Water Air Soil Pollut (2013) 225:1791

DOI 10.1007/s11270-013-1791-y

Occurrence of Micro-pollutants in a SoilRadish System

Irrigated with Several Types of Treated Domestic Wastewater
I. Petousi & M. S. Fountoulakis & N. Tzortzakis &
S. Dokianakis & E. I. Stentiford & T. Manios

Received: 2 August 2013 / Accepted: 17 October 2013

# Springer Science+Business Media Dordrecht 2013

Abstract During this study, the effect of applying

several types of treated domestic wastewater on the
translocation and accumulation of organic and inorganic
micropollutants in soil and radish plants (Raphanus
sativus L.) was examined. Primary (PTW), secondary
(STW) and tertiary (TTW) treated wastewater as well as
tap water (TW) were used for the irrigation of radish
plants for a period (transplantating and harvesting) of
67 days. Higher concentrations of polycyclic aromatic
hydrocarbons (PAHs) were observed in soils irrigated
with PTW. The concentration of PAHs in radish roots
ranged between 107.612.1 g/kg for plants irrigated
with TTW and 124.117.7 g/kg for plants irrigated
with PTW. The root concentration factors (RCFs)
expressed as the ratio of PAH concentration in the root
mass (dry weight) to the residual concentration in the
soil varied from 1.6 to 1.9 indicating a higher
accumulation of PAHs in the edible part of radishes than
soil. Heavy metals were not detected in the wastewaters
I. Petousi (*) : M. S. Fountoulakis : S. Dokianakis :
T. Manios
School of Agricultural Technology, Technological
Educational Institute of Crete,
Heraklion 71004, Greece
e-mail: ioannapetousi@staff.teicrete.gr
I. Petousi : E. I. Stentiford
Department of Civil Engineering, University of Leeds,
Leeds LS2 9JT, UK
N. Tzortzakis
Department of Agricultural Sciences, Biotechnology and
Food Science, Cyprus University of Technology,
Limassol 3603, Cyprus

utilised and, as a result, no accumulation was found in

either the soil or plants in comparison with tap water.
RCFs for heavy metals were calculated between 0.91 and
0.99, 0.49 and 0.66, 0.004 and 0.005 for Cu, Zn and Ni,
respectively. The results showed that radishes have the
ability to concentrate PAHs when they are present in the
wastewater and this could have associated health risks.
Keywords Polycyclic aromatic hydrocarbons . Heavy
metals . Macroelements . Soil pollution . Irrigation

1 Introduction
In recent years, the use of marginal quality water for
crop irrigation has gained increased importance in
water-scarce regions. One of the major types of marginal
quality water is wastewater, which has been reused in
agriculture for centuries; however, the amount recycled
has greatly increased in the last decade (Bhogal et al.
2003; Kalavrouziotis et al. 2008; Qadir et al. 2010). The
types of wastewater used for recycling into agriculture
include primary (Zabalaga et al. 2007), secondary
(Pedrero and Alarcn 2009) and tertiary treated
wastewater (Pollice et al. 2004).
In general, treated wastewater reuse has several
advantages which include: (a) a reduced amount of fresh
water being used for irrigation; (b) an improvement in
farm productivity in water-constrained systems
(Raschid-Sally et al. 2005; Huibers and Van Lier
2005); (c) a reduced discharge of nutrients into surface
waters; and (d) a decrease in the cost of wastewater
treatment by eliminating the need for nutrient removal

