Agriculture, Ecosystems and Environment 181 (2013) 50–57

Contents lists available at ScienceDirect

Agriculture, Ecosystems and Environment

journal homepage: www.elsevier.com/locate/agee

Microbial impact of different types of municipal wastewaters used

to irrigate nectarines in Southern Italy
Gaetano Alessandro Vivaldi ∗ , Salvatore Camposeo, Pietro Rubino, Antonio Lonigro
Dipartimento di Scienze Agro-Ambientali e Territoriali, Università degli Studi di Bari Aldo Moro, Via Amendola 165/A, 70126 Bari, Italy

a r t i c l e i n f o a b s t r a c t

Article history: This research was performed in order to assess the microbiological impact of three different sources
Received 21 March 2013 of treated municipal wastewater on the tree-soil system of a nectarine orchard. Wastewaters coming
Received in revised form 27 August 2013 from two different tertiary treatment processes [conventional tertiary water (TW) and lagoon simplified
Accepted 3 September 2013
tertiary water (LW)] and a secondary treatment process (SW) were compared with fresh water (FW). A
Available online 17 October 2013
two-year field experiment (2011 and 2012) was carried out monitoring Total Coliforms, Fecal Coliforms,
E. coli and Salmonella spp. concentrations on source waters, root zone soil and fruits. The microbial con-
Keywords:
centration mainly depended on the treatment system of the source water but in most cases it was always
Prunus persica (L.) Batsch
Fecal indicators
over the current Italian threshold (10 CFU 100 mL−1 ; D. No. 152/2006). In general, the SW showed the
Fruit contamination highest level of contamination for both years followed by LW, TW and FW. Although soil plots irrigated
Water recycling with the SW were heavily contaminated as expected, nevertheless the soil was able to greatly reduce of
Tertiary treatment 2.6 log10 CFU 100 g−1 the fecal contamination. The soils irrigated with LW, TW and FW showed a lower
fecal bacteria contamination. Total Coliforms were confirmed as ubiquitous in this agricultural environ-
ment and contamination by E. coli on fruits surfaces was not negligible when plots were irrigated with
SW for both years. This study supports the hypothesis that very low quality of irrigation waters cannot
be used safely in a nectarine orchard because it represents a risk for human infection.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction waters (Toze, 1997,1999). To break down or otherwise reduce the

By 2050, food production is projected to increase by about 70%
globally and nearly 100% in developing countries (FAO, 2011). This
increased demand for food will place unprecedented pressure on
many agricultural production systems across the world, increas-
ing the water demand for agriculture. As such wastewater reuse
will need to provide more water for irrigated areas and to make
available water in dry areas (Angelakis et al., 1999).
A large number of studies (Bastos and Mara, 1995; El Hamouri
et al., 1996; Lopez et al., 2006, 2010; Ndiaye et al., 2011; Palese et al.,
2009; Petterson et al., 2011; Pollice et al., 2004) have shown that
microbiological contamination remains a crucial issue to insure the
safe use of municipal wastewater in agriculture. The risk is rep-
resented by a many kinds of microorganisms, including viruses,
bacteria and pathogenic protozoan/helminthes; the bacteria are
the most common of the microbial pathogens found in recycled
Abbreviations: FW, fresh water; TW, conventional tertiary water; LW, lagoon
simplified tertiary water; SW, secondary water.
∗ Corresponding author. Tel.: +39 080 5442982; fax: +39 080 5442982.
E-mail addresses: gaetano.vivaldi@uniba.it (G.A. Vivaldi),
salvatore.camposeo@uniba.it (S. Camposeo), pietro.rubino@agr.uniba.it (P. Rubino),
antonio.lonigro@uniba.it (A. Lonigro).
contamination of the municipal wastewater and then minimize
the risk of crops contamination, high-technology tertiary treat-
ments and disinfection systems, such as activated carbon, reverse
osmosis, membrane filtration, chlorination, ozonation, UV irradia-
tion (Asano and Levine, 1998) and tertiary lagoons (or maturation
ponds) are essential to insure microbial populations remain below
critical levels. The latter process has generally been viewed as an
effective, low-cost method of removing pathogens from wastewa-
ter (Maynard et al., 1999), while the other treatment systems are
often prohibitively expensive, particularly in developing Countries,
where only about 10% of wastewater undergoes treatment of any
kind (Homsi, 2000). The low operation and maintenance costs of
tertiary lagoons, coupled with the effective percentage pathogen
removed reported in literature (Pearson et al., 1987a,b; Mara and
Pearson, 1987; Mara et al., 1992a,b), have made them a popular
choice for wastewater treatment in such economically stressed
areas (Maynard et al., 1999).
Different legislative approaches are used in the world to deter-
mine the level of contamination of the wastewater that can be used
in agriculture. The WHO guidelines (Blumenthal et al., 2000), using
empirical epidemiological evidence, classified the reuse in three
category: Category A – “unrestricted irrigation” recommended
the level of Fecal Coliforms less than 1.000 CFU 100 mL−1 , usable
for vegetable and salad crops eaten uncooked, sport fields and

