Environ Monit Assess (2014) 186:4857–4870

DOI 10.1007/s10661-014-3743-4

Assessment of long-term wastewater irrigation impacts

on the soil geochemical properties and the bioaccumulation
of heavy metals to the agricultural products
Anastasis Christou & Elena Eliadou & Costas Michael &
Evroula Hapeshi & Despo Fatta-Kassinos

Received: 24 October 2013 / Accepted: 18 March 2014 / Published online: 1 April 2014
# Springer International Publishing Switzerland 2014

Abstract An extensive field survey was employed for
assessing the impacts of long-term wastewater irrigation
of forage crops and orange orchards in three suburban
agricultural areas in Cyprus (areas I, II, and III), as
compared to rainfed agriculture, on the soil geochemical
properties and the bioaccumulation of heavy metals (Zn,
Ni, Mn, Cu, Co) to the agricultural products. Both
ryegrass fields and orange orchards in areas I and II
were continuously wastewater irrigated for 10 years,
whereas clover fields in area III for 0.5, 4, and 8 years.
The results revealed that wastewater reuse for irrigation
caused a slight increase in soil salinity and Cl− content in
areas I and II, and a remarkable increase, having strong
correlation with the period in which wastewater irriga-
tion was practiced, in area III. Soil salinization in area III
was due to the high electrical conductivity (EC) of the
wastewater applied for irrigation, attributed to the influx
of seawater to the sewage collection network in area III.
In addition, the wastewater irrigation practice resulted in
a slight decrease of the soil pH values in area III, while a
subtle impact was identified regarding the CaCO3, Fe,
A. Christou (*)
Agricultural Research Institute, P.O. Box 22016,
1516 Nicosia, Cyprus
e-mail: anastasis.christou@ari.gov.cy
E. Eliadou : C. Michael : E. Hapeshi : D. Fatta-Kassinos
Department of Civil and Environmental Engineering,
University of Cyprus, P.O. Box 20537, 1678 Nicosia, Cyprus
C. Michael : E. Hapeshi : D. Fatta-Kassinos
NIREAS-International Water Research Center, University of
Cyprus, P.O. Box 20537, 1678 Nicosia, Cyprus
and heavy metal content in the three areas surveyed. The
heavy metal content quantified in the forage plants’
above-ground parts was below the critical levels of
phytotoxicity and the maximum acceptable concentra-
tion in dairy feed, whereas heavy metals quantified in
orange fruit pulp were below the maximum permissible
levels (MPLs). Heavy metal phytoavailability was con-
fined due to soil properties (high pH and clay content),
as evidenced by the calculated low transfer factor (TF).
Keywords Electrical conductivity . Forage crops .
Heavy metal . Orange . Public health . Wastewater
irrigation
Introduction
Climate change and global warming effects are widely
recognized during the recent decades and therefore wa-
ter availability and management issues are of special
significance in all arid and semi-arid regions worldwide
(Hanjra and Qureshi 2010; Ostfeld et al. 2012). Water
scarcity already affects every continent, and it is among
the main problems to be faced by many societies and the
world in the twenty-first century (March et al. 2012; UN
2013). This is particularly true in the Mediterranean
areas of both southern Europe and North Africa, where
water resources are limited and unequally distributed in
both space and time (Milano et al. 2012). Water supply
and demand imbalance in the Mediterranean region is
further exacerbated by the increasing population and
living standards, the development of irrigated
4858
agriculture, and the increasing industry and tourism
activities (García-Ruiz et al. 2011; Milano et al. 2012).
Therefore, a great number of integrated water resource
management schemes are already being implemented,
or are under consideration (Hering et al. 2010; Iglesias
et al. 2011; Choukr-Allah et al. 2012), with particular
focus being given to the exploitation of existing water
resources and the utilization of nonconventional water
resources, such as treated wastewater (García-Ruiz et al.
2011; Milano et al. 2012). As a result, wastewater reuse,
mainly for irrigation, is already a well-established prac-
tice in all arid and semi-arid areas around the
Mediterranean countries (Bixio et al. 2006; Angelakis
and Durham 2008; Pedrero et al. 2010).
Although wastewater reuse for irrigation may in-
crease the agricultural production and expand irrigated
agriculture, while an equivalent amount of water for
other sectors may be conserved, many contaminants in
wastewater can be sequestered in the soil and conse-
quently may pose environmental problems with health
implications (Muchuweti et al. 2006; Li et al. 2009;
Kalavrouziotis 2011). Soil salinization due to wastewa-
ter reuse has been extensively reported during the recent
years (Mohammad Rusan et al. 2007; Klay et al. 2010;
Morugán-Coronado et al. 2011), with salinity being
recognized as a major factor reducing crop productivity
and a major cause of the abandonment of lands and
aquifers for agricultural purposes (Dodd and Pérez-
Alfocea 2012). In addition, many studies worldwide
have emphasized the risk of heavy metal accumulation
in wastewater irrigated topsoil (Mapanda et al. 2005;
Rattan et al. 2005; Ingwersen and Streck 2006; Li et al.
2009; Xu et al. 2010; Morugán-Coronado et al. 2011;
Kalavrouziotis and Koukoulakis 2012; Zheng et al.
2012). Importantly, heavy metal accumulation to toxic
concentrations in cultivated topsoil may pose serious
limitations to plant growth (Shanker et al. 2005; Di
Salvatore et al. 2008; Aycicek et al. 2011), while soil-
to-plant-to-human or soil-to-plant-to-livestock-to-human
translocation of heavy metals represents the main path-
ways of human exposure to soil contamination
(Rajaganapathy et al. 2011). Several studies have dem-
onstrated that wastewater irrigated plants may absorb and
accumulate heavy metals in concentration greater than
the maximum permissible limits (MPLs) with serious
public health implications (Mapanda et al. 2005;
Muchuweti et al. 2006; Khan et al. 2008; Singh et al.
2010; Chung et al. 2011; Kalavrouziotis et al. 2011;
Keser and Buyuk 2012; Keser 2012). Thus, information
Environ Monit Assess (2014) 186:4857–4870
about heavy metal concentrations due to wastewater
irrigation, both in cultivated soils and agricultural prod-
ucts, are very important for assessing the risks to both the
environmental sustainability and public health.
Cyprus, a semi-arid Mediterranean country, is facing
long-term water imbalances. Therefore, increasing ef-
forts are being made in order to mitigate water scarcity.
The maximum utilization of treated urban wastewater,
which can replace equal amounts of good quality pota-
ble water, is in the front line of such efforts. According
to the Water Development Department (WDD) of the
Republic of Cyprus, 22 million cubic meters (MCM) of
tertiary-treated wastewater per year are being currently
used for irrigation and aquifer recharge. Wastewater
reuse is already a common practice in Cyprus, and the
environment and public health is protected through a
comprehensive guideline and a code of good agricultur-
al practices. However, little is known about how long-
term wastewater reuse for irrigation in Cyprus has af-
fected soil quality and sustainability, as well as the
agricultural products safety. Therefore, the most typical
areas in three provinces of Cyprus, where wastewater
irrigation of forage crops and orange orchards is being
practiced for some years now, were selected for
assessing the effects of wastewater irrigation, as com-
pared to rainfed agriculture, on the soil physicochemical
parameters and the bioaccumulation of heavy metals to
the agricultural products.
Material and methods
Wastewater irrigation and site description
Cyprus has a typical Mediterranean climate which is
best described by the hot, dry summers and the mild,
wet winters. As a result, only irrigated agriculture can be
applied in the most agricultural sites of the island. One
inland and two coastal suburban areas in Cyprus were
selected for field sampling. The surveyed areas denoted
thereafter as areas I, ΙΙ, and III are located in the prov-
inces of Nicosia, Limassol, and Larnaka, respectively,
and have been irrigated with tertiary-treated effluent
from the municipal wastewater treatment plants
(MWTPs) serving the respective nearby cities (Fig. 1).
Particle size analysis of sieved soils revealed that soil in
areas I and II are similar in texture, once both showed
characteristics consistent with a sandy clay loam, with
sand and silt content ranging from 54 to 60 and 16 to
Environ Monit Assess (2014) 186:4857–4870
Fig. 1 Map indicating the sampling wastewater irrigated sites at the three areas surveyed in Cyprus. Within each marker, dotted area
represents wastewater irrigated forage field or orange orchard, while white area represents adjacent neighboring control rainfed field
24 %, respectively, while clay content varied from 21 to
26 %. The soil of area III is dominated by silty loam,
with sand, silt, and clay contents ranging between 20
and 30, 50 and 55, and 20 and 30 %, respectively (data
not shown). Treated wastewater in areas I and II has
been continuously applied for 10 years for the irrigation
of orange orchards and ryegrass fields, whereas in area
III for 0.5, 4, and 8 years for the irrigation of clover
fields. It is worth noting that treated wastewater was the
sole irrigation water source available in the surveyed
sites, since tubewell water abstraction is too costly,
compared with the subsidized wastewater. Crops were
irrigated with pressurized sprinkler systems based upon
their water requirements (~10,000 m3 ha−1) (Metochis
1997) and precipitation. Sites and treatment and disin-
fection processes applied at the three MWTPs are ana-
lytically described in Table 1. The physicochemical
characteristics and microbial load of the three MWTPs
effluents applied for irrigation in the three surveyed
areas are presented in Table 2.
4859
Soil and agricultural products sampling procedure
During the pre-rainy period of 2012, soil and agricultur-
al product samples from ryegrass fields and orange
orchards in areas I and II, as well as from clover fields
in area III, were collected (Table 1). Soil samples from
ryegrass fields and orange orchards in areas I and II,
where wastewater irrigation was practiced continuously
for 10 years, were obtained with an auger sampler from
the 0–25 cm topsoil layer as composite samples,
consisting of approximately 20 randomly collected sub-
samples. Sampling in area III was performed in three
clover fields where wastewater irrigation was practiced
continuously for 0.5, 4, and 8 years, respectively.
Samples were obtained from four soil profile layers
(0–25, 26–50, 51–75, and 76–100 cm) with a hydraulic
soil sampling system as composite samples, consisting
of approximately 10 randomly collected subsamples.
The soil profile depth sampled in area III reached 1 m
depth in order to examine the effects of wastewater
4860 Environ Monit Assess (2014) 186:4857–4870