1791, Page 2 of 8

(Rosenqvist et al. 1997). At the same time, with the

increasing reuse of wastewater there is an increasing
concern regarding the fate of micro-pollutants in the
treated wastewater (Song et al. 2006; Fatta-Kassinos
et al. 2011). It is well known that domestic wastewater
contains persistent organic and inorganic pollutants
including heavy metals, nutrients, polycyclic aromatic
hydrocarbons (PAHs), polychlorinated biphenyls, and
pthalic acid esters (Voutsa and Samara 1998; Mapanda
et al. 2005; Kalavrouziotis et al. 2012).
Nowadays, PAHs are of particular interest because: (a)
they are persistent pollutants in the environment; (b) they
have the lowest metabolism and degradability by most
living organisms; and (c) due to their low water solubility
and high hydrophobicity, PAHs are adsorbed onto solid
particles. The main sources for PAHs in sewers are
municipal and industrial wastes, rainfall and storm runoff
waters, particularly from road surfaces (Mangas et al.
1998). Several PAHs have been shown to be potentially
highly carcinogenic and mutagenic (Blanchard et al.
1999) and, as a result, have been listed by regulatory
agencies such as the USEPA as top priority pollutants.
Vegetables grown in soils irrigated with wastewater
may take up PAHs in sufficient quantities to cause
negative effects on consumers. The buildup of PAHs
in plants depends on soil concentrations, plant species,
and microbial population (Kapusta 2004). Previous
works reported the accumulation of high concentrations
of PAHs in plants cultivated on PAH-contaminated soils
(Zohair et al. 2006; Khan et al. 2008; Cai et al. 2008).
Different mechanisms may be responsible for the tranfer
of organic contaminants from soil to plants, including
sorption, uptake through transpiration or volatilization
and subsequent deposition on leaves (Wild et al. 2004).
The accumulation of inorganic contaminants in
wastewater depends on many local factors such as the
type of industries in the area, the traffic environment,
building materials and people's ways of life (Sorme and
Legerkvist 2002). The occurrence of these macro- and
microelements in treated wastewater may have adverse
effects on human health if they accumulate in the edible
part of plants (Mapanda et al. 2005). Indeed, previously
studies have found significant concentrations of heavy
metals in vegetables grown in soils irrigated with
wastewater (Rattan et al. 2005; Kalavrouziotis et al.
2008; Khan et al. 2008; Gupta et al. 2010).
The goal of the present study was to investigate the
possible accumulation of PAHs and inorganic pollutants
(macro- and microelements) in the soil and radish roots

Water Air Soil Pollut (2013) 225:1791

following the application of three different qualities of

treated wastewater: (a) primary (following primary
sedimentation), (b) secondary (following secondary
sedimentation after a treatment with compact packed
bed biofilter), and (c) tertiary (following sand filtration
and a chlorination process). While the accumulation of
PAHs has been studied, as stated above, in relation to
PAH-contaminated soils (sewage sludge deposition or
long-term irrigation), it has not been examined in
relation to wastewater quality. Furthermore, to our
knowledge there are no previous studies for either PAHs
or inorganic contaminants comparing the effect of
wastewater treatment on the fate of these pollutants in
soil and plant tissues.

2 Materials and Methods

2.1 Chemical Analysis
Water samples were analyzed for chemical oxygen
demand (COD) using the closed reflux colorimetric
method, and for total suspended solids (TSS) by the
glass fibre method, while pH was measured with a pH
meter (WTW, 3110) and electrical conductivity (EC)
with a conductimeter (Hanna, 8333) in accordance with
Standard Methods (APHA 1995). Total nitrogen (TN)
and total phosphorus (TP) were determined
spectrophotometrically by use of standard test kits
(Hach-Lange). The analysis of macroelements (K, Ca,
Mg), boron (B) and heavy metals (Cr, Cu, Ni, Zn) in
water, soil and plant samples was carried out by
inductively coupled plasma mass spectroscopy (ICPMS-Agilent 7500-CX). PAHs were recovered from
water samples using liquidliquid extraction with
hexane as an extraction solvent, as described by Manoli
and Samara (1996). For soil and plant samples, the
extraction of PAHs was performed according to USEPA
method 3541 (USEPA 1994) with some modifications.
Dried plant and soil samples were transferred into
precleaned cellulose extraction thimbles and extracted
with 50 ml of acetone/hexane (1:1) by a Soxhlet system
(SER148, Velp Scientific) for 2 h. Chysene-d12 was
used as the internal standard solution. All extracts were
filtered through 0.45-m PTFE membrane filters and
analyzed using High Pressure Liquid Chromatography
(HPLC-Agilent 1200 Series) with programmable
Fluorescence Detection by injecting 20 l into the
system. In total, the PAHs determined were: fluorene

