Accepted Manuscript

Fate of the fecal indicator Escherichia coli in irrigation with partially treated
wastewater

P. Vergine, R. Saliba, C. Salerno, G. Laera, G. Berardi, A. Pollice

PII: S0043-1354(15)30152-4
DOI: 10.1016/j.watres.2015.08.001
Reference: WR 11448

To appear in: Water Research

Received Date: 6 May 2015

Revised Date: 28 July 2015
Accepted Date: 1 August 2015

Please cite this article as: Vergine, P., Saliba, R., Salerno, C., Laera, G., Berardi, G., Pollice, A., Fate of
the fecal indicator Escherichia coli in irrigation with partially treated wastewater, Water Research (2015),
doi: 10.1016/j.watres.2015.08.001.

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 ACCEPTED MANUSCRIPT

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1 Fate of the fecal indicator Escherichia coli in irrigation with partially treated wastewater

2 P. Vergine1, R. Saliba2,3, C. Salerno1, G. Laera1, G. Berardi1, A. Pollice1*

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3 IRSA CNR, Viale F. De Blasio, 5 - 70132 Bari, Italy
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4 CIHEAM IAMB, Via Ceglie, 9 - 70010 Valenzano (Bari), Italy
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5 Università degli Studi di Napoli Parthenope - Via Amm. F. Acton, 38 - 80133 Napoli, Italy

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6 email: pollice@irsa.cnr.it

7 Abstract

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8 Treated wastewater reuse is increasing in semi-arid regions as a response to the effects of climate

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9 change and increased competition for natural water resources. Investigating the fate of bacterial indicators

10 is relevant to assess their persistence in the environment and possible transfer to groundwater or to the

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11 food chain. A long-term field-scale experimental campaign and a soil column test were carried out to

12 evaluate the fate of the fecal indicator Escherichia coli (E. coli) in a cultivated soil when contaminated
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13 water resources are used for irrigation. For field experiments, fecal contamination was simulated by

14 dosing the indicator to the effluent of a membrane bioreactor, thus simulating a filtration system’s failure,
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15 and irrigating a test field where grass was grown. The presence of E. coli on grass and topsoil samples

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was monitored under different scenarios. For evaluating the fate of the same indicator in the subsoil, a set

17 of soil columns was installed next to the field, operated, and monitored for E. coli concentration over time
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18 and along depth. Real municipal wastewater was used in this case as source of fecal contamination.

19 Results showed that short- and medium-term effects on topsoil were strongly dependent on the
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20 concentration of E. coli in the irrigation water. Limited persistence and no relevant accumulation of the

21 indicator on the grass and in the topsoil were observed. Watering events performed after fecal
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22 contamination did not influence significantly the decay in the topsoil, which followed a log-linear model.
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23 The trend of the E. coli concentrations in the leaching of the soil columns followed a log-linear model as

24 well, suggesting bacterial decay as the dominant mechanism affecting the underground indicator’s

25 concentration.

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27 Keywords: Water reuse; Escherichia coli; Irrigation; Agricultural soil; Wastewater; Fecal

28 contamination.

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29

30 1. Introduction

31 Water stress in semi-arid areas calls for solutions that should promptly provide responses to

32 the effects of climate change and consequent decrease of available resources. Two approaches

33 to be combined in order to properly tackle water scarcity include (i) the improvement of

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34 resource management practices by balancing demand and supply, and (ii) the adoption of

35 alternative water resources (e.g. desalination, wastewater reuse, rainwater harvesting, etc.)

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36 (GWP, 2012). Water quality and the impairment of rivers, streams, groundwater and soils is a

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37 direct menace to the livelihood of growing populations. According to USEPA (2008), the major

38 cause of impairment of rivers and streams is excessive levels of pathogen bacteria. In temperate

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39 climates, peak irrigation demand generally coincides with the time at which surface water
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40 systems are at their greatest risk for microbial contamination (Jamieson et al., 2003). To

41 mitigate the effects of soil water deficits, producers of high value crops may be forced to use
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42 water containing indicator organisms exceeding recommended levels (Wood et al., 2010).

43 Countries facing the Mediterranean Sea have different priorities and constraints with respect
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44 to the improvement of freshwater availability, and also different regulations governing treated
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45 wastewater reuse (Kellis et al., 2013). With specific reference to the hygienic safety of treated

46 effluents, most national regulations base their approach on one of the two guidelines for
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47 wastewater reuse proposed by the WHO and the State of California, respectively (WHO, 1989;

48 State of California, 1978). These two guidelines include different requirements and indications
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49 towards the quality of effluents that can be used for irrigation, and in particular they set the
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50 limits of 1000 fecal coliforms per 100 mL and 2.2 total coliforms (MPN) per 100 mL,

51 respectively. The adoption of one of these two approaches implies very different requirements

52 in terms of treatment technologies, which reflect on the costs of produced water, and finally

53 determine the sustainable applicability of treated wastewater reuse in the different countries.

54 Therefore the knowledge of the real persistence of the fecal contamination in the environment is

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55 a crucial aspect in order to evaluate the appropriateness of current standards for wastewater

56 reuse in agriculture.

57 When cultivated fields are irrigated with fully or partially treated municipal wastewater, the

58 highest risk for human health occurs in the short-term period. Then, natural processes (die-off,

59 solar disinfection, rain, and transfer to the lower layers) progressively reduce the presence of

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60 pathogenic organisms in the topsoil and on the vegetables, but at the same time may cause

61 contamination of subsurface water resources (Sjorgen, 1995; Naclerio et al., 2009). The possible

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62 persistence of fecal contamination in soil depends on its characteristics, moisture content and on

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63 the origin of the fecal contamination itself (i.e. waste type) (Lang and Smith 2007). However,

64 the knowledge of processes affecting persistence and the movement of fecal contamination

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65 through the soil is still scarce. In this context, the identification of suitable indicators of fecal
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66 contamination is also critical. The enteric bacterium Escherichia coli (E. coli) is normally

67 considered a good and specific indicator of this type of contamination, and it is adopted in most
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68 European regulations as a basic tool to assess the microbiological quality of treated municipal

69 wastewater (Kellis et al., 2013). Indeed it represents an easily detectable indicator of the
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70 presence of microorganisms responsible of enteric diseases ranging from mild or bloody