Table 1 Surveyed sites, MWTPs’ treatment process and disinfection, and soils and agricultural product samples’ description

Surveyed Province MWTP treatment process Cultivated crops and Soil layer sampled Agricultural
area location and disinfection period of WW irrigation depths (cm) products sampled

I Nicosia Tertiary (MBR)-UV Ryegrass, orange trees 0–25 Ryegrass (stems and leaves)
(10 years) orange fruits
II Limassol Tertiary (SSF)-chlorination Ryegrass, orange trees 0–25 Ryegrass (stems and leaves)
(10 years) orange fruits
III Larnaka Tertiary (SSF)-chlorination Clover (0.5, 4, and 8 years) 0–25 Clover (stems and leaves)
26–50
51–75
76–100

MWTP Municipal Wastewater Treatment Plant, WW wastewater, MBR membrane bioreactor, UV ultraviolet disinfection, SSF slow sand
filtration

irrigation on the salinization of the soil profile, since the
wastewater applied in that area is characterized by high
salinity (Table 2). Soil samples from adjacent neighbor-
ing rainfed fields, where wastewater irrigation or
tubewell water irrigation was never applied before, were
collected and used as control samples providing refer-
ence “background” concentrations.
Agricultural products samples included orange fruits
and the above-ground parts of ryegrass and clover plants.
Approximately, 100 ripened orange fruits randomly col-
lected from nearly all trees in each orange orchard, and
the above-ground parts (stems and leaves) from ryegrass
and clover plants, constituted the composite samples
from the orange orchards and forage fields, respectively

Table 2 Physicochemical characteristics and microbial load of the treated effluent from the three MWTPs applied for irrigation in the
surveyed areas