0167-8809/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.agee.2013.09.006
 G.A. Vivaldi et al. / Agriculture, Ecosystems and Environment 181 (2013) 50–57
public parks; Category B – “restricted irrigation” recommended the
level of Fecal Coliforms less than 100.000 CFU 100 mL−1 usable for
cereal crops, industrial crops, fodder crops, pasture and trees (in the
case of fruit trees, irrigation should stop two weeks before fruit is
picked, and no fruit should be picked off the ground. Spray/sprinkler
irrigation should not be used); Category C – localized irrigation of
crops in category B if exposure of workers and the public does
not occur (WHO, 2006). Although the WHO guidelines are quite
flexible, many Countries have preferred to use internal regulations
more restricted. The state of California allows for food crop irri-
gation, recycled water with 2.2 CFU 100 mL−1 (7-day med), 23 CFU
100 mL−1 (not more than one sample exceeds this value in 30 days)
and 240 100 mL−1 (max) of Total Coliforms (USEPA, 2012). In
Italy the guidelines for crop irrigation allow the use of munici-
pal wastewater with a contamination less than 10 CFU 100 mL−1
of E. coli (Decree of Ministry for Environment, No. 152/2006). These
thresholds could be considered very restrictive because, the risk
of contracting an infectious disease after the consumption of spe-
cific vegetables is low when the contamination of the irrigation
source not exceeded 103 CFU 100 mL−1 . On the other hand, millions
of hectares of irrigated lands across the world use raw, treated,
and/or partially diluted wastewater (Palese et al., 2009). In fact,
many farmers regularly use untreated wastewater unlawfully to
satisfy crop water needs under extreme water shortage conditions
(Ait Melloul et al., 2001; Capra and Scicolone, 2004; Campos et al.,
2002), thereby subjecting the consumer to a high risk of disease
outbreak. For this reason further studies are need to better clar-
ify the acceptable level of contamination in treated wastewaters,
specific for each crop, in such a way as to encourage low cost
treatment systems and at the same time to guarantee the public
health.
The main purpose of this study was to monitor the micro-
bial impact of drip irrigation with four different quality municipal
wastewaters on the soil-tree system of a nectarine orchard in
Southern Italy. Total Coliforms, Fecal Coliforms and E. coli were
selected as indicators of fecal contamination, whereas Salmonella
spp. was selected as pathogenic bacteria.
2. Materials and methods
2.1. Site description and climate patterns
A two year study (2011–2012) was carried out in a com-
mercial nectarine grove (Prunus persica L. Batsch.) cv Big Top
grafted on GF 677. The grove, located at Trinitapoli (Apulia Region,
Southern Italy), was planted in 2008. The planting density was
400 trees ha−1 with 5.0 m spacing between rows and 5.0 m between
trees; the trees were vase-shaped trained. The soil was sandy
loam classified as Vertisol-Gleysols (FAO); the main physical and
chemical characteristics of the soil are reported in Table 1. The
site is characterized by a typical Mediterranean climate, with a
long-term average annual rainfall of 560 mm (mean 1976–2006),
two thirds concentrated from fall to winter. The total global
radiation, monthly rainfall, minimum and maximum tempera-
ture and ET0 were monitored during the experimental period of
both years (Table 2). The agro-climatic data were supplied by
ASSOCODIPUGLIA (www.agrometeopuglia.it) and recorded at the
nearest station, few kilometers far from the experimental site. The
highest level of total global radiation (Rs MJ m−2 ) was during July
for both years of experiment. During the 2011 irrigation season, the
maximum temperature monthly means were lower than 2012 and
the rainfall monthly were higher by over 27 mm than 2012 values,
concentrated in the last year especially in the month of September
(Table 2). Consequently the monthly ET0 values were 719 mm in
2011 and 878 mm in 2012.
51
2.2. Water sources, irrigation treatments and other cultural
practices
Four water sources were used in the experiment. The first water
source was a secondary-treated municipal wastewater (SW) orig-
inated from a public plant. After screening and grit removal, the
wastewater flows to primary clarifiers followed by activated sludge
process and partial aerobic stabilization of the sludge, finally the
chemical precipitation of phosphorus, denitrification and chlorina-
tion. The second water source was a tertiary-treated wastewater
made by a simplified lagoon treatment pilot plant (LW) included
a big tank reversed pyramid-shaped, measuring 25 × 7 m at the
bottom and 33 × 12 m at the top, 1.8 m deep, covered with a
black polyethylene film. The last treatment was a tertiary-treated
wastewater (TW) made by membrane filtration public plant located
near the experimental site, where the water was primarily collected
in a tank of 180 m3 and pumped to the sand filter section, including
five tanks with the following filling materials: anthracite 1150 kg,
quartz sand 4500 kg, and gravel support with different diameters
2040 kg (Fig. 1). The second phase of treatment was represented
by ultra-filtration module equipped with hollow fiber membranes
(nominal porosity 0.2 ␮m) with cellulose triacetate double wall
(0.8 mm diameter) at an internal pressure of 0.8–1.0 bar. Period-
ically all the lines were automatically cleaned by back flushing. All
these treatment were compared with fresh water source (FW) from
the Marana Capacciotti dam which served as the control. Finally,
the reclaimed wastewater has been reused under controlled flow
rate and distribution conditions specifically aimed at avoiding the
contamination of bordering fields and underlying groundwater.
Drip irrigation was adopted with the drip lines suspended along
rows on the tree at 1.70 m from the soil. Each tree was provided
with two self-compensating drippers that delivered water at 32 L
h−1 . The distance of the drippers from the trunk was 1.0 m. Because
wastewater quality strongly influences dripper efficiency (Capra
and Scicolone, 2004; Taylor et al., 1995) a 5 cm filter grit, equipped
with a flushing system, was used and each flow meter was equipped
with 2.5 cm cartridge filters frequently cleaned. At the beginning
of each irrigation season, the irrigation system was cleaned and
the emitters were checked periodically throughout the irrigation
season. The irrigation interval was performed following the local
farmers common practices: twice a week from the end of May
to the end of July and once a week from August to the middle of
September. The irrigation volume has been calculated by the water
balance method, with restitution of 100% crop evapotranspiration
(ETc ) lost in each irrigation interval. ETc was calculated using Eq.
(1) recommended by FAO (Allen et al., 1998):
ETc = Kr Kc ET0 (1)
were Kr is reduction coefficient, Kc is crop coefficient, ET0 is ref-
erence evapotranspiration. Kc = 0.80Kcini , 1.15Kcmid , 0.852Kcend ;
Kr = 0.75; ET0 was calculated by Penman–Monteith equation and
directly provided by ASSOCODIPUGLIA (www.agrometeopuglia.it).
All the other agricultural practices (fertilization, weed and pest
control, etc.) were those followed by the local farmers. In particular,
pest and disease control was performed according to the regional
service recommendations for commercial nectarine orchard. Fertil-
izers were applied taking into account, peach requirements, trees
potential yield and soil chemical analyses.
2.3. Water, soil and fruit sampling
Water samples were taken nine times for each experimental
year and collected randomly in a four replicates using a 1 L sterile
glass bottles and stored at +4 ◦ C before microbiological analysis.
Soil samples were collected in quadruplicate at the end each
irrigation season of at 10 cm depth. This depth was chosen because
52 G.A. Vivaldi et al. / Agriculture, Ecosystems and Environment 181 (2013) 50–57