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71 diarrhea to the deadly hemolytic uremic syndrome, which affects humans of all ages (Paton and

72 Paton, 1998). Moreover, E. coli does not duplicate appreciably outside of its natural habitat,
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73 therefore in non-enteric environments its population rapidly tends to decrease after an event of

74 fecal contamination (Odonkor and Ampofo, 2013). The survival period of enteric bacteria in
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75 general, and E. coli in particular, in the soil is influenced by various soil and environmental
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76 variables, such as soil texture, organic matter, pH, temperature, moisture content and nutrients

77 (Semenov et al., 2008). Under field conditions, solar radiation, temperature and dryness may

78 also affect the survival of fecal indicators (Oliveira et al., 2012). According to Oliveira and

79 coworkers (2012) environmental factors play a big role in the determination of the survival time

80 in soil samples. While mobilization represents the dominant factor influencing the fecal

81 microbial dynamics in streams and rivers (McDonald et al., 1982; Hunter et al., 1992), many

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82 research outcomes highlighted the importance of die-off of the fecal indicator as the key process

83 behind its survival or transport to other media (Wilkinson2011).

84 In Apulia (South East Italy), a region whose economy is based on agriculture, the combination

85 of dry weather conditions, absence of permanent rivers and natural lakes, and progressive

86 groundwater salinization requires the adoption of non-conventional resources (Lopez et al.,

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87 2006). Several activities were carried out over the last decades to test and demonstrate the

88 feasibility of wastewater recycling for producing a dedicated resource to activities requiring

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89 continuous water availability during the dry season (e.g. agriculture) (Bixio et al., 2006). In

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90 particular, previous experimental activities have shown the long term feasibility of adopting

91 tertiary membrane filtration as a suitable tool for the upgrade of municipal effluents to comply

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92 with the stringent local standards for reuse (Pollice et al., 2004; Lopez et al., 2010). However, in
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93 the presence of membrane failures, bacterial post-contamination/regrowth in storage basins or

94 ineffective disinfection, a relevant fecal contamination may be transferred to the cultivated soil
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95 through irrigation.

96 Aim of this work is to identify the main factors that affect the fecal contamination of a
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97 cultivated soil (and the underlying groundwater) irrigated with partially treated wastewater.
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98 Results are reported of a field-scale experimental campaign and a soil column test aimed at

99 assessing the fate of E. coli when water containing high concentrations of this indicator is used
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100 for irrigation. The short-term effects of a single contamination event on topsoil (first 10 cm) and

101 grass were firstly investigated at the field-scale. Then, the potential accumulation of a series of
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102 contamination events, and the persistence of E. coli after the contamination were also studied.
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103 Finally, the potential migration of the target bacteria through the lower soil layers was evaluated

104 in the soil column experiment.

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106 2. Materials and methods

107 2.1 Field experiments

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108 A small experimental field was cultivated with grass (Paspalum vaginatum) and divided into

109 two identical plots, defined as control plot and test plot. Plots had dimensions of 4x4 m each,

110 were separated by a 2 m wide aisle paved with plastic blocks, and were equipped with 4

111 sprinklers placed in the corners and oriented towards the center of the plot. A waterproof curtain

112 having height of 1.2 m was also placed between the two plots to prevent cross-contamination in

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113 case of wind. The control plot was irrigated with tap water, while the test plot was irrigated with

114 the effluent of a pilot scale membrane bioreactor (MBR) placed next to the field and operated

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115 for organic carbon removal and nitrification of a synthetic municipal wastewater. The main

116 characteristics of both tap water and MBR effluent are reported in Table 1. Before being used

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117 for irrigating the test plot, the MBR effluent went continuously into a 480 L reservoir and the

118 overflow was discharged. Inocula of E. coli were spiked into the reservoir to simulate various

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scenarios of fecal contamination. Therefore the level of E. coli in the irrigation water varied
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120 along the experiment. Table 1 also shows the characteristic of the raw wastewater used for the

121 soil column test described in section 2.2. Inocula of E. coli were prepared by dissolving E. coli
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122 pellet (ATCC® 25922TM, Liofilchem) in a Lysogeny broth (10 g of tryptone, 5 g of yeast extract
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123 and 5 g of sodium chloride in 1 L of distilled water, autoclaved at 121 °C for 15 min) and
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124 incubated at 37 °C for 24 hours.

125 The soil was made of sand (49%), silt (26%) and clay (25%) and corresponded to a sandy clay
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126 loam in the USDA classification (USDA, 1987). Water content at saturation, field capacity and

127 permanent wilting point were determined using the Saxton pedotransfer function (Saxton et al.,
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128 1986) and their values were 43%, 28% and 16%, respectively.
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129 Several field experiments were performed during an overall experimental period of

130 approximately one year. Average specific irrigation volume was 9.7 (±0.9) mm and the watering

131 events were scheduled in relation to the soil water deficit in the root zone. The latter, estimated

132 according to evapotranspiration criterion, was approximately 35% of the total available water.

133 In practice, irrigations were performed daily during the warmer season (May 1st to September

134 30th) and three times per week during the rest of the year. Climate variables were measured by a

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135 weather station installed next to the field (Associazione Consorzi di Difesa della Puglia,

136 www.agrometeopuglia.it). The average temperature in the topsoil (first 10 cm) was 25.2 (±3.6)

137 °C during the warmer season and 13.0 (±4.9) °C during the rest of the year. The total rainfall

138 during the year of testing was 581 mm.

139 As a consequence of the continuous transition of the MBR effluent through the reservoir and

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140 the natural die-off of E. coli, the concentration of E. coli in the irrigation water used for the test

141 plot varied as follows: it had a peak after each E. coli addition and then decreased by about half

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142 order of magnitude every day until the subsequent addition. The following types of fecal

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143 contamination scenarios were simulated:

144 a. Multiple irrigation events with decreasing fecal contamination. It consisted in a single

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145 spike of E. coli into the reservoir, followed by several irrigations with decreasing
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146 concentration of E. coli. The concentrations of E. coli in the topsoil and on the grass were

147 measured five hours after the first irrigation following the spike. This scenario simulated
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148 the failure of the MBR filtration system, repaired after one day.