MWTP in area I MWTP in area II MWTP in area III Permissible levelb

Parameter Mean valuea Mean valuea Mean valuea

pH 7.91±0.05 8.12±0.05 7.70±0.16 6.5–8.5

−1
EC (mS cm ) 1.77±0.04 1.69±0.16 3.60±0.44 <2.2
BOD5 (mg O2 L−1) 3.58±0.41 3.83±0.65 3.06±0.21 <10.00
COD (mg O2 L−1) 21.38±3.52 20.83±3.19 43.00±5.65 <70.00
SS (mg L−1) 7.00±0.12 7.00±0.03 12.00±3.56 <10.00
E. coli (cfu 100 mL−1) 1.20±0.11 NDd ND <5.00
−1
Total N (mg L ) 6.25±1.51 7.38±1.49 18.86±8.17 <15.00
Total P (mg L−1) 0.92±0.11 1.40±0.27 3.16±1.08 <10.00
Cl− (mg L−1) 314.00±7.52 209.00±12.34 897.00±56.44 <300.00
Fe (mg L−1) 0.09±0.01 0.12±0.03 0.10±0.03 –
Zn (mg L−1) 0.02±0.00 0.04±0.01 0.02±0.00 <0.10
Mn (mg L−1) 0.03±0.01 0.01±0.00 0.03±0.00 <0.10
Ni (mg L−1) 0.02±0.00 0.02±0.00 0.02±0.01 <0.20
Cu (mg L−1) b.l.q.c 0.03±0.01 b.l.q. <0.10
Co (mg L−1) 0.012±0.00 0.03±0.00 0.02±0.00 <0.10
a
Mean values are the average of 10 samples taken during 2010 and 2011
b
As set by the Cyprus guideline for wastewater reuse for irrigation
c
Below limit of quantification
d
Not detected
Environ Monit Assess (2014) 186:4857–4870
(Table 1). Ryegrass plants in area I and clover plants in
area III reached the ripening stage, while ryegrass plants
in area II reached the early heading stage. Respective
control samples were unable to be collected, since all
cultivated orange and forage crops in the sampling areas
are irrigated with treated wastewater.
Sample preparation and analytical methods
All soil samples were allowed to dry laid in a glasshouse,
ground, and sieved (<10 mesh). From the sieved sam-
ples, soil texture, pH, electrical conductivity (EC),
CaCO3, Cl−, Fe, as well as heavy metal (Mn, Co, Cu,
Zn, Ni) content, were determined. Selection of heavy
metals for analyses was based upon their concentration
quantified in treated wastewater effluents applied for
irrigation, since a number of heavy metals (Cd, Pb, Hg,
Cr) in wastewater were below the limit of quantification.
Soil texture was determined following the hydrometer
method (Day 1965), while soil pH was determined with a
pH meter (Thermo Scientific, Orion 4 Star) in a 1:2
sample to Milli-Q H2O suspension, as described by
Datta et al. (1997). The Bernard calcimeter was used for
the assay of the percentage of total CaCO3. Soil EC was
measured in the saturated soil-paste extract using the
Hanna HI4321 benchtop conductivity meter. Chloride
anions were determined following the Mohr’s method
(APHA 1998). For soil Fe and heavy metal content deter-
mination, 0.5 g of ground sieved soil samples were accu-
rately weighed directly into pre-cleaned microwave PTFE
vessels, and 9 mL concentrated HNO3 and 3 mL concen-
trated HCl were added. Samples were digested in the
MARS Xpress Microwave Digestion System in a total
digestion procedure equivalent to the USEPA Method
3051A (U.S. Environmental Protection Agency 1998).
Samples were then allowed to cool, filtered (Whatman
42 Filter Paper) into polypropylene tubes, and diluted to
50 mL with Milli-Q H2O. A blank digest was run in the
same way in all digestion events. Metal concentrations in
soil digests were determined using a Thermo Scientific
SOLLAR/AA flame atomic absorption spectrophotome-
ter (AAS). A Certified European sandy soil Reference
Material (Code ERM-CC018 trace elements in contami-
nated sandy soil) purchased from BAM Federal Institute
for Material Research and Testing was used to check
analytical accuracy and precision regarding soil metal
content determination. The analysis of CRM metal con-
tent revealed that elemental recoveries ranged from 92 to
102 % (Table 3).
4861
Table 3 Results of the Certified European sandy soil Reference
Material (Code ERM-CC018 trace elements in contaminated
sandy soil) analysis for checking the analytical accuracy of the
trace elements quantification method
Element Certified value Quantified value Recovery (%)
(mg kg−1) (mg kg−1)
Zn 313±13 305.81±19.16 98
Fe 22,600±400 22,922.80±372.74 101
Mn 509±6 469.37±33.38 92
Ni 55.8±1.8 56.89±3.93 102
Cu 80±4 80.62±4.22 101
Co 5.9±0.4 6.03±0.46 102
Both orange fruits and above-ground parts of rye-
grass and clover plants were washed with Milli-Q H2O
in order to remove any soil particles and then blotted
dried with tissue paper. Subsequently, orange pulp and
ryegrass and clover stems and leaves were weighed to
obtain fresh mass and dried in a preheated oven at 80 °C
for 72 h, until constant weight. Dry samples were
weighed again to obtain dry mass, ground, and sieved
(30 mesh). Dried ground agricultural product samples
(0.4 g) were dissolved in 8 mL mixture of concentrated
HNO3 and H2O (1:1 v/v) in a microwave PTFE vessels.
Samples’ decomposition was accelerated by microwave
digestion in the MARS Xpress Microwave Digestion
System, in a program consisting of an 8-min ramp to
175 °C, a 15-min step at 175 °C, and then a ventilated
cooling period. Solution samples were transferred to
polypropylene centrifuge tubes and diluted with Milli-
Q H2O to 25 mL. Centrifugation (7,000×g, 2 min) was
followed in order to obtain clear supernatant. A blank
digest was run in the same way in all digestion events.
Metal concentrations in agricultural products digests
were determined using a Thermo Scientific SOLLAR/
AA flame atomic absorption spectrophotometer (AAS).
The soil-to-plant heavy metal transfer factor was
calculated as the ratio of the concentration of heavy
metal to the agricultural products (on dry weight basis)
to its respective total content in the sampled topsoil
layer, as proposed by Cui et al. (2004).
Statistical analysis
Statistical analysis was carried out using the software
package SPSS v21.0 (SPSS Inc., Chicago, USA), and
the comparison of averages of each treatment was based
on the analysis of variance (one-way ANOVA)
4862 Environ Monit Assess (2014) 186:4857–4870

