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WHO, World Health Organization. (2006). WHO guidelines for the safe use of

708

wastewater, excreta and grey water. Wastewater use in agriculture. Available

709

from: http://whqlibdoc.who.int/publications/2006/9241546832_eng.pdf. Accessed

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5 July 2017.

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Zhang, S., Ye, C., Lin, H., Lv, L. & Yu, X. (2015). UV disinfection induces a VBNC

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state in Escherichia coli and Pseudomonas aeruginosa. Environ. Sci. Technol.,

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49, 1721-1728.

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ACCEPTED MANUSCRIPT Figure Legends

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Figure 1. Initial and residual concentrations of chlorine dioxide (ClO2) in treated water

716

from the secondary effluent of a wastewater treatment plant (ClO2W) used for irrigation

717

of lettuce cultivated in a greenhouse. The figure shows only the data obtained in days

718

when lettuce and water samples for microbiological and physicochemical analyses were

719

taken.

720

Figure 2. Boxplot representing E. coli counts (log CFU/100 mL) of water from the

721

secondary effluent of a wastewater treatment plant untreated (SW) and ClO2 treated

722

(ClO2W) used for irrigation of lettuce cultivated in a greenhouse. (A) Counts obtained

723

by conventional plate count method. (B) Counts obtained by PMA–qPCR molecular

724

quantification method. In a boxplot, the bottom and top of the boxes represent the

725

quartiles (25th and 75th percentile), with the line inside the box representing the

726

median. Different letters indicate significant differences (p<0.05).

727

Figure 3. Boxplot representing E. coli counts (log CFU/g) of baby lettuce irrigated with

728

water from the secondary effluent of a wastewater treatment plant untreated (SW) and

729

ClO2 treated (ClO2W) and cultivated in a greenhouse. (A) Counts obtained by

730

conventional plate count method. (B) Counts obtained by PMA–qPCR molecular

731

quantification method. In a boxplot, the bottom and top of the boxes represent the

732

quartiles (25th and 75th percentile), with the line inside the box representing the

733

median. Different letters indicate significant differences (p<0.05).

734

Figure 4. Boxplot representing E. coli counts (log CFU/100 mL) in the subset of water

735

samples with either absence or presence of pathogens in the water from the secondary

736

effluent of a wastewater treatment plant untreated (SW) and ClO2 treated (ClO2W) used

737

for irrigation of lettuce cultivated in a greenhouse. (A) E. coli counts obtained by

738

conventional plate counting method. (B) E. coli counts obtained by PMA–qPCR

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30

ACCEPTED MANUSCRIPT quantification method. In a boxplot, the bottom and top of the boxes represent the

740

quartiles (25th and 75th percentile), with the line inside the box represents the median.

741

Different letters indicate significant differences (p<0.05).

742

Figure 5. Chlorate concentration in water samples (mg/L) and baby lettuce (mg/kg)

743

irrigated with secondary effluent of a wastewater treatment plant untreated (SW) and

744

ClO2 treated (ClO2W) for irrigation during cultivation in a greenhouse.

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ACCEPTED MANUSCRIPT Figure 1

Initial ClO2 Residual ClO2

9

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6

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5 4 3 2 1 0 20

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17

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ClO2 (mg/L)

7

13

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Days before harvest

6

4

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ACCEPTED MANUSCRIPT Figure 2

6 a

E. coli (Log cfu/ 100 mL)

5 4

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E. coli (Log cells/100 mL)

6 5

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SW

ClO2W

Irrigation water types

BB

ACCEPTED MANUSCRIPT Figure 3

A

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2

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E. coli (Log cfu/g)

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E. coli (Log cells/g)

4

1

0

SW

ClO2W

Irrigation water types

B

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A 7

a a

4

0

a

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E. coli (Log cells/100 mL)

A

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7

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Pathogen: Presence Absence

0

SW

ClO2W Irrigation water types

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SW Water ClO2W Water SW Lettuce ClO2W Lettuce

9.0 8.0

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18

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11

6

Days before harvest

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ACCEPTED MANUSCRIPT Table 1. pH, temperature, oxidation reduction potential (ORP), and UV254 of secondary effluent of a wastewater treatment plant untreated (SW) and ClO2 treated (ClO2W) for irrigation of baby lettuce cultivated in a greenhouse. SW pH

Temperature ORP (°C)

(mV)

pH

Temperature

ORP

(°C)

(mV)

19.1

477

16.7

493

6.57

20.0

460

7.75

-

-

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Days before harvest

ClO2W

6.43

19.3

472

6.36

18

6.34

16.7

482

6.38

13

6.31

19.2

463

11

7.60

-

-

6

8.13

16.5

431

7.95

16.9

432

4

8.01

15.2

514

7.89

15.3

791

0

7.58

16.9

450

7.51

17.0

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Table 2. Presence and absence of pathogen microorganisms in water samples and baby lettuce irrigated with secondary effluent of a wastewater

Samples type

Salmonella

STEC

Genedisc®

Confirmed

Genedisc®

SW

5/8

1/5

7/8

ClO2W

1/8

0/1

Lettuce

Genedisc®

Confirmed

SW

2/15

0/2

ClO2W

2/15

0/2

E. coli O157:H7

Genedisc®

Confirmed

6/7

6/8

0/6

1/8

1/1

1/8

0/1

Genedisc®

Confirmed

Genedisc®

Confirmed

2/15

1/2

1/15

0/1

1/15

0/1

1/15

0/1

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Water

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treatment plant untreated (SW) and ClO2 treated (ClO2W) for irrigation during cultivation in a greenhouse.

ACCEPTED MANUSCRIPT Highlights

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ClO2 is a suitable treatment for irrigation water ClO2 reduced levels of cultivable E. coli in water and in irrigated lettuce Levels of viable by no-cultivable E. coli were higher than cultivable E. coli Plate count might led to overestimation of the efficacy of the treatment. Irrigation with ClO2-treated water led to the accumulation of chlorate in the crop

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