149 b. Multiple irrigation events with constant fecal contamination. It consisted in a series of
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150 daily spikes of E. coli into the reservoir, in order to have an approximately constant
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151 concentration of E. coli in the irrigation water during a period of 7 days. The

152 concentration of E. coli in the topsoil and on the grass was measured five hours after the
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153 first and the last irrigation of the series. This scenario simulated the failure of the filtration

154 system, repaired after one week. It allowed the evaluation of the potential accumulation or
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155 re-growth of E. coli in the test plot.

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156 c. Single irrigation event and dry monitoring. It consisted in performing a single irrigation

157 after the spike of E. coli into the reservoir and measuring the concentrations of E. coli in

158 the topsoil and on the grass five hours after the watering event. Then the irrigation of the

159 test plot was stopped and the presence of E. coli in topsoil and on the grass was monitored

160 four times per week. This scenario allowed the study of the survival of E. coli after a

161 single fecal contamination event.

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162 In order to avoid accumulation effects on soil and grass, sufficient time was left between the

163 end of a contamination test and the beginning of a new one. The concentration of E. coli in the

164 topsoil and on the grass was monitored, and a new test was started only when these were below

165 1 CFU/g. No rain events occurred during any of the tests performed, but only between different

166 tests.

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167

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168 2.2 Soil column experiment

169 Twelve identical soil columns were set-up and drip-irrigated with identical water volumes and

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170 frequency. Soil samples were collected along the experiment. Each sampling was performed in

171 a different column and corresponded to its destruction (Hekman et al., 1994). This procedure

172
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allowed to consider all the soil samples as belonging to a unique column and was chosen to
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173 avoid the disturbance that sampling would have had on the transport of E. coli through the soil,

174 if a single column was used for multiple sampling. Soil columns consisted of cylindrical
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175 containers of 16 L volume (diameter: 23 cm; height: 40 cm), with undulated walls (ring-like
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176 with amplitude of 5 mm and wavelength of 25 mm) to minimize preferential water flows along

177 the column’s edge (Corwin, 2000). The columns were packed with 19.26 (±0.03) kg of soil for a
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178 depth of 36 cm and were installed vertically on a steel structure and separated by a distance of

179 50 cm between each other. To reduce the lateral heating and possibly simulate an in-situ
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180 situation, each column was wrapped in aluminum foil and the whole set was protected with a
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181 lateral curtain for shielding from wind and direct sunlight. A porous cylinder, 40 cm long and 2
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182 cm wide, was stuck in the center of one of the columns to allow the use of a soil moisture probe

183 (PR2, Delta-T). The outflow (percolated water) was collected from the bottom of each column

184 in sterile bottles.

185 Columns were filled with soil extracted close to the control plot used in field experiments, the

186 profile of which was reproduced by the separation of every 12 cm layer before filling the

187 columns. To allow for the compaction of the soil, prior to the start of the experiment soil

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188 columns were irrigated with tap water for one month, during which overall irrigation and

189 rainfall were 145 and 112 mm, respectively.

190 The experiment took place outside, during the warmer season (from May 13th to June 24th).

191 The specific irrigation volumes applied are presented in Figure 1, together with the climate

192 variables, measured by a weather station installed in a field next to the columns. The average

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193 temperatures in the topsoil (first 10 cm) and the subsoil (from 10 to 36 cm depth) were 21.7

194 (±2.7) °C and 21.0 (±2.4) °C, respectively. The average values of soil water content were 0.20

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195 (±0.04) m3/m3 and 0.28 (±0.05) m3/m3 for topsoil and subsoil, respectively. Irrigation volumes,

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196 higher than in the field experiments, were chosen in order to emphasize the potential downward

197 movement of E. coli.

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198 The pollution scenario consisted of a single fecal contamination event, which simulated a
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199 sewage spill or a supplemental irrigation with raw wastewater, and subsequent irrigations with

200 conventional water (tap water). Initially the soil columns were irrigated with 48 mm of raw
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201 wastewater, taken from a local municipal wastewater treatment plant. For one of the columns, 2

202 gNaCl/L were dissolved in the wastewater to allow the comparative study of the transport of E.
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203 coli and salt through the soil. The main characteristics of both raw wastewater and tap water are
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204 reported in Table 1. The salt addition increased the electrical conductivity of the wastewater

205 from 1020 µS/cm to 4270 µS/cm, without influencing significantly the concentration of E. coli,
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206 which are tolerant to salt at concentrations up to 50 gNaCl/L or more (Hrenovic and Ivankovic,

207 2009), and the other characteristics.

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The concentrations of E. coli in the topsoil, the subsoil and outflow were monitored along the
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209 experiment. Soil samples were collected at days 0, 1, 6 8, 14, 22 and 42. At both days 8 and 14

210 two samplings were performed, one before and one after the watering event. One column was

211 destroyed every soil sampling, meaning that after the sampling the column was not used for the

212 experiment anymore. For two of the columns, one of which corresponding to the sodium

213 chloride addition during the pollution event, samples of outflow were collected after each

214 irrigation until the specific percolating flow was lower than 0.01 mm/d. The frequency of the

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215 outflow sampling was proportional to the speed of the percolating flow (about every 10 min

216 during the first hour, every 30 min during the second hour and then another two-three samples

217 until the end of flow).

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219 2.3 Sampling and analyses

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220 Samples of grass were collected by cutting bundles of leaves, and were immediately carried to

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221 the lab. In field experiments, homogeneity of soil and grass samples with respect to irrigation

222 was optimized by collecting the samples from the center of the plots, in the zone reached by

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223 water coming from all the 4 sprinklers. For the soil column experiment, each soil layer was

224 homogenized before the collection of soil samples.