Table 1
Main physical and chemical soil characteristics. Values represents means and standard errors (each value represent a mean of 4 replicates).

Depth Texture USDA pH OM (%) N Kjeldahl (g kg−1 ) Total P (mg kg−1 ) K exchangeable (mg kg−1 )

0–60 cm Sandy loam 8.0 ± 0.01 1.76 ± 0.20 1.09 ± 0.24 7.60 ± 6.25 945 ± 25.55

Table 2
Total global radiation, minimum and maximum temperature, rainfall and ET0 of 2011 and 2012. The numbers represent a monthly value.

May 2011 June 2011 July 2011 August 2011 September 2011 May 2012 June 2012 July 2012 August 2012 September 2012

Rs (MJ m−2 ) 22.9 23.1 24.9 24.9 16.7 20.6 25.6 24.6 23.2 15.8
TMax (◦ C) 25.0 28.9 31.7 31.5 30.5 26.9 34.8 37.1 36.8 28.7
TMin (◦ C) 11.6 15.0 17.5 18.8 16.6 11.1 17.0 20.6 19.6 17.2
Rainfall (mm) 44 31 39 0 14 20 0 9 1 71
ET0 (mm) 131 165 193 117 113 158 223 231 191 75

the wastewater distribution along the irrigation season slightly
affected soil hygienic features especially in the first 10 cm (Palese
et al., 2009) and 10 days after the end of irrigation season. Soil
samples were placed in sterile plastic bags, brought to the labora-
tory and stored at +4.0 ◦ C. Microbiological analysis was done within
24 h from sample collection. The analyses were performed on sub-
samples collected at every plot after each crop cycle, under the
drippers.
The fruits were harvested by hand from the crown portion near-
est the drippers (the worst case condition in terms of potential
contamination), stored at +4.0 ◦ C and transported to the labora-
tory in sterile plastic bags. Using a sterile knife 200 g of nectarine
peel was aseptically weigh into a sterile jar and added 500 mL of
sterile water. The jars were shaken by machine for 15 min and the
solutions were examined for analysis. To minimize possible risks
to farm workers and researchers, common protective clothing such
as disposable gloves and boots were adopted. Any potential risk to
the public was avoided by destroying the fruits after sampling as
recommended by the law.
2.4. Microbiological analyses
Microbiological analyses, including Total Coliforms, Fecal Col-
iforms, E. coli and Salmonella, were carried out on water, soil
and fruit samples according to standard methods (Woomer,
1994; Scharf, 1966). The densities of colonies were expressed
as log10 CFU 100 mL−1 for waters, log10 CFU 100 g−1 for soils and
CFU g−1 for fruits. The determination for Salmonella spp. was per-
formed as reported by APAT, IRSA-CNR (2003).
2.5. Experimental design and data analysis
The experimental site included 16 plots (4 treatments × 4 repli-
cates), organized in a randomized block design. Each plot was 25 m
long and included 5 trees; each block was separated from others
by a not treated row. The 3 middle trees were used for measure-
ments (sampling area). Data were analyzed by analysis of variance
(ANOVA) followed by Tukey’s post hoc test for significant values.
Standard error (SE) was also calculated. Values of p < 0.05 were con-
sidered statistically significant. All the analyses were performed
using the R 2.15.0 software (R Foundation for Statistical Computing
Vienna, Austria).
3. Results
3.1. Microbiological quality of the water treatments
Irrigation water contamination varied considerably among
the different water sources used. For both years SW showed
the highest levels of Total Coliforms (Fig. 2), Fecal Col-
iforms (Fig. 3) and E. coli (Fig. 4), varying from a minimum of
5.4 log10 CFU 100 mL−1 to a maximum of 6.3 log10 CFU 100 mL−1 ,
from 4.7 to 5.6 log10 CFU 100 mL−1 and from 4.7 and
5.4 log10 CFU 100 mL−1 , respectively. Salmonella spp. was not
detected in wastewater samples, except once in June 2011 and
once at the end of July 2012. After 3–4 days of storing, the
LW Total Coliforms, Fecal Coliforms and E. coli ranged from
a maximum of 4.1 and a minimum of 3.0 log10 CFU 100 mL−1 ,
3.9–2.1 log10 CFU 100 mL−1 and 3.3–2.0 log10 CFU 100 mL−1 ,

Fig. 1. Sketch of the SW (secondary treatment), TW (conventional tertiary treatment), LW (lagoon simplified tertiary water), FW (fresh water) and the experimental test
field.
 G.A. Vivaldi et al. / Agriculture, Ecosystems and Environment 181 (2013) 50–57 53

Fig. 2. Total Coliforms (log10 CFU 100 mL−1 ) recorded in the water used for the experiment. For SW (secondary treatment), TW (conventional tertiary treatment), LW (lagoon
simplified tertiary water) and FW (fresh water) the values are from 18 samples (each point represent a means of four replicates) collected from May to September 2011 and
2012.