Water Air Soil Pollut (2013) 225:1791

Page 3 of 8, 1791

(Fl), phenanthrene (Phe), fluoranthene (Flu), anthracene

(Ant), pyrene (Pyr), chrysene (Chr), benzo(a)anthracene
(Baa), benzo(a)pyrene (Bap), benzo(b)fluoranthene
(Bbf) and benzo(k)fluoranthene (Bkf).
2.2 Plant Cultivation
The experiment was carried out in greenhouse, at the
School of Agricultural Technology at the Technological
Educational Institute of Crete, Greece, during the winter
period, using 32 experimental white polyurethane
containers (width: 39 cm, length: 49 cm, height:
20 cm). Twenty seeds of radish (Raphanus sativus L.)
were sown in each container and after seed

germination/emergence the seedlings were thinned until

four uniform seedlings were established in each
container. The substrate used for the experiment was
local soil, mixed thoroughly and passed through a 5-mm
sieve. The treatments consisted of the control (tap water)
and the three qualities of treated wastewater (primary,
secondary and tertiary). Eight replicate containers each
containing four plants were used for every treatment.
Plants were grown over 67 days (from 8 November to
14 January) and harvested at the end of the experiment,
when soil samples were collected from each pot.
Overall, tap water and treated wastewaters were applied
13 times during the growth period at a rate of 1 l per pot
at each irrigation, or 680 mm/ha.

Table 1 Physicochemical characteristics of several types of water sources used in the experiment
Parameter

Tap water (control)

Primary treated wastewater

Secondary treated wastewater

Tertiary treated wastewater

Mean

SD

Mean

SD

Mean

SD

Mean

SD

pH

7.8

0.3

7.3

0.2

7.5

0.2

7.5

0.2

EC (mS/cm)

0.7

0.3

1.6

0.3

1.2

0.4

1.1

0.3

COD (mg/l)

16

12

224

87

27

17

24

14

TSS (mg/l)

2.6

0.7

37.0

20

9.2

4.2

9.1

3.9

TN (mg/l)

4.7

1.1

64

12

22

13

20

13

TP (mg/l)

0.2

0.1

11.4

2.1

8.2

0.9

8.0

0.8
0.6

B (g/l)

16.4

1.2

250.1

1.6

254.6

1.8

251.2

Mg (mg/l)

19.1

5.1

50.5

1.0

52.9

1.6

51.2

1.3

Ca (mg/l)

60.5

5.8

120.1

1.5

119.1

2.2

123.0

0.4

K (mg/l)

n.d

27.2

1.0

26.6

1.9

28.5

1.5

10PAHs (g/l)

n.d

2.10

1.65

0.84

0.65

0.81

0.37

Fl

n.d

0.27

0.13

0.1

0.08

0.07

0.11

Phe

n.d

0.93

0.47

0.37

0.21

0.36

0.07

Ant

n.d

0.07

0.09

0.03

0.06

0.03

0.09

Flu

n.d

0.63

0.28

0.26

0.14

0.28

0.10

Pyr

n.d

0.14

0.08

0.05

0.13

0.05

0.07

Baa

n.d

0.04

0.12

0.01

0.08

0.01

0.04

Chr

n.d

0.02

0.09

0.01

0.07

0.01

0.04

Bbf

n.d

n.d

n.d

n.d

Bkf

n.d

n.d

n.d

n.d

Bap

n.d

n.d

n.d

n.d

Heavy metals (g/l)

Cr

n.d

n.d

n.d

n.d

Cu

n.d

1.4

1.1

n.d

n.d

Ni

n.d

n.d

n.d

n.d

Zn

n.d

27.5

17.2

7.2

5.3

7.0

6.1

SD standard deviation, n.d. not detected

Water Air Soil Pollut (2013) 225:1791

physicochemical characteristics of the treated

wastewaters used in the experiment are summarized in
Table 1.

Roots
Shoots

-1

Mean dry weight (g plant )

1791, Page 4 of 8

6
5

2.4 Data Analysis

Statistical analyses were peformed with MicroCal

Origin 7.0 (OriginLab). The data were analyzed through
one-way analysis of variance (ANOVA) to compare the
effect of each irrigation type on the concentration levels
of PAHs, heavy metals and macroelements in soils and
radish roots. To detect the statistical significance of
differences (p<0.05) between means of treatments, the
Tukey test was performed.

3
2
1
0
TW

PTW

STW

TTW

Irrigation type
Fig. 1 Plant biomass (dry mass of roots and shoots) of Raphanus
sativus at the end of experiment (mean values and standard errors).
TW tap water, PTW primary treated wastewater, STW secondary
treated wastewater, TTW tertiary treated wastewater

3 Results and Discussion

3.1 Plants

2.3 Wastewater Treatment

Figure 1 shows the dry mass of radish roots and shoots
for the four treatments at the end of the experiment.
Radishes irrigated with tap water had a mean dry mass
production of 3.20.8 g for roots and 1.60.3 g for
shoots. All types of wastewater used showed a similar
effect on dry mass production