225
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Analyses of pH, electrical conductivity (EC), chemical oxygen demand (COD), suspended
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226 solids (SS), total nitrogen, ammonia, nitrite, nitrate and phosphate were performed according to

227 Standard Methods (APHA, 2005). The content of sodium chloride in the outflow of the soil
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228 column that underwent the sodium chloride addition was estimated by measuring the electrical
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229 conductivity.
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230 For the enumeration of E. coli, water samples were filtered through 0.45 µm nitrocellulose

231 membranes, then the membranes were placed on TBX (Tryptone Bile X-glucuronide) agar and
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232 incubated at 37◦C for 24 h. To extract the bacteria from soil and grass samples, 10 g of each

233 sample were dispersed in 100 mL of sterile water and centrifuged at 1000 rpm; then the
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234 supernatant was analyzed for E. coli. Comparative analyses (plate counts) of E. coli on soil
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235 samples (500 g) spiked with wastewater (50 mL) at different concentrations and on samples of

236 the same wastewater confirmed satisfactory recovery of bacteria from soil (> 70%).

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238 3. Results and discussion

239 3.1 Occurrence of E. coli in the topsoil and on the grass (field experiments)

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240 3.1.1 Background concentrations

241 During the field experiments, the existence of a source of microbiological contamination

242 (animal feces) different from the irrigation water was noticed. Its effect was more pronounced

243 on grass than on topsoil. The presence of E. coli was detected in 12% (3/26) of topsoil samples

244 and 23% (7/31) of grass samples irrigated with tap water (having concentration of E. coli<1

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245 CFU/100mL). These results are in agreement with Forslund et al. (2010; 2012) and Lang and

246 Smith (2007), which observed the presence of E. coli in samples of control soil (i.e., soil not

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247 exposed to irrigation water or amendment containing fecal contamination) and argued that other

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248 environmental fecal contamination sources, like wild animals and birds, could have caused it.

249 Généreux et al. (2015) also detected E. coli on strawberry fruits before the irrigation with

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250 contaminated water. Considering only the values beyond the detection limit (1 CFU/g), the
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251 average concentrations of E. coli in the topsoil and on the grass in the control plot were 4 (±2)

252 CFU/g and 51 (±65) CFU/g, respectively. Results of the present study confirm that it is not
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253 possible to consider the background concentrations of E. coli as zero in a cultivated field. This

254 aspect should be taken into account when planning standard limits for treated wastewater reuse
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255 in agriculture.
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256

257 3.1.2 Short-term effects of a single pollution event

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258 To evaluate the short-term effects of a single pollution event, for each scenario applied during
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259 the field experiments, the concentrations of E. coli in the topsoil (E. colits) and on the grass (E.
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260 colig) were measured five hours after the initial pollution event. Results are displayed in Error!

261 Reference source not found. versus the concentration of E. coli in the irrigation water (E.

262 coliw) and refer to a specific irrigation volume of 9.7 (±0.9) mm. One irrigation with water

263 containing up to 70 CFU/100mL of E. coli did not produce a detectable presence of E. coli in

264 the topsoil, neither on the grass (E. coli<1 CFU/g). On the contrary, a considerable occurrence

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265 of E. coli was always observed both in the topsoil and on the grass when irrigating with water

266 containing 3000 CFU/100mL of E. coli or more.

267 Figure 2 shows the concentrations of E. coli in the topsoil (first 10 cm, Figure 2A) and on the

268 grass (Figure 2B), observed five hours after each pollution event, versus the concentration of E.

269 coli in the irrigation water. E. colits was observed to increase with E. coliw according to a linear

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270 relationship between the common logarithms of the two variables:

271 log10(E. colits)=0.733·log10(E. coliw)-2.442,

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272 with R2=0.858 and p-value<0.001 for F-test for regression, which means that a power-law curve

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273 fits the observed values (E. colits=0.0036·E. coliw0.733). On the contrary, Forslund and coworkers

274 (2012) found a weak association between the concentrations of E. coli in the irrigation water

275

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and in the irrigated soil. However, in their case the collection of soil samples did not occur
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276 always on the day of irrigation and the range of concentrations of E. coli in the irrigation water

277 that they tested was lower and smaller than in the present study, thus a greater influence of
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278 external contaminations may be expected. The power coefficient (0.733) lower than 1 indicates

279 that the percentage of the E. coli transferred with the irrigation that was retained in the topsoil
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280 decreased for increasing values of E. coliw, thus suggesting that also in the subsoil the spreading
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281 of E. coli depends on the concentration in water. One observation performed during soil column

282 experiment (SC) is also reported in Figure 2A. The concentration of E. coli in the topsoil after
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283 the pollution event could have been approximately estimated using field experiments results,
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284 despite the higher specific irrigation volume applied (48 mm) in soil column experiments. This

fact, together with the existence of the power relationship in Figure 2A itself, highlights the
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286 preeminence of the concentration of E. coli in the irrigation water over the other factors (such as

287 temperature and amount of water irrigated) that are known to influence the retention of E. coli

288 in the soil after a pollution event. On the contrary, the values of E. colig observed five hours

289 after the pollution events did not show any correlation with the values of E. coliw (Figure 2B). In

290 this case, other factors, like temperature, solar radiation or other sources of contamination, had

291 probably a dominant role (Muela et al., 2000).

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292

293 3.1.3 Medium-term accumulation due to a series of pollution events

294 The potential accumulation of E. coli in the topsoil and on the grass due to a series of

295 pollution events was investigated in field experiments by irrigating with an approximately

296 constant value of E. coliw during a period of seven days (scenario b. Multiple irrigation events

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297 with constant fecal contamination). Table 2 shows the results of the three series of seven daily

298 irrigations, 9.7(±0.4) mm each, performed with approximately constant E. coliw. The

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299 concentration of E. coli in the first 10 cm of soil (E. colits) and on the grass (E. colig) was

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300 measured five hours after the first and the last irrigation of each series. As it can be seen from

301 results of run 1 reported in Table 2, medium-term effects of irrigating with water containing on

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302 average 89 (±49) CFU/100mL of E. coli were not detectable for both topsoil and grass. For the
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303 other two runs, the values of E. colits generated by each series of seven pollution events were

304 slightly higher than those observed after the first event of the series, suggesting very limited
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305 accumulation under the tested conditions. These preliminary results suggest that in the first 10

306 cm of soil other phenomena, such as the natural die-off and the movement to the subsoil,
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307 drastically contrasted the effects of both accumulation and possible re-growth of E. coli. As for
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308 the grass, where also solar radiation may have had a relevant effect, no accumulation was

309 observed.
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310
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311 3.2 Persistence of E. coli (field and soil column experiments)