respectively. In many samples LW Fecal Coliforms and E. coli
contaminations were lower than FW or null. For LW, TW and FW
Salmonella spp. was never detected during the monitoring period.
TW showed values of Total Coliforms less than SW and
LW 3.9 and 1.6 log10 CFU 100 mL−1 (maximum and mini-
mum, respectively). The TW values were higher by almost
0.1 log10 CFU 100 mL−1 than LW only for the third and the ninth
samples. The TW contamination of Fecal Coliforms and E. coli was
always lower than SW and LW in both years. In addition, FW was
the source with the lowest Total Coliforms contamination, with
a maximum of 1.9 log10 CFU 100 mL−1 ; in most of cases this con-
tamination was absent. The same results were obtained for Fecal
Coliforms and E. coli, although in four cases the FW contamination
was higher than TW for Total Coliforms and three times for E. coli.
3.2. Microbiological quality of the soil
As expected, the soils of the plots irrigated with the SW were
heavily contaminated by Total Coliforms, with values ranging from
2.7 log10 CFU 100 g−1 to 3.5 log10 CFU 100 g−1 in the first year and
from 2.9 log10 CFU 100 g−1 to 3.5 log10 CFU 100 g−1 in the second
one (Fig. 5a and b). Instead, the contamination values of soil irri-
gated with LW varied between 1.8 and 2.6 log10 CFU 100 g−1 in the
2011 and between 2.1 and 2.5 log10 CFU 100 g−1 in 2012. The results
of post hoc test revealed that the soils treated with LW and SW
differed statistically (p < 0.05), although SW was much more con-
taminated than LW.
Different contamination levels have been detected in the
plots irrigated with TW between years (p < 0.001). Indeed, in
the first year, the level of Total Coliforms ranged from 2.0
to 2.9 log10 CFU 100 g−1 , while in the second year the contam-
ination values ranged from 1.5 to 1.9 log10 CFU 100 g−1 . The
FW treated soils were characterized by Total Coliform levels
from 1.8 to 2.4 log10 CFU 100 g−1 in the 2011 and from 1.3 to
2.2 log10 CFU 100 g−1 in the 2012. For this treatment, a statistically
significant difference was found for both years in comparison with
the SW treatment with p-values respectively of p < 0.001 and of
p < 0.001 for the 2011 and 2012 years.
The contamination by Fecal Coliforms of the soil irrigated
with different water sources, followed the same behavior of
Total Coliforms (Fig. 6c and d). The highest level of contam-
ination has been identified in the plot irrigated with SW for

Fig. 3. Fecal Coliforms (log10 CFU 100 mL−1 ) recorded in the water used for the experiment. For SW (secondary treatment), TW (conventional tertiary treatment), LW
(lagoonsimplified tertiary water) and FW (fresh water) the values are from 18 samples (each point represent a means of four replicates) collected May–September 2011 and
May–September 2012.
54 G.A. Vivaldi et al. / Agriculture, Ecosystems and Environment 181 (2013) 50–57

Fig. 4. E. coli (log10 CFU 100 mL−1 ) recorded in the water used for the experiment. For SW (secondary treatment), TW (conventional tertiary treatment), LW (lagoonsimplified
tertiary water) and FW (fresh water) the values are from 18 samples (each point was means of 4 replicates) collected May–September 2011 and 2012.

Fig. 5. Total Coliform (CFU g−1 ) measured in the soil according to the experimental years 2011 (a) and 2012 (b) and the different treatments: SW (secondary treatment), TW
(conventional tertiary treatment), LW (lagoonsimplified tertiary water) and FW (fresh water). Capital letters represent significant difference between treatments (p < 0.05).