Table 4 Geochemical properties of the top 0-25 cm soil profile layer of the sampled ryegrass fields and clover orchards irrigated with wastewater for 10 years, along with their adjacent

305.13±53.29*

926.97±20.56
Citrus orchard

1.50±0.11*
according to Duncan’s multiple range test at signifi-

23.65±1.13

27.22±0.69

21.57±0.43
69.21±1.27
8.48±0.04

70.41±1.04
82.31±1.79
cance level 5 % (P<0.05). Data are means±standard
error (SE) of five replications.

Results and discussion

892.59±18.55
1.16±0.03
23.52±2.05
8.42±0.04

184.78±6.73
27.52±0.50

20.79±1.60
69.91±4.46
65.14±3.44
74.23±2.62
Control
Wastewater applied for irrigation

Treated wastewater applied for irrigation in both areas I

and II was fully complied with the MPLs, except for Cl−

2.14±0.05*

445.73±9.89*
26.06±0.31*

22.16±0.56*

61.38±0.78*
2048.3±63.68
Ryegrass field
concentration in wastewater applied in area I, whereas

12.83±0.88
8.29±0.04

61.71±2.13
84.49±0.61

*Indicates the control and wastewater irrigation pair values that are significantly different according to Duncan’s multiple range test (P<0.05)
the treated effluent from the MWTP in area III failed to
meet MPLs regarding EC, SS, TN, and Cl− content
(Table 2). The high values of EC and Cl− content of
the treated effluent observed in area III are attributed to
the influx of seawater to the sewage collection network,

2001.94±63.71
8.38±0.12

11.33±1.67
188.55±3.18
24.88±1.10

15.84±0.12
60.79±3.99
75.29±4.74
48.07±1.45
1.16±0.1
since the area served, as a coastal city (Larnaka) with
underground water level at a small depth from the sea

Control
Area II
level (0.5–6 m), enables seawater intrusion. Overall, the
treated effluent applied for irrigation in the surveyed
sites was slightly alkaline in reaction, since the mean

157.01±13.11*
pH values ranged between 7.70 and 8.12, with the

1.66±0.03*

206.73±19.42

1189.74±39.62
Citrus orchard

8.23±0.05

47.74±0.55

25.70±0.26
5.98±1.14

104.40±2.13

44.25±0.45
normal range of pH for irrigation water ranging between
6.50 and 8.40 (Ayers and Westcot 1985). Furthermore,
heavy metal contents (Zn, Mn, Ni, Cu, Co) in all
MWTPs effluents applied for irrigation were below the
MPLs set for the use of treated wastewater for irrigation, 204.23±10.72

1227.06±19.69
by both the Cyprus guideline and the FAO guidelines
8.16±0.03

46.69±1.12

26.35±0.22
107.20±3.41
114.95±4.31
43.52±0.29
5.35±0.56
1.16±0.11

(Pescod 1992). Also, the average total nitrogen and total

Control

phosphorous content in the studied treated effluents

strengthen their high fertigation value for field crops
production.
170.25±10.14*

1226.73±21.62*
1.56±0.04*

37.29±0.73*

18.86±0.58*
90.85±5.23*

Effect of the wastewater irrigation on the soil pH

8.16±0.02

120.22±4.57
61.14±0.38
20.98±0.98
Ryegrass field

and CaCO3 content

neighboring control rainfed fields, in areas I and II

As shown in Table 4, CaCO3 content at area I ranged

from nearly 21 % at the ryegrass sampled field to just
Data are means±SE of five replications

around 6 % at orange orchard, whereas the correspond-

127.81±12.61
838.88±11.89
8.13±0.02
0.86±0.06

118.03±8.24
32.01±0.42

14.29±0.42
64.89±2.49

65.41±0.38
20.32±2.21

ing values at area II were 12 and 24 %, respectively.

Moreover, CaCO3 content at area III varied depending
Control
Area I

on the field location and the profile depth, ranging from

21 to 50 % (Table 5). No significant differences were
observed regarding the CaCO3 content between waste-
water irrigated fields and adjacent neighboring control
EC (mS cm−1)

Mn (mg kg−1)
Cl− (mg kg−1)

Co (mg kg−1)
Cu (mg kg−1)
Zn (mg kg−1)
Ni (mg kg−1)
CaCO3 (%)

Fe (g kg−1)

fields in all the sites surveyed. The high CaCO3 content

Parameters

gives further support to the alkaline pH values observed

pH

(pH ranging from 8 to 8.80). It is noteworthy that

Table 5 Chemical (CaCO3 and Fe content) and heavy metal content analysis of soil in the sampled clover fields at area III, irrigated with wastewater for 0.5, 4 and 8 years, along with their
adjacent neighboring control fields

Soil Period of CaCO3 (%) Fe (g kg−1) Zn (mg kg−1) Ni (mg kg−1) Mn (mg kg−1) Cu (mg kg−1) Co (mg kg−1)
profile irrigation
Environ Monit Assess (2014) 186:4857–4870

(cm) (years) Control Wastewater Control Wastewater Control Wastewater Control Wastewater Control Wastewater Control Wastewater Control Wastewater
irrigation irrigation irrigation irrigation irrigation irrigation irrigation