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312 3.2.1 Under dry conditions

313 The persistence of E. coli in the experimental field after a fecal contamination event was firstly

314 investigated under dry conditions, i.e., with no irrigation and no rain (scenario c. Single

315 irrigation event and dry monitoring). Under these conditions, the possible movement of E. coli

316 to the subsoil is strongly limited, thus the disappearance of E. coli from topsoil occurs mainly

317 for the natural die-off. After the pollution event (specific irrigation volume of 10 mm, E. coliw

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318 of 5.0E06 CFU/100mL), the concentrations of E. coli in the topsoil and on the grass were

319 monitored until both reached values below1 CFU/g. Results are presented in Figure 3. The

320 survival rate was much lower on the grass than in the topsoil. In the first two days after the fecal

321 contamination event, the concentration of E. coli on the grass decreased by more than two

322 orders of magnitude. During the same period, the reduction observed in the topsoil was less than

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323 half order of magnitude. Assuming the absence of E. coli for values below 1 CFU/g, the

324 survival time of E. coli was between 10 and 13 days in the topsoil and between 3 and 6 days on

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325 the grass. Compared with the latter, a similar result (i.e., survival time lower than 7 days) is

326 reported for the pathogenic bacteria E. coli 0157:H7 on lettuce (Moyne et al., 2011). A very low

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327 persistence of E. coli on plants was also observed by Généreux et al (2015), which monitored

328 the presence of E. coli on strawberry irrigated with naturally contaminated water. However the

329
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survival time of specific E. coli strains on plants may be much longer, as shown by the two
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330 months survival of E. coli 0128:B12 on grass (Sjorgen, 1995).
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331 The mortality of E. coli observed in topsoil can be described by a log-linear model (log10(E.

332 colits)=3.7-0.248·d, with d=number of days from the pollution event and R2 of 0.971). Also the
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333 mortality of E. coli on the grass seemed to follow the same type of trend. However, in this case,
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334 the limited number of observations higher than 1 CFU/g was considered not to be statistically

335 relevant for suggesting a model.

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336
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337 3.2.2 In the presence of rain and irrigation

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338 The study of the persistence of E. coli in the topsoil and in the subsoil in the presence of rain

339 and irrigation was conducted within the soil column experiment. After the pollution event, the

340 concentrations of E. coli in the topsoil, the subsoil and in the outflows collected at the bottom of

341 the soil columns were monitored until all reached values below the detection limit (1 CFU/g for

342 soil samples, 1 CFU/100 mL for water samples). Between day zero and day 14, the mortality of

343 E. coli in the topsoil (Figure 4) followed a log-linear model (log10(E. colits)=4.7-0.249·d, with R2

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344 of 0.991), the slope of which is very close to the one found for the mortality in the topsoil under

345 dry conditions (Figure 3). This indicates that, compared with the effect of the natural die-off, the

346 overall effect of the watering events of day 3 and day 8 on the persistence of E. coli in the

347 topsoil was very limited.

348 After 14 days from the pollution event, the concentrations of E. coli in the topsoil and in the

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349 subsoil reached values of 1 CFU/g or lower. Nevertheless, a detectable presence of E. coli in the

350 outflows was observed for longer. This means that values of E. coli in the soil lower than 1

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351 CFU/g have still to be considered with regards to the potential leaching to groundwater.

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352 The mortality of E. coli in the topsoil after day 14 may have followed a different pattern from

353 the one observed until day 14. Indeed, the values of E. colits are compatible with a two-stage

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354 log-linear decay and with a Weibull survival model (a parametric survival model where the
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355 survival time is assumed to follow a Weibull distribution) as well. Both of these models were

356 successfully used in previous studies to describe the mortality of specific strains of E. coli (E.
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357 coli NAR and E. coli O157:H7) and of Fecal coliforms (Zhai et al., 1995; Franz et al., 2008;

358 Semenov et al. 2011; Farhangi et al., 2013). The possibility that the decay rate in the topsoil
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359 decreased in the second part of the experiment is supported by the die-off of E. coli observed in
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360 a sample of the wastewater used for the pollution event that was stored in the lab at 20.2 (± 1.5)

361 °C (Figure 4, “bottle”). In this case, a two-stage log-linear decay fitted the observed values of E.
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362 coli concentrations in the wastewater (E. colib): log10(E. colib)=7.2-0.258·d for d≤7 (R2 of 0.997)

363 and log10(E. colib)=5.4-0.049·d for d≥7 (R2 of 0.945). Blaustein and coworkers (2013) recently
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364 reported that this kind of pattern, i.e. a fast mortality followed by a slow mortality, is the most
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365 prevalent among 170 datasets of E. coli survival in water found in the literature.

366

367 3.3 Transport of E. coli through the soil (soil column experiment)

368 Comparing the concentrations of E. coli in the topsoil and in the subsoil, there was no

369 evidence of a continuous movement downward of E. coli from the topsoil to the underlying

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370 layers during the experiment. A small movement downward seemed to occur only in

371 correspondence to the irrigations, as suggested by the increase of the concentration of E. coli in

372 the subsoil (E. coliss) observed after the irrigation performed at day 8 (Figure 4).

373 In correspondence to all the irrigations performed (including the pollution event), a relevant

374 presence of E. coli was found in the percolated water. The leaching of water from the soil

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375 columns started about 20 minutes after the beginning of each irrigation and ended (i.e., specific

376 percolating flow lower than 0.01 mm/d) after about 24 hours. However, the leaching of E. coli

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377 practically ended after five hours from the beginning of each irrigation, when more than 99.8 %

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378 of the amount of E. coli collected after 24 hours had already leached out. After the pollution

379 event (48 mm, 2.E+07 CFU/100mL), the load of E. coli that would have reached a groundwater

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380 table situated at a depth of 36 cm was 2.E+07 CFU/m2 (29 mm, 8.E+04 CFU/100mL), which
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381 corresponded to less than 1% of the amount of E. coli supplied with the irrigation. Due to the

382 natural die-off of E. coli in the soil, the leaching of E. coli decreased at every watering event
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383 and, compared with the leaching generated by the pollution event, it was already one order of

384 magnitude smaller in correspondence to the irrigation performed at day 8.