Fig. 6. Fecal Coliform (CFU g−1 ) measured in the soil according to the experimental years 2011 (c) and 2012 (d) and the different treatments: SW (secondary treatment), TW
(conventional tertiary treatment), LW (lagoonsimplified tertiary water) and FW (fresh water). Capital letters represent significant difference between treatments (p < 0.05).
 G.A. Vivaldi et al. / Agriculture, Ecosystems and Environment 181 (2013) 50–57
Fig. 7. E. coli (CFU g−1 ) measured in the soil according to the experimental years 2011 (e) and 2012 (f) and the different treatments: SW (secondary treatment), TW
(conventional tertiary treatment), LW (lagoonsimplified tertiary water) and FW (fresh water). Capital letters represent significant difference between treatments (p < 0.05).
both years. In 2011 the minimum and maximum value detected
were 2.5 and 3.3 log10 CFU 100 g −1 , while in 2012 were 2.5
and 3.1 log10 CFU 100 g−1 , respectively. The hygienic quality of
the soil irrigated with LW (minimum and maximum values
of 1.5 and 1.9 log10 CFU 100 g−1 for the 2011 and 1.4 of and
2.0 log10 CFU 100 g−1 for 2012, respectively) was lower than SW
and statistically different for 2011 and for 2012 (p < 0.04). For the
first year, considering the high pollution of the TW, the soil con-
tamination by Fecal Coliforms, ranged from a minimum of 1.5 to a
maximum of 2.1 log10 CFU 100 g−1 these values were not statisti-
cally different from SW and LW. In 2012 the TW soil contamination
by fecal contamination was statistically lower (respectively a
minimum of 0.3 and a maximum of 1.0 log10 CFU 100 g−1 ) than the
soil irrigated with LW and SW (p < 0.02 and p < 0.001, respectively).
Finally very low contamination was detected in the plots irrigated
with FW for both years of the trial, resulting in statistically lower
values with respect to TW, LW, and SW treated soils for the 2011
(p < 0.001, p < 0.007 and p < 0.001 respectively) and LW and SW
treated soils in 2012 (p < 0.04 and p < 0.001, respectively).
As observed for Total Coliforms and Fecal Coliforms, the high-
est contamination of E. coli was recorded in the plots irrigated
with SW (minimum and maximum of 2.0 and 3.1 log10 CFU 100 g−1
for 2011, 2.2 and 3.0 log10 CFU 100 g−1 for 2012, respectively)
(Fig. 7e and f). The effect of the LW on the soil was similar for
both years, reaching from a minimum of 0.6 to a maximum of
1.9 log10 CFU 100 g−1 for 2011 and from a minimum of 1.2 to a max-
imum of 1.9 log10 CFU 100 g−1 for 2012. For soil irrigated with TW
in 2011, E. coli was detected with a minimum of 0.9 and a maxi-
mum of 1.9 log10 CFU 100 g−1 , due to the high contamination of the
water; while in 2012 the E. coli was never detected. Finally for both
years E. coli pollution was never detected in the plots irrigated with
FW. No Salmonella spp. contamination was recorded for any of the
treatments, either years.
3.3. Microbiological quality of fruits
The fruits collected from the trees irrigated by SW, showed the
highest Total Coliforms values in both years (Table 3). Similar level
was observed in fruit from LW and TW treated plots but not for FW,
which always showed the lowest values. Only for trees irrigated
with SW was detected contamination by Fecal Coliforms for both
years, with a maximum of 250 CFU g−1 in 2011 and of 30 CFU g−1
in 2012. E. coli was not detected on surface of the fruits by the trees
irrigated with FW, TW and LW, but not for SW; in the last case the
values reached a maximum of 90 CFU g−1 in 2011 and 10 CFU g−1
55
in 2012. During second year a very weak Fecal Coliforms and E. coli
contamination was observed on the fruits irrigated with FW. Obvi-
ously, no Salmonella spp. contamination was recorded for all the
irrigation treatments.
4. Discussion
In this study, the E. coli water concentrations of SW and LW were
always over the current Italian threshold (10 CFU 100 mL−1 (Decree
of Ministry for Environment, No. 152/2006). However, the level of
contamination by E. coli in LW was 2 unit log10 lower than SW. Even
the Fecal Coliforms densities detected in SW and LW were always
over the WHO threshold (103 CFU 100 mL−1 ). Anyway, the LW had
less contamination than SW, determining a very low soil contam-
ination and no fruits pollution. The TW exhibited contamination
by Fecal Coliforms and E. coli 4 times out of 9 in 2011 and 2 times
out of 9 in 2012. These values are probably due to both a higher
contamination of the wastewater incoming in 2011 than 2012 and
to a bad management of the TW plant. This study demonstrated
that values of E. coli and Fecal Coliforms in FW frequently exceed
those observed in reclaimed irrigation water. In fact the FW exhib-
ited fecal contamination 3 times out of 9 both in 2011 and 2012.
Moreover, in 4 cases during the entire experiment, the FW fecal
contamination was higher than TW. Data suggested that open chan-
nel water is exposed to microbiological contamination and this
situation can be presumably ascribed to some illegal discharge of
untreated wastewater into the underlying aquifer and/or to uncon-
trolled leakage from the local sewage network (Lopez et al., 2010).
These episodic contaminations emphasize the need to perform
periodic microbiological analysis of the irrigation water supplies,
regardless the water source, in order to minimize negative public
health impacts (WHO, 2006).
The plots irrigated with SW showed the highest soil pollution
values with respect to the other treatments. Moreover, compar-
ing the E. coli contamination level, in both water and soil, a
notable reduction of 2.6 log10 CFU 100 g−1 has been observed in
the latter. Very similar behavior has been reported by Palese et al.
(2009), using an irrigation water with Coliform contamination up to
78.000 CFU 100 mL−1 , drip irrigation method and sandy loam soil
texture. Only after 10 days from the end of irrigation season, the
soil was able to reduce the fecal contamination as demonstrated by
Campos et al. (2000b) which reported a soil fecal pollution reduc-
tion up to four logarithmic units, one day after. At the end of both
irrigation seasons, E. coli contamination breakdown made by soil