0–25 0.5 40.00±2.12 36.50±3.34 35.41±0.90 33.63±1.60 126.04±5.17 134.22±7.85 42.36±5.31 42.30±1.76 1138.32±26.74 1211.48±48.49 90.72±3.60 94.38±0.80 21.32±0.33 20.83±0.45
4 50.00±2.44 50.00±2.03 36.56±0.22 37.60±1.20 174.70±4.64 177.87±6.84 42.58±0.25 43.90±1.28 1322.86±7.70 1225.92±38.62 84.01±2.17 91.30±2.58 26.17±0.10 25.17±0.66
8 21.50±1.78 21.00±1.23 16.03±0.66 20.71±1.33* 67.03±6.80 62.33±6.36 52.42±2.34 46.85±0.21 774.38±27.35 849.51±45.27 38.36±1.48 51.08±3.57* 19.13±0.30 20.34±0.15*
26–50 0.5 40.00±2.03 37.50±2.34 27.10±1.72 32.87±4.01 121.35±6.90 121.14±0.34 36.25±3.07 40.32±1.15 1163.11±44.57 1178.62±51.72 88.17±5.47 83.75±6.11 20.19±0.61 21.05±0.60
4 50.00±0.89 50.00±2.45 32.51±0.48 39.17±2.30* 121.51±5.95 125.90±5.03 41.83±0.68 41.69±2.55 1085.52±17.03 1129.20±52.93 79.84±0.72 82.09±2.61 23.99±0.41 25.69±1.80
8 27.00±3.03 24.00±1.11 19.71±0.75 17.81±1.14 71.91±6.24 79.69±2.06 49.63±2.60 51.60±2.30 871.30±20.01 836.33±32.56 51.29±2.29 43.02±3.07 18.22±0.53 20.37±0.28*
51–75 0.5 41.00±2.67 36.00±3.11 30.79±2.50 33.68±2.52 101.54±7.57 104.11±6.05 29.96±2.34 30.82±3.80 1099.87±87.07 1124.00±81.26 78.72±3.29 87.01±3.48 20.85±0.58 22.05±1.16
4 50.00±1.88 50.00±1.98 33.31±0.63 31.29±1.26 117.12±1.05 123.40±7.50 41.92±1.46 42.32±2.13 1074.96±14.94 990.54±9.97* 76.39±1.49 77.03±3.71 25.78±0.89 25.52±0.66
8 30.00±3.01 29.50±1.34 16.89±0.48 21.37±0.87* 57.77±0.85 66.29±1.31* 59.75±4.05 50.48±0.68 987.84±45.30 930.01±38.20 43.84±2.19 49.38±2.06 21.17±1.10 20.94±1.06
76–100 0.5 36.00±2.11 39.00±2.45 33.30±2.58 27.45±1.49 75.85±2.44 77.36±3.41 26.60±0.88 29.03±2.85 1008.37±44.15 875.02±34.44 84.52±7.00 69.57±3.21 22.08±0.38 18.62±1.02*
4 50.00±3.24 48.00±4.11 34.68±1.10 34.39±1.08 125.92±9.17 124.48±8.92 32.05±0.84 32.59±1.14 1059.10±31.03 1050.32±33.18 86.14±2.43 78.43±2.98 23.53±0.54 26.04±0.38*
8 36.50±4.21 30.00±3.78 18.51±1.50 22.61±0.05 68.74±0.98 72.18±1.34 50.01±1.59 51.68±0.52 961.04±15.86 963.06±4.29 45.97±3.92 54.71±0.65 19.67±0.63 20.07±0.27