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385 The concentration of E. coli in the leaching water that reaches the groundwater table
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386 (supposed to be at a depth of 36 cm) depends on several parameters, such as the die-off rate, the

387 leaching coefficient, soil porosity and the natural movement of E. coli through the soil. Even if
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388 these parameters can vary over time and were not individually determined in this study, it was

389 observed that the overall trend of the E. coli concentrations in the leaching water (Figure 4) may
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390 be described by a log-linear model. Indeed, the concentration of E. coli in both outflows (E.
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391 coliout1 and E. coliout2) followed log-linear models (log10(E. coliout1)=4.9-0.130·d, with R2 of

392 0.996; log10(E. coliout2)=4.6-0.136·d, with R2 of 0.995). The same pattern was observed by

393 Gagliardi and Karns (2000) for the E. coli O157:07 in the leaching of various types of soils

394 amended with animal manures.

395 During the five hours following the pollution event and the other irrigations performed during

396 the soil column experiment, samples of the outflow of the soil column that underwent the

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397 addition of sodium chloride were collected, and the cumulative amounts of water, salt and E.

398 coli in the outflow were determined (see Table 3). For each parameter, the cumulative

399 percentage collected in the outflow over time was determined and displayed in Figure 5. After

400 about one hour from the beginning of each irrigation, when approximately half of the total

401 amount of water that will be collected after five hours had been drained, the flow dropped

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402 significantly. Simultaneously, the percolation of both salt and E. coli decreased too. This result

403 indicates that under transient flow the transport of both salt and E. coli depended on the flow

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404 passing through the column and confirms the results reported by Shang and coworkers (2009),

405 who showed that the drainage induces the mobility of irregularly shaped particles with diameter

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406 < 2µm, as E. coli.

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407 Comparing the behavior of salt and E. coli, the latter showed a stronger affinity with the soil.
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408 Indeed, as it can be seen from Figure 5, for each irrigation the curve representing the cumulative

409 percentage of E. coli in the outflow overlied the curve representing the cumulative percentage of
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410 salt. This information may be useful for modeling the transport of E. coli through a sandy clay

411 loam soil by integrating data of tracer tests, performed with salt, with measurements of E. coli.
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412
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413 4. Conclusions
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414 Field and soil column experiments were carried out in Apulia (South East Italy) in order to

415 investigate the fate of the fecal contamination indicator E. coli present in relevant concentrations
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416 in water used for irrigation. The following conclusions can be drawn for a sandy clay loam soil
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417 under Mediterranean climate:

418 • The main factors affecting the contamination of soil irrigated with partially treated

419 wastewater are: (i) the concentration of E. coli in the irrigation water and (ii) the natural dye-

420 off of E. coli. The first mainly influences the extent of contamination in the short-term

421 period. The second is the dominant process affecting the persistence of E. coli in the topsoil

422 and the level of contamination of the water leaching through the soil column. A power

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423 relationship was found between the concentration of E. coli in the irrigation water and in the

424 topsoil (first 10 cm), as measured five hours after a single contamination event. Persistence

425 of E. coli in the topsoil after a pollution event followed a log-linear model (dye-off rate of

426 0.25 d-1) both under dry conditions and in the presence of rain and irrigations.

427 • In terms of potential contamination of subsurface water resources, less than 1% of the

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428 amount of E. coli present in the irrigation water passed through a 36 cm deep soil column

429 after the pollution event (48 mm; 2.E+07 CFU/100mL of E. coli).

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430 • When several watering events followed a pollution event, the overall trend of the E. coli

431 concentrations in the leaching water (at 36 cm depth) was described by a log-linear model.

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432 • Under the tested conditions, total disappearance of the E. coli in the leaching water was

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433 observed after 35-40 days. AN
434 The behavior of E. coli in topsoil and subsoil can be useful for evaluating the environmental

435 consequences of discharging effluents with microbiological characteristics beyond compliance

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436 standards. Further research is required to investigate the dynamics of other microbiological

437 pollution indicators in order to gain a more detailed picture and assure safer and more efficient
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438 reuse of marginal water resources.

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439

Acknowledgements
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440

441 The results reported were obtained as partial fulfillment of the national project PRIN
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442 “Innovative treatments for agricultural wastewater reuse” funded by the Italian Ministry of
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443 Education, University and Research and the Euro-India collaborative project “Water4Crops”

444 (UE Contract n. FP7 KBBE 311933).

445

446 REFERENCES

447 APHA, AWWA, WEF, 2005. Standard methods for the examination of water and wastewater,

448 21st edition. Washington: American Public Health Association.

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449 Bixio, D., Thoeye, C., De Koning, J., Joksimovic, D., Savic, D., Wintgens, T., Melin, T.,

450 2006. Wastewater reuse in Europe. Desalination 187 (1-3), 89-101.

451 Blaustein, R.A., Pachepsky, Y., Hill, R.L., Shelton, D.R., Whelan, G., 2013. Escherichia coli

452 survival in waters: temperature dependence. Water Research 47 (2), 569-78.

453 Corwin, D.L., 2000. Evaluation of a simple lysimeter-design modification to minimize

PT
454 sidewall flow. Journal of Contaminant Hydrology 42 (1), 35-49.

RI
455 Forslund, A., Ensink, J.H.J., Battilani, A., Kljujev, I., Gola, S., Raicevic, V., Jovanovic, Z.,

456 Stikic, R., Sandei, L., Fletcherg, T., Dalsgaard, A., 2010. Faecal contamination and hygiene

SC
457 aspect associated with the use of treated wastewater and canal water for irrigation of potatoes

458 (Solanum tuberosum). Agricultural Water Management 98 (3), 440-450.

459
U
Forslund, A., Ensink, J.H.J., Markussen, B., Battilani, A., Psarras, G., Gola, S., Sandei, L.,
AN
460 Fletcher, T., Dalsgaard, A., 2012. Escherichia coli contamination and health aspects of soil and

461 tomatoes (Solanum lycopersicum L.) subsurface drip irrigated with on-site treated domestic
M

462 wastewater. Water Research 46 (18), 5917-5934.

D

463 Farhangi, M.B., Safari Sinegani, A.A., Mosaddeghi, M.R., Unc, A., Khodakaramian, G.,
TE

464 2013. Impact of calcium carbonate and temperature on survival of Escherichia coli in soil.