irrigated with LT and TW has been 1.4 and 0.3 log10 CFU 100 g−1 . It is
56 G.A. Vivaldi et al. / Agriculture, Ecosystems and Environment 181 (2013) 50–57
Table 3
Total Coliforms, Fecal Coliforms and E. coli (CFU g−1 ) on nectarine fruits irrigated with SW (secondary treatment), TW (conventional tertiary treatment), LW (lagoonsimplified
tertiary water) and FW (fresh water) in 2011 and 2012.
Years Treatment Total Coliforms
a a
Minimum Maximum P/T
(CFU g−1 ) (CFU g−1 )
2011 FW 0 60 2/4
TW 0 570 3/4
LW 0 210 3/4
SW 0 720 3/4
2012 FW 0 30 1/4
TW 0 80 2/4
LW 0 110 2/4
SW 0 800 3/4
a
Ratio between positive samples and total samples.
well known that microrganism survival in the soil is function of soil
texture, organic matter, moisture, irrigation regime, pH and chemi-
cal fertilizer (Mawdsley et al., 1995; Cools et al., 2001; Ogden et al.,
2001; Estrada et al., 2004; Nicholson et al., 2005; Pourcher et al.,
2007; Bernstein, 2009). In general, higher clay contents usually
provide a more protective environment, support a larger bacterial
biomass overall and extend the survival period of enteric bacteria
than a sandy or loam soil (Fenlon et al., 2000; Cools et al., 2001).
However, Lang and Smith (2007) observed an opposite situation
as the fine textured soil contained fewer E. coli than the sandy
loam soil type. Considering the soil texture in our study (sandy
loam), we confirm the results obtained by Cools et al. (2001). These
authors reported that the sandy soil allowed the best E. coli survival,
while the loamy sand and loamy soils with low organic matter rep-
resent the worst condition for exogenous bacterial survival. This
suggested that others soil properties may have greater influence
than soil texture on enteric microorganisms within the background
microbial community. The low level of organic matter of the soil
detected in our experiment (1.76%) may have created disadvanta-
geous conditions for fecal bacteria. In fact organic matter promotes
the survival, and in some instances, the regrowth of enteric bacteria
(Gerba et al., 1975), increasing the nutrients retention, providing
a carbon for bacterial species and improving moisture reten-
tion properties (Jamieson et al., 2002). In our soil conditions, the
conventional irrigation management used (4–7 days of interval and
total restoring of water loss) allowed a very fast reduction of bacte-
rial persistence, as observed for Salmonella with a similar irrigation
management (Bernstein et al., 2007b). The occasional contamina-
tion of soil irrigated with FW, could be attributed to irrigation water
contamination and to other factors, such as roaming wild animals
and birds and runoff (Mawdsley et al., 1995; Venglovsky et al.,
2006). In some cases, it can also happen that the plots irrigated with
conventional water could result more polluted than plots irrigated
with treated municipal wastewater (Lopez et al., 2006).
Regardless of water source quality, Total Coliforms were always
detected on fruits, although with different contamination levels.
This result is not alarming because it is well known that Total
Coliforms are ubiquitous in agricultural environments (Materon,
2003). According to WHO guideline 2000, orchard wastewater irri-
gation should be stopped two weeks before harvest and no fruit
should be picked up off the ground. In our case, the scalar har-
vest of nectarine did not allow the irrigation to stop during the
fruit ripening period, so increasing the probability to having fruits
contaminated over a longer period. Despite this situation, for both
years, no Fecal Coliform and E. coli contamination has been detected
on fruits irrigated whit TW and LW, because of lack of contact
between water and canopy, with a very low risk of contamination
of fruits. It is in line with what reported by Palese et al. (2009)
for olive orchard irrigated with municipal wastewater, using the
same irrigation method. Consequently, drip irrigation combined
Fecal Coliforms E. coli
a
Minimum Maximum P/T Minimum Maximum P/T
(CFU g−1 ) (CFU g−1 ) (CFU g−1 ) (CFU g−1 )
0 0 0/4 0 0 0/4
0 0 0/4 0 0 0/4
0 0 0/4 0 0 0/4
0 250 3/4 0 90 3/4
0 20 1/4 0 2 1/4
0 0 0/4 0 0 0/4
0 0 0/4 0 0 0/4
0 30 2/4 0 10 2/4
with wastewater reuse may offer the most effective and efficient
way to cope with water shortage for fruit tree crops and protect the
environment receiving wastewater (Capra and Scicolone, 2007).
Moreover, for trees irrigated with SW, fruits surface contamina-
tion with fecal bacteria was nearly always recorded. This result
suggests that the reuse of municipal wastewater without tertiary
treatment may increase the health risks for human consumption
of edible fruits. Finally, Fecal Coliform and E. coli pollution of fruits
irrigated with FW is likely due to the environmental pollution, a
secondary source of contamination or to an accidental contamina-
tion occurring during sampling (Palese et al., 2009). In some cases,
as in greenhouse, following routine chlorination of SW, no E. coli
were detected in the water, soilless media and rose leaf surfaces,
but bacteria accumulated in the leachates (Bernstein et al., 2008).
We can also assume that the environmental condition of our exper-
iment may have influenced the survival of microorganism on the
fruits surface. The nectarine ripening started in June and finished
at the end of July with the harvest. During this period, different
agents may cause the mortality of microorganisms on fruit crop
surfaces. First of all, as reported by Minhas et al. (2006), the high
level of solar radiation in our experiment could have contributed
to a reduction in the bacteria density. Rose et al. (2001) verified
that weather influences the transport and dissemination of micro-
bial agents via rainfall and runoff and the survival and/or growth
through such factors as temperature. The temperature during the
harvest period may have a positive effect on reducing the level of
contamination of fruits as observed by Nicholson et al. (2000).
5. Conclusions
Although the reuse of municipal wastewater represent interest-
ing alternative for agriculture, the microbial risk and the efficiency
of tertiary treatment are aspects need more attention. Our results