Data are means±SE of five replications

*Indicates the control and wastewater irrigation pair values that are significantly different according to Duncan’s multiple range test (P<0.05)
4863
4864
10 years of continuous wastewater irrigation caused no
significant modulation on soil pH at the topsoil profile
layer of both ryegrass fields and orange orchards in the
studied areas I and II (Table 4), whereas a slight decrease
was registered in some of the deeper soil profile layers
sampled in area III, as compared to the respective values
observed in the adjacent neighboring control fields
(Fig. 2a). The alkalinization of soils in area III due to
wastewater irrigation ranged from 0.17 to 0.68 in pH
units. Furthermore, the significant nitrogen fertilization
being extensively practiced in clover cultivation in
Cyprus may partly contribute to soil alkalinization in
the sampled fields in area III. Results in areas I and II are
in accordance with previous findings recording that 2, 5,
and 10 years of wastewater irrigation of forage crops in
Jordan did not consistently affect soil pH (Mohammad
Rusan et al. 2007). Results in area III, highlighting a
decrease in soil pH due to wastewater irrigation, are in
line with the findings of Rattan et al. (2005), who
reported that soil pH dropped on an average by 0.40
units as a result of long-term wastewater irrigation.
Similarly, Xu et al. (2010) reported an average soil pH
decrease by 1.08 units in the soil profile (0–150 cm)
irrigated with treated wastewater for 20 years.
Effect of the wastewater irrigation on the soil EC
and Cl− content
Prolonged wastewater irrigation in areas I and II resulted
in the slight accumulation of salts in the topsoil layer,
since soil EC was found to increase in a range lower or
equal to 0.70 and 0.98 mS cm−1 in the ryegrass fields
and orange orchards, respectively, as compared to the
adjacent neighboring control fields. Noteworthy, the Cl−
pH
a 0
8.0 8.5 9.0 9.5 b0
0.5 years
4 years
20 8 years 20
Soil depth (cm)
40 * * 40
60
60 *
* *
80 80
* *
* *
100 100
Fig. 2 Soil pH (a), electrical conductivity (b), and Cl− content (c)
in the 1-m profile depth in clover fields in area III, as affected by
wastewater irrigation (0.5, 4, 8 years). Vertical solid line represents
the average value of the three representative adjacent neighboring
Environ Monit Assess (2014) 186:4857–4870
content in the topsoil profile layer in both the ryegrass
field and orange orchard continuously irrigated with
wastewater for 10 years in surveyed area I was less than
or equal to 206.73 mg kg−1, whereas the corresponding
value in area II was 445.73 mg kg−1 (Table 4). Similar
results, reporting an increase in soil salinity due to
wastewater irrigation were also reported for Tunisia,
South East Australia, Brazil (Sao Paulo State), USA
(California), and Spain (Leal et al. 2009; Klay et al.
2010; Xu et al. 2010; Morugán-Coronado et al. 2011;
Muyen et al. 2011).
In contrast to the findings in areas I and II, wastewa-
ter irrigation in area III caused a marked increase in soil
salinity, as compared to the adjacent neighboring control
rainfed fields. Interestingly, soil salinity increase in the
clover fields at area III was proportional to the period of
wastewater irrigation and soil depth. More precisely,
clover fields where wastewater irrigation was practiced
continuously for 8 years appeared to have the highest
soil EC, followed by those irrigated for 4 and 0.5 years.
The EC of the 0–25 cm cultivated topsoil profile layer,
in which the shallow clover root system functions for the
water uptake and the nutrition of plants, was found to be
5.56, 5.29, and 7.46 mS cm−1 in the fields irrigated with
wastewater for 0.5, 4, and 8 years, respectively. In turn,
the EC values in the 76–100 cm soil profile layer were
8.05, 7.70, and 12.19 mS cm−1 in the fields irrigated
with wastewater for 0.5, 4, and 8 years, respectively.
Noteworthy, the soil EC in all sampled soil profile layers
in the three adjacent neighboring control fields did not
exceed 1.83 mS cm −1 (Fig. 2b). The findings of
Mohammad Rusan et al. (2007), demonstrating a re-
markably higher accumulation of soluble salts and EC
increase in the deeper profile layers (20–40 and 40–
EC (mS cm-1) Cl- content (mg kg-1)
5 10 15 c0 1000 2000 3000 4000
* * * 0.5 years * * * 0.5 years
4 years 4 years
20 8 years
8 years
* * *
40
* * *
* 60
* * * *
80
* * *
*
100
control clover fields, whereas dashed lines represent correspond-
ing standard errors. * indicates mean values that are significantly
different from the control according to Duncan’s multiple range
test (P<0.05)
Environ Monit Assess (2014) 186:4857–4870
60 cm) than the surface (0–20 cm) layer of soil being
irrigated with wastewater in the long-term, further corrob-
orate our findings. A similar trend to the EC was identified
regarding the soil Cl− content, ranging from 1,043 to
3,188 mg kg−1 in the wastewater irrigated fields in area
III. On the contrary, Cl− content did not exceed
220 mg kg−1 in the adjacent control rainfed fields
(Fig. 2c). The strong correlation between the salinity levels
and Cl− content at all sampled layers of the soil profile in
the wastewater irrigated fields at sampled area III can be
explained by the intrusion of seawater to the sewage
collection network serving the study area.
Considering the length of the period that treated
wastewater has been reused for irrigation, it seems un-
likely that salinity will be a serious problem in the long
term, regarding ryegrass crop productivity in areas I and
II, once ryegrass is considered to be moderately tolerant
to salinity, with a threshold of soil EC beyond which
yield starts to decline at 5.60 mS cm−1. Conversely, the
EC in wastewater irrigated orange orchards almost
reached the salinity threshold of the sensitive to salinity
orange tree (1.70 mS cm−1) (Ayers and Westcot 1985).
Though, taking into account that sampling took place
during the pre-rainy period and that the subsequent
winter precipitation will facilitate salt leaching and soil
EC mitigation, soil salinity is not expected to consider-
ably impact orange orchards’ productivity in the long
term, given that continuous monitoring of soil EC will
be carried out.
On the other hand, soil salinization due to wastewater
irrigation in the study area III poses serious problems
regarding clover crop productivity. According to Maas
and Hoffman (1977), clover yield starts to decline at soil
EC of 1.50 mS cm−1, whereas 12 % decrease in yield per
unit increase in salinity beyond threshold may occur. In
the present study, the EC in the topsoil layer of waste-
water irrigated fields, where the shallow root system of
clover plants exists, ranged from 5.29 to 7.46 mS cm−1.
This suggests that the yield of clover in the wastewater
irrigated fields in area III is far beyond the expected
optimum and that excess water beyond plant water
demands should be applied, in order to leach salts in
depths greater than the respective ones of plants’ root
system. Though, despite the high infiltration rate and the
adequate leaching fraction due to the soil structure in
area III, special attention should be given to the depth of
groundwater table and the amount of salt that could be
stored between the root zone and the water table.
According to the Geological Survey Department of the
4865
Republic of Cyprus, area III is lying above an alluvium
unconfined aquifer, with the unsaturated zone ranging
from 7 to 10 m depth, thus enabling salt leaching in the
long term. Considering all the findings together, it is
apparent that only salt tolerant plants, such as barley,
wheat, cotton, sugar beet, and sorghum, should be
grown in fields irrigated with wastewater in area III
and that special agricultural practices should be applied
in order to alleviate salt built up in the root zone of these
crops. In this context, winter period precipitation is
fundamental for salt leaching and soil EC mitigation.
This is particularly true, since sampling the 0–25 cm
topsoil profile layer after heavy precipitation few days
following our survey revealed a significant salt leaching
and mitigation of soil EC (1.98, 2.84, and 2.44 mS cm−1
in the fields irrigated with wastewater for 0.5, 4, and
8 years, respectively; data not shown). Additionally, the
sealing of the collecting sewage network serving the
studied area, in order to prevent seawater influx to it
and facilitate the mitigation of treated effluent EC,
seems to be the ideal solution that could prevent soil
salinization and permit the long-term use of wastewater
for irrigation in area III.
Effect of the wastewater irrigation on the soil Fe content
Soil Fe content showed a slight increase in the waste-
water irrigated ryegrass fields in areas I and II, as com-
pared to the reference control fields, whereas no impacts
were observed in citrus orchards (Table 4). In addition,
soil Fe content was found to be slightly higher in some
of the soil profile layers sampled in clover fields irrigat-
ed with wastewater for 4 and 8 years in area III (Table 5).
Results showing no consistent effects on soil Fe content
due to long-term wastewater irrigation were also report-
ed by Mohammad Rusan et al. (2007).
Effect of the wastewater irrigation on the soil heavy
metal content
The total concentrations of the examined heavy metals
(Zn, Ni, Mn, Cu, Co) in all soil profile layers sampled
indicated that the changes registered in response to the
wastewater irrigation were in general not conspicuous.
Interestingly, no significant effects of the long-term
(10 years) wastewater irrigation on heavy metal content
in orange orchards in both areas I and II, except for an
observed 36.5 % higher Zn concentration in wastewater
irrigated orange orchard in area I, compared to control,
4866 Environ Monit Assess (2014) 186:4857–4870

were determined. In contrast to this, higher contents of
some of the heavy metals examined were registered in
some of the soil profile layers sampled in the wastewater
irrigated ryegrass and clover fields, as compared to the
representative heavy metal content in the control refer-
ence soil profile layers sampled (Tables 4 and 5). The
higher heavy metal content in the ryegrass and clover
fields of all the three sampled areas could be attributed
to soil heterogeneity, rather than to wastewater reuse for
irrigation, since no adverse effects on the soil heavy
metal content were identified in the orange orchards
being continuously irrigated with wastewater for
10 years in areas I and II, despite having similar to
forage crops yearly water demands (~10,000 m3 ha−1).
In addition to this, taking into consideration the heavy
metals’ concentration in the wastewater effluents used
for irrigation (maximum concentration 0.04 mg L−1)
and the yearly irrigation water demands of the crops,
as well as the bulk density of the soil in the surveyed
areas, not more than 1.20 mg of a single heavy metal can
be added to a 25-cm soil profile layer of any of the three
areas surveyed, after a continuous 10-year-period of
wastewater irrigation. Importantly, the observed concen-
trations for Cu, Zn, Ni, and Co in the soil at all surveyed
sites were below the MPLs set by the European Union
(EEC 1986) (Directive 86/278/EEC, last updated 2002),
which are 140, 300, 75, and 50 mg kg−1, respectively, in
soil pH ranging from 6 to 7, while the Mn concentration
exceeded the limit value of 500 mg kg−1. However, this
high Mn concentration can be attributed to the compo-
sition of the parent rock, rather than to wastewater reuse,
as evidenced from the quantified Mn concentrations in
the reference control fields. These results are consistent
with previous findings from Smith et al. (1996) and
Mohammad Rusan et al.(2007), reporting no built up
of heavy metal content in long-term wastewater irrigated
soils. Similarly, only Zn was found to be accumulated in
soil irrigated for 17 years with treated wastewater, com-
pared to the nearby nonirrigated control area, in Tunisia
(Belaid et al. 2012).
Effect of the wastewater irrigation on the agricultural
products heavy metal content
Considerable variations of heavy metals concentrations
were registered in both the orange fruit pulp and the
ryegrass stems and leaves in areas I and II, despite being
wastewater irrigated for the same period (10 years). The
concentrations of Mn, Co, Cu, Zn, and Ni in orange fruit
pulp and ryegrass stems and leaves were consistently
higher in the samples taken from area I, compared to
those collected from area II (Table 6). However, the
concentrations of Cu and Zn found in the orange fruit
pulp were lower than the MPLs proposed by the joint
WHO/FAO Expert Committee on Food Additives
(WHO/FAO 2007) (40 and 60 mg kg−1 DW for Cu
and Zn, respectively). In turn, the observed higher heavy
metal concentration in ryegrass plants grown in area I, in

Table 6 Heavy metal concentrations (mg kg−1 dry weight) in sampled agricultural products from citrus orchards and forage fields irrigated
with wastewater for various periods of time

Crop Orange fruit pulp Ryegrass stems and leafs Clover stems and leafs

Sampled I II I II III(0.5) III(4) III(8)

area
MC (%) 87.2 89.2 74.9 76.3 76.5 81.7 80.6

Mn 5.192±0.083 a 2.452±0.056 b 56.560±0.385 a 54.416±0.452 a 76.834±0.200 a 54.812±0.200 c 65.206±0.374 b

Co 2.768±0.136 a 0.978±0.035 b 1.444±0.295 a 0.507±0.036 b 1.550±0.212 a 1.428±0.125 a 1.364±0.111 a
Cu 4.352±0.033 a 3.582±0.169 b 10.852±0.273 a 9.283±0.195 b 9.356±00.160 b 9.116±0.182 b 11.340±0.220 a
Zn 4.150±0.403 b 5.927±0.161 a 54.052±2.503 a 35.141±0.173 b 22.034±0.023 b 21.201±0.374 b 28.408±0.245 a
Ni 2.524±0.215 a 1.189±0.047 b 4.000±0.126 a 2.709±0.069 b 3.196±0.094 b 3.004±0.133 b 4.822±0.179 a

Data are means±SE of five replications

Mean values with different letters in a single agricultural product category are significantly different according to Duncan’s multiple range
test (P<0.05)
Numbers in parenthesis indicate the period in years in which wastewater irrigation was practiced in area III
MC moisture content
Environ Monit Assess (2014) 186:4857–4870 4867

0.1258±0.0038 b

Data are means±SE of five replications; Mean values with different letters in a single agricultural product category are significantly different according to Duncan’s multiple range test
Table 7 Heavy metal transfer factor (TF) from the topsoil (0–25 cm) of citrus orchards and ryegrass and clover fields in the three surveyed areas irrigated with wastewater for different

0.0757±0.0019 a
0.0697±0.0075 a

0.0465±0.0519 c
0.1033±0.0074 a
comparison to those grown in area II, might be attribut-
ed to the fact that plants in area I reached the ripening
stage earlier than those in area II. The heavy metals

III (8)
concentrations observed in the ryegrass above-ground
parts were in the order of Mn > Zn > Cu > Ni > Co.
Variations in heavy metal content were also registered in
clover aerial parts sampled in area III, irrigated for

0.1223±0.0062 b
0.0694±0.0051 b
0.0449±0.0014 c
0.0613±0.0094 a
0.1829±0.0101 a
different periods of time with wastewater. The concen-
tration of Cu, Zn, and Ni was higher in the clover above-
ground parts sampled in the field irrigated with waste-

III (4)
water for 8 years, as compared to the representative
samples taken from fields irrigated for 0.5 and 4 years,

Clover stems and leafs

while that of Mn was lower. Otherwise, no changes were

0.0635±0.0027 b

0.0725±0.0032 b
0.0691±0.0132 a
0.1001±0.0015 c
0.1650±0.0096 a
recorded regarding the Co content in the clover stems
and leaves, regardless of the period of wastewater irri-
gation (Table 6). In a previous study, Rattan et al. (2005)

(P<0.05). Numbers in parenthesis indicate the period in years in which wastewater irrigation was practiced in sampled area III
III (0.5)
reported similar values for Zn in wastewater irrigated
sorghum (Sorghum vulgare Pers.) and oats (Avena sativa
L), as well as for Cu in wheat (Triticum aestivum L.) and
oats, while higher Ni and lower Mn values were found

0.0267±0.0009 b
0.0230±0.0022 b

0.0452±0.0007 b
0.1479±0.0056 a
0.4163±0.0055 a
periods of time to the orange fruit pulp and ryegrass and clover plants above-ground parts (in dry weight basis)
in the same crops. Additionally, higher values of Zn, Cu,
and Ni and lower values of Mn were reported in maize
(Zea mays L.) and Egyptian clover (Trifolium
alexandrinum L.) in the same work. Similar observa-
II
Ryegrass stems and leafs

tions were also documented in wastewater-irrigated sor-

ghum (Yadav et al. 2002). Noteworthy, the heavy metal
0.1189±0.0054 b
0.0463±0.0007 a
0.0768±0.0156 a

0.4530±0.0353 a
0.0667±0.0009 a
content observed in the above-ground parts of forage
plants in all surveyed areas was below the critical limits
of phytotoxicity, as defined by Macnicol and Beckett
(1985). Also, the Zn and Cu contents in the aerial parts
I

of both ryegrass and clover plants, used as dairy feed,

were within the typical ranges reported in the European
Union (European Commission 2003) (500 and
0.0459±0.0036 b
0.0026±0.0001 b

0.0144±0.0001 b
0.0518±0.0029 a
0.0848±0.0034 a

100 mg kg−1 of dry mass for Zn and Cu, respectively).

Heavy metal transfer factor (TF) from the topsoil

to agricultural products
II

The TF calculated for the ryegrass aerial parts was

0.0417±0.0001 b
0.0281±0.0038 b
0.1124±0.0027 a
0.0043±0.0001 a

0.0542±0.0048 a
Orange fruit pulp

higher than that of orange fruit pulp, with regard to

Mn, Cu, and Zn, while the TF calculated for the Co
exhibited an opposite trend. Variations between the two
plant species regarding the TF for Ni were less pro-
I

nounced. Similar variations concerning the TF were

registered regarding the period of wastewater irrigation
Sampled area

in clover plants above-ground parts in area III. More

precisely, the TF for Mn and Ni was higher in the clover
field irrigated with wastewater for 8 years, whereas TF
Crop

Mn
Co
Cu
Zn
Ni

for Cu and Zn was found to be higher in the fields

4868
irrigated with wastewater for 4 and 0.5 years, respec-
tively (Table 7). The TF, being recognized as an indica-
tor of heavy metal mobility and phytoavailability in soil,
is affected by various parameters, like the type of heavy
metal, the soil properties (texture, pH, temperature, or-
ganic matter), and the plant species (Lin et al. 2008;
Kalavrouziotis et al. 2012). The soil in the examined
areas is alkaline (pH ranges from 8 to 8.80) with high
CaCO3 (ranging from 6 to 50 %) and clay content
(ranging from 16 to 30 %), which in turn facilitates the
adsorption of heavy metals to soil particles, especially
clay, and the precipitation of metal hydroxides and
carbonates, or the formation of insoluble organic com-
plexes (Smith et al. 1996). The TF values calculated for
both ryegrass and clover aerial parts were lower to those
previously reported in forage crops (maize, sorghum,
oats, and clover) irrigated in the long term with waste-
water (Rattan et al. 2005; Khan et al. 2008). These
results indicate that the accumulation of heavy metal
due to wastewater irrigation to the agricultural products
in the studied sites is unlikely to reach undesirable levels
for public health safety.
Conclusions
Wastewater irrigation of forage crops and orange or-
chards is of pivotal importance for the Cyprus agricul-
tural sector, since water shortages restrict any other
water resource for being applied for this purpose.
Wastewater irrigation of ryegrass fields and orange or-
chards in areas I and II caused no significant modula-
tions in soil geochemical properties and heavy metal
content, except for an increase of soil EC by an average
equal or lower than 0.98 mS cm−1, which could not
affect soil sustainability and ryegrass plants and orange
trees productivity in the long term. On the other hand,
wastewater irrigation of clover fields modified some of
the soils’ physicochemical properties evaluated in the
surveyed area III, as compared to the adjacent neighbor-
ing rainfed fields. The most pronounced impact of
wastewater irrigation was the salinization of the irrigated
sites, since soil EC and Cl− content increased consider-
ably. Soil salinization in area III may pose serious threats
to clover crop production and economic sustainability,
once clover is considered as a moderately sensitive
forage crop. Area III should be considered as a special
case that needs to be handled properly, since the sealing
of the sewage collection network could alleviate treated
Environ Monit Assess (2014) 186:4857–4870
wastewater’s salinity and allows the long-term waste-
water irrigation of forage crops without considerable
economic losses. In addition, the low heavy metal
phytoavailability, due to the soil properties of the sur-
veyed sites, appears to ensure agricultural products’
safety. This statement is further corroborated by the
low heavy metal content quantified in the agricultural
product samples, complied with the MPLs set, as well as
by the low TF estimated. Overall, proper management
and monitoring are required in order to ensure the long-
term environmental sustainability and public health
safety of wastewater irrigation in Cyprus.
Acknowledgments The authors wish to thank all the personnel
of the Cyprus Agricultural Research Institute involved in the study
for their excellent technical assistance. This study was undertaken
in collaboration with Nireas-International Water Research Center
of the University of Cyprus and supported by Cyprus Agricultural
Research Institute Internal Grant to Anastasis Christou.
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