465 Journal of Environmental Management 119,13-19.

EP

466 Franz, E., Semenov, A.V., Termorshuizen, A.J., de Vos, O.J., Bokhorst, J.G., van Bruggen,

467 A.H., 2008. Manure-amended soil characteristics affecting the survival of E. coli O157:H7 in 36
C

468 Dutch soils. Environmental Microbiology 10 (2), 313-27.

AC

469 Gagliardi, J.V., Karns, J.S., 2000. Leaching of Escherichia coli O157:H7 in diverse soils

470 under various agricultural management practices. Applied and Environmental Microbiology

471 Journal 66 (3), 877-883.

18
 ACCEPTED MANUSCRIPT
472 Généreux, M., Grenier, M., Côté, C., 2015. Persistence of Escherichia coli following

473 irrigation of strawberry grown under four production systems: Field experiment. Food Control

474 47, 103-107.

475 GWP (2012). Water Demand Management: The Mediterranean experience. Global Water

476 Partnership, Technical focus paper, pp. 76, www.gwp.org (accessed 29.04.15)

PT
477 Hekman, W.E., Heijnen, C.E., Trevors, J.T., van Elsas, J.D., 1994. Water flow induced

478 transport of Pseudomonas fluorescens cells through soil columns as affected by inoculant

RI
479 treatment. FEMS Microbiology Ecology 13 (4), 313-326.

SC
480 Hunter, C., McDonald, A.T., Beven, K.J., 1992. Input of faecal coliform bacteria to an upland

481 stream channel in the Yorkshire Dales. Water Resources Research 2 (8), 1869-1876.

482
U
Jamieson, R.C., Gordon, R.J., Tattrie, S.C., Stratton, G.W., 2003. Sources and persistence of
AN
483 fecal coliform bacteria in a rural watershed. Water Quality Research Journal of Canada 38 (1),

484 33–47.
M

485 Hrenovic J., Ivankovic T., 2009. Survival of Escherichia coli and Acinetobacter junii at
D

486 various concentrations of sodium chloride. Eurasian Journal of Biosciences 3, 144-151.

TE

487 Kellis, M., Kalavrouziotis, I.K., Gikas, P., 2013. Review of wastewater reuse in the

488 Mediterranean countries, focusing on regulations and policies for municipal and industrial
EP

489 applications. Global NEST Journal 15 (3), 333-350.

Lang, N.L., Smith, S.R., 2007. Influence of soil type, moisture content and Biosolids
C

490

491 application on the fate of Escherichia coli in agricultural soil under controlled laboratory
AC

492 conditions. Journal of Applied Microbiology 103 (6), 2122-2131.

493 Lopez, A., Pollice, A., Lonigro, A., Masi, S., Palese, A.M., Cirelli, G.L., Toscano, A.,

494 Passino, R., 2006. Agricultural wastewater reuse in southern Italy. Desalination 187 (1-3), 323-

495 334.

19
 ACCEPTED MANUSCRIPT
496 Lopez, A., Pollice, A., Laera, G., Lonigro, A., Rubino, P., 2010., Membrane filtration of

497 municipal wastewater effluents for implementing agricultural reuse in Southern Italy. Water

498 Science & Technology 62 (5), 1121-1128.

499 McDonald, A.T., Kay, D., Jenkins, A., 1982. Generation of faecal and total coliform surges by

500 stream flow manipulation in the absence of normal hydrometeorological stimuli. Applied and

PT
501 Environmental Microbiology 44 (2), 292-300.

502 Moyne, A.L., Sudarshana, M.R., Blessington, T., Koike, S.T., Cahn, M.D., Harris, L.J., 2011.

RI
503 Fate of Escherichia coli O157:H7 in field-inoculated lettuce. Food Microbiology 28 (8), 1417-

SC
504 1425.

505 Muela, A., García-Bringas, J.M., Arana, I., Barcina, I., 2000. The Effect of Simulated Solar

506

U
Radiation on Escherichia coli: The Relative Roles of UV-B, UV-A, and Photosynthetically
AN
507 Active Radiation. Microbial Ecology 39 (1), 65-71.

508 Naclerio, G., Nerone, V., Bucci, A., Allocca, V., Celico, F., 2009. Role of organic matter and
M

509 clay fraction on migration of Escherichia coli cells through pyroclastic soils, southern Italy.
D

510 Colloids Surfaces B: Biointerfaces 72 (1), 57-61.

TE

511 Oliveira, M., Viñas, I., Usall, I., Anguera, M., Abadias, M., 2012. Presence and survival of

512 Escherichia coli O157:H7 on lettuce leaves and in soil treated with contaminated compost and
EP

513 irrigation water. International Journal of Food Microbiology 156 (2), 133-140.

514 Odonkor, S.T., Ampofo, J.K., 2013. Escherichia coli as an indicator of bacteriological quality
C

515 of water: an overview. Microbiology Research 4(1), 5-11.

AC

516 Paton, J.C., and Paton, A.W., 1998. Pathogenesis and diagnosis of Shiga toxin–producing

517 Escherichia coli infections. Clinical Microbiology Reviews 11 (3), 450-479.

518 Pollice, A., Lopez, A., Laera, G., Rubino, P., Lonigro, A., 2004. Tertiary filtered municipal

519 wastewater as alternative water source in agriculture: a field investigation in southern Italy.

520 Science of the Total Environment 324 (1-3), 201-210.

20
 ACCEPTED MANUSCRIPT
521 Saxton, K.E., Rawls, W.J., Romberger, J.S., Papendick., R.I., 1986. Estimating generalized

522 soil water characteristics from texture. Soil Science Society of America Journal, 50 (4), 1031-

523 1036.

524 Semenov, A.V., Franz, E., van Overbeek, L., Termorshuizen, A.J., van Bruggen, A.H.C.,

525 2008. Estimating the stability of Escherichia coli O157:H7 survival in manure amended soils

PT
526 with different management histories. Environmental Microbiology 10 (6), 1450–1459.