suggest that the microbial contamination of the tree-soil system
is a function of both (i) the irrigation water pollution level and (ii)
the pedological and climatic conditions. The water coming from the
lagoon simplified tertiary treatment resulted in low soil contamina-
tion which then translated into no fruit contamination. On the other
hand, conventional tertiary treatment could cause water pollution
if not well managed. Considering the general high level of microbial
pollution of the waters used, further investigations need to under-
stand the removal level of other contamination indicators, such
as Giardia cysts, Cryptosporidium oocysts and viruses, by different
treatment plants and under different soil/climate conditions. More-
over, considering that in the last years several studies have stated
that human pathogens can also penetrate into the plant tissues via
the root, translocate and survive inside the vegetables (Bernstein
et al., 2007a,c; Gorbatsevich et al., 2013), it could be extremely
interesting to investigate this topic on the fruit tree crops.
 G.A. Vivaldi et al. / Agriculture, Ecosystems and Environment 181 (2013) 50–57
Acknowledgements
The authors wish to thank the Italian Ministry of University
and Research (MIUR) for its financial support under the Project
In. T.e.R.R.A.(contract No 01 01480) co-funded within the Italian
Program “PON/Ricerca e Competitività 2007-2013”.
References
Allen, R.G., Pereira, L.S., Raes, D., Smith, M., 1998. Crop evapotranspiration. Guide-
lines for computing crop water requirements. FAO Irrigation and Drainage Paper
No. 56.
Ait Melloul, A., Hassani, L., Rafouk, L., 2001. Salmonella spp. contamination of
vegetables irrigated with untreated wastewater. World Journal Microbiology
Biotechnology 17, 207–209.
Angelakis, A.N., Marecos do Monte, M.H.F., Bontoux, L., Asano, T., 1999. The status
of wastewater reuse practice in the Mediterranean basin: need for guidelines.
Water Research 10, 2201–2217.
APAT, IRSA-CNR, 2003. Metodi analitici per le acque. Manuali e linee guida 29/2003.
Asano, T., Levine, A.D., 1998. Wastewater reclamation, recycling, and reuse: an intro-
duction. In: Asano, T. (Ed.), Wastewater Reclamation and Reuse, vol. 10. CRC
Press, Boca Raton, Fla, pp. 1–56.
Bastos, R.K.X., Mara, D.D., 1995. The bacterial quality of salad crops drip and fur-
row irrigated with waste stabilization pond effluent: an evaluation of the WHO
guidelines. Water Science and Technology 12, 425–430.
Bernstein, N., Sela, S., Neder-Lavon, S., 2007a. Assessment of contamination potential
of lettuce by Salmonella enterica serovar Newport added to the plant growing
medium. Journal of Food Protection 70, 1717–1722.
Bernstein, N., Sela, S., Neder-Lavon, S., 2007b. Effect of irrigation regimes on per-
sistence of Salmonella enterica serovar Newport in small experimental pots
designed for plant cultivation. Irrigation Science 26, 1–8.
Bernstein, N., Sela, S., Pinto, R., Ioffe, M., 2007c. Evidence for internalization of
Escherichia coli into the aerial parts of maize via the root system. Journal of
Food Protection 70, 471–475.
Bernstein, N., Guetsky, R., Friedman, H., Bar-Tal, A., Rot, I., 2008. Monitoring bac-
terial populations in agricultural greenhouse production system irrigated with
reclaimed wastewater. Journal of Horticultural Science and Biotechnology 83,
821–827.
Bernstein, N., 2009. Contamination of soils with microbial pathogens originating
from effluent water used for irrigation. In: Contaminated Soils: Environmental
Impact, Disposal and Treatment. Nova Science Publishers, NY, USA.
Blumenthal, U.J., Mara, D.D., Peasey, A., Ruiz-Palacios, G., Stott, R., 2000. Guide-
lines for the microbiological quality of treated wastewater used in agriculture:
recommendations for revising WHO guidelines. Bulletin of the World Health
Organization 78 (9), 1104–1116.
Campos, C., Oron, G., Salgot, M., Gillerma, L., Casals, G., 2000b. Attenuation of
microorganism in the soil during drip irrigation with water stabilization pond
effluent. Water Science and Technology 10-11, 387–392.
Campos, C., Guerrero, A., Cardenas, M., 2002. Removal of bacterial and viral fecal
indicator organisms in a waste stabilization pond system in Choconta, Cundina-
marca (Colombia). Water Science and Technology 1, 61–66.
Capra, A., Scicolone, B., 2004. Emitter and filter tests for wastewater reuse by drip
irrigation. Agricultural Water Management 68, 135–149.
Capra, A., Scicolone, B., 2007. Recycling of poor quality urban wastewater by drip
irrigation systems. Journal of Cleaner Production 15 (16), 1529–1534.
Cools, D., Merckx, R., Vlassak, K., Verhaegen, J., 2001. Survival of E. coli and Enterococ-
cus spp derived from pig slurry in soils of different texture. Applied Soil Ecology
17, 53–62.
Decree of Ministry for Environment, No. 152, 03/04/2006, Gazzetta Ufficiale, No. 88,
Rome April 14, 2006. Italian Technical Guidelines for Wastewater Reuse.
El Hamouri, B., Handouf, A., Mekrane, M., Touzani, M., Khana, A., Khallayoune, K.,
Benchokroun, T., 1996. Use of wastewater for crop production under arid and
saline conditions: yield and hygienic quality of the crop and soil contaminations.
Water Science Technology 10-11, 327–334.
Estrada, I.B., Aller, A., Aller, F., Gomez, X., Moran, A., 2004. The survival of
Escherichia coli, Fecal Coliforms and enterobacteriaceae in general in soil treated
with sludge from wastewater treatment plants. Bioresource Technology 93,
191–198.
FAO, 2011. The state of the world’s land and water resources for food and
agriculture. Summary Report, pp. 1–47. http://www.fao.org/nr/water/docs/
SOLAW EX SUMM WEB EN.pdf
Fenlon, D.R., Ogden, I.D., Vinten, A., Svoboda, I., 2000. The fate of Escherichia coli and
E. coli O157 in cattle slurry after application to land. Journal Applied Microbiol-
ogy Symposium Supplement 88, 149S–156S.
Gerba, C.P., Wallis, C., Melnick, J.L., 1975. Fate of wastewater bacteria and viruses in
soil. Journal of Irrigation Drainage Engineering 101, 157–174.
Gorbatsevich, E., Sela, S., Pinto, R., Bernstein, N., 2013. Root internalization, transport
and in-planta survival of Salmonella enterica serovar Newport in sweet basil.
Environmental Microbiology Reports 5 (1), 151–159.
Homsi, J., 2000. The present state of sewage treatment. International Report Water
Supply 18, 325–327.
Jamieson, R.C., Gordon, R.J., Sharples, K.E., Stratton, G.W., Madani, A., 2002. Move-
ment and persistence of fecal bacteria in agricultural soils and subsurface
drainage water: a review. Canadian Biosystems Engineering 44, 11–19.
57
Lang, N.L., Smith, S.R., 2007. Influence of soil type, moisture content and
biosolids application on the fate of Escherichia coli in agricultural soil under
controlled laboratory conditions. Journal of Applied Microbiology 103 (6),
2122–2131.
Lopez, A., Pollice, A., Lonigro, A., Masi, S., Palese, A.M., Cirelli, G.L., Toscano, A.,
Passino, R., 2006. Agricultural wastewater reuse in southern Italy. Desalination
187, 323–334.
Lopez, A., Pollice, A., Laera, G., Lonigro, A., Rubino, P., 2010. Membrane filtration of
municipal wastewater effluents for implementing agricultural reuse in southern
Italy. Water Science & Technology 625, 1121–1128.
Mara, D.D., Pearson, H.W., 1987. Waste Stabilization Ponds: Design Manual for
Mediterranean Europe, EUR/ICP/CWS 053. WHO, Copenhagen.
Mara, D.D., Alabaster, G.P., Pearson, H.W., Mills, S.W., 1992a. Waste Stabilization
Ponds: A Design Manual for Eastern Africa. Lagoon Technology International
Ltd, Leeds.
Mara, D.D., Mills, S.W., Pearson, H.W., Alabaster, G.P., 1992b. Waste stabilization
ponds: a viable alternative for small community treatment systems. Water and
Environment Journal 6 (3), 72–78.
Materon, L.A., 2003. Survival of Escherichia coli 0157:H7 applied to cantaloupes and
the effectiveness of chlorinated water and lactic acid as disinfections. World
Journal of Microbiology & Biotechnology 19 (8), 867–873.
Mawdsley, J.L., Bardgett, R.D., Merry, R.J., Pan, B.F., Theodorou, M.K., 1995. Pathogens
in livestock waste, their potential for movement through soil and environmental
pollution. Applied Soil and Ecology 2, 1–5.
Maynard, H.E., Ouki, S.K., Williams, C., 1999. Tertiary lagoons: a review of removal
mechanisms and performance. Water Research 33 (1), 1–13.
Minhas, P.S., Sharma, N., Yadav, R.K., Joshi, P.K., 2006. Prevalence and control of
pathogenic contamination in some sewage irrigated vegetable, forage and cereal
grain crops. Bioresource Technology 97, 1174–1778.
Ndiaye, M.L., Niang, S., Pfeifer, H.R., Peduzzi, R., Tonolla, M., Dieng, Y., 2011. Effect
of irrigation water and processing on the microbial quality of lettuces produced
and sold on markets in Dakar (Senegal). Irrigation Drainage 60, 509–517.
Nicholson, F.A., Hutchison, M.C., Smith, K.A., Keevil, C.W., Chamber, B.J., Moore, A.,
2000. A study on farm manure application to Agri land and an assessment of
risks of pathogen transfer into the food chain. Project Report FS 2526. Ministry
of Agriculture, Fisheries and Food, London.
Nicholson, F.A., Groves, S.J., Chambers, B.J., 2005. Pathogen survival during live-
stock manure storage and following land application. Bioresource Technology
96, 135–143.
Ogden, I.D., Fenlon, D.R., Vinten, A.J.A., Lewis, D., 2001. The fate of Escherichia coli O-
157 in soil and its potential to contaminate drinking water. International Journal
of Food Microbiology 66, 111–117.
Palese, A.M., Pasquale, V., Celano, G., Figliuolo, G., Masi, S., Xiloyannis, C., 2009. Irriga-
tion of olive groves in Southern Italy with treated municipal wastewater: effects
on microbiological quality of soil and fruits. Agricultural Water Management
129, 43–51.
Pearson, H.W., Mara, D.D., Mills, S.W., Smallman, D.J., 1987a. Factors determining
algal populations in waste stabilization ponds and the influence of algae on
pond performance. Water Science Technology 19 (12), 131–140.
Pearson, H.W., Mara, D.D., Mills, S.W., Smallman, D.J., 1987b. Physico-chemical
parameters influencing fecal bacterial survival in waste stabilization ponds.
Water Science Technology 19 (12), 145–152.
Petterson, S.R., Ashbolt, N.J., Sharma, A., 2011. Microbial risks from wastewater irri-
gation of salad crops: a screening-level risk assessment. Water Environment
Research 73 (6), 667–672.
Pollice, A., Lopez, A., Laera, G., Rubino, P., Lonigro, A., 2004. Tertiary filtered municipal
wastewater as alternative water source in agriculture: a field investigation in
Southern Italy. Science of Total Environment 324, 201–210.
Pourcher, A., Picard-Bonnaud, F., Ferré, V., Gosinska, A., Stan, V., Moguedet, G., 2007.
Survival of fecal indicators and enteroviruses in soil after land-spreading of
municipal sewage sludge. Applied Soil Ecology 35, 473–479.
Rose, J.B., Epstein, P.R., Lipp, E.K., Sherman, B.H., Bernard, S.M., Patz, J.A., 2001. Cli-
mate variability and change in the United States: potential impacts on water-
and foodborne diseases caused by microbiologic agents. Environmental Health
Perspectives 109 (2), 211–221.
Scharf, J.M., 1966. Recommended Methods for the Microbiological Examination of
Foods, 2nd edition. APHA, New York.
Toze, S., 1997. Microbial pathogens in wastewater. Literature review for urban water
systems multi-divisional research program CSIRO LAND and WATER Techical
Report No. 1/97.
Toze, S., 1999. PCR and the detection of microbial pathogens in water and wastew-
ater. Water Research 33 (17), 3545–3556.
Taylor, H.D., Bastos, R.K.X., Person, H.W., Mara, D.D., 1995. Drip irrigation with waste
stabilization pond effluents: solving the problem of emitter fouling. Water Sci-
ence Technology 12, 417–424.
USEPA, United States Environmental Protection Agency, 2012. Guidelines for water
reuse 2012 EPA/600/R-12/618.
Venglovsky, J., Martinez, J., Placha, I., 2006. Hygienic and ecological risks connected
with utilization of animal manures and biosolids in agriculture. Livestock Sci-
ence 102, 197–203.
World Health Organization (WHO), 2006. Wastewater use in agriculture. In: Guide-
lines for the Safe Use of Wastewater, Excreta and Greywater. WHO, Geneva, pp.
100.
Woomer, P.L., 1994. Microbiological and biochemical properties. In: Weaver, R.W.,
et al. (Eds.), Methods of Soil Analysis Part 2. Soil Science Society of America Inc,
Madison, WI.