527 Semenov, A.V., van Overbeek, L., Termorshuizen, A.J., van Bruggen, A.H., 2011. Influence

RI
528 of aerobic and anaerobic conditions on survival of Escherichia coli O157:H7 and Salmonella

SC
529 enterica serovar Typhimurium in Luria-Bertani broth, farm-yard manure and slurry. Journal of

530 Environmental Management 92 (3), 780-787.

531

U
Shang, J., Flury, M., Deng, Y., 2009. Force measurements between particles and the air-water
AN
532 interface: Implications for particle mobilization in unsaturated porous media. Water Resources

533 Research 45 (6), W06420.

M

534 Sjogren, R.E., 1995. Thirteen-year survival study of an environmental Escherichia coli in
D

535 field mini-plots. Water, Air, and Soil Pollution 81(3-4), 315-335.State of California, 1978.
TE

536 Wastewater reclamation criteria. Berkeley, CA, Dept. of Health Services. California

537 administrative code, Title 22, Division 4, Environmental Health.

EP

538 U.S. DA (Dept. of Agriculture), 1987. Soil mechanics - Level I. Module 3 - USDA Textural

539 soil classification. Study guide. United States Department of Agriculture, Soil Conservation
C

540 Service.
AC

541 U.S. EPA (Environmental Protection Agency), 2008. National Coastal Condition Report III.

542 EPA/842-R-08-002. U.S. Environmental Protection Agency, Office of Research and

543 Development and Office of Water, Washington, DC.

544 World Health Organization, 1989. Health guidelines for the use of wastewater in agriculture

545 and aquaculture. Report of a WHO Scientific Group. Geneva, WHO Technical Report Series n.

546 778.

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547 Wilkinson, R.J., McKergow, L.A. , Davies-Colley, R.J. , Ballantine, D.J. and Young, R.G.,

548 2011. Modelling storm-event E. coli pulses from the Motueka and Sherry Rivers in the South

549 Island, New Zealand. New Zealand Journal of Marine and Freshwater Research, 45 (3), 369-

550 393.

551 Wood, J.D., Bezanson, G.S., Gordon, R.J., Jamieson, R., 2010. Population dynamics of

PT
552 Escherichia coli inoculated by irrigation into the phyllosphere of spinach grown under

553 commercial production conditions. International Journal of Food Microbiology 143, 198-204.

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554 Zhai, Q., Coyne, M.S., Barnhisel, R.I., 1995. Mortality rates of fecal bacteria in subsoil

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555 amended with poultry manure. Bioresource Technology 54 (2), 165-169.

556

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557 FIGURES CAPTIONS

558 Figure 1. Soil column experiment: experimental conditions. Specific irrigation volumes

559 applied and weather conditions observed.

560 Figure 2. Field experiments: short-term effects of a single pollution event (specific irrigation

561 volume of 9.7 (±0.9) mm).

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562 Figure 3. Field experiments: survival of E. coli in the topsoil (first 10 cm) and on the grass

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563 (scenario c. Single irrigation event and dry monitoring). Average temperature in the topsoil:

564 23.3 (±0.9) °C.

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565 Figure 4. Soil column experiment: concentrations of E. coli in the topsoil (first 10 cm), the

566 subsoil (from 10 cm to 36 cm depth), and in the outflows of two soil columns. At day 0 columns

567
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568 dotted lines represent rain and irrigation events. The benchmark is a wastewater sample stored

569 in a bottle in the lab (“bottle”).

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570 Figure 5. Soil column experiment: cumulative percentages of water, salt and E. coli over time
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571 in the outflow of the soil column that underwent the addition of sodium chloride.
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Table 1. Main average (± standard deviation) characteristics of the irrigation waters used in the

experiments. Inocula of E. coli were added to the MBR effluent before using it for irrigation.

Parameter MBR effluent (for field Raw wastewater (for soil Tap water (for both
experiments)a column experiment) experiments)a
Suspended solids (mg/L) <5 264 <5
Chemical Oxygen 10.3 (±7.9) 760 <5
Demand (mgO2/L)

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Total Nitrogen (mgN/L) 27.5 (±4.7) 60.3 1.2 (±0.1)
Ammonia (mgN/L) <1 42.0 <1

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Nitrite (mgN/L) <1 <1 <1
Nitrate (mgN/L) 20.3 (±5.7) <1 <1

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Phosphate (mgP/L) <2 16.8 <1
pH (-) 7.3 (±0.3) 6.1 7.9 (±0.1)
Electrical Conductivity 599 (±40) 1020 499 (±121)

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E. Coli (CFU/100mL) <1 1.0E+07 <1
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a
41 samples of MBR effluent, 6 samples of tap water
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Table 2. Field experiments: scenario b. Multiple irrigation events with constant fecal contamination.

Run average E. coliw

E. colits (CFU/10g) after: E. colig (CFU/10g) after:
(CFU/100mL)
1 event 7 events 1 event 7 events
Run 1 8.9E01 (±4.9E01) <1 <1 <1 <1
Run 2 9.9E03 (±4.0E03) 4.7E01 6.0E01 9.3E01 9.3E01
Run 3 1.0E05 (±8.1E04) 8.7E01 1.6E02 1.1E02 8.6E01

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Table 3. Water, salt and E.coli in the inflows and the outflows of the soil column that underwent the

addition of sodium chloride. For each parameter, outflow values represent the cumulative amounts collected

after about five hours from the beginning of the watering event.

Experimental Event Water (mL) Salt (mg) E. coli (CFU)

time (d)
inflow outflow inflow outflow inflow outflow

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0 P* 2000 1264 4000 100 2.0E+08 3.6E+04
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8 I1 2000 1101 0 598 0 3.6E+03

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14 I2* 2000 1123 0 773 0 3.1E+02
27 I3* 2000 686 0 537 0 8.4E+00
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P = Pollution event; I1-I3 = Irrigation with tap water

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HIGHLIGHTS

1. Fate of E. coli after irrigation with contaminated water was assessed with field and soil column tests.

2. E. coli concentration in irrigation water mainly influences short-term soil contamination.

3. Dye-off is the dominant process affecting E. coli concentration in topsoil and leaching water.

4. Persistence models of E. coli in topsoil and leachate after pollution are log-linear.

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5. Less than 1% of E. coli in irrigation water is transferred through the soil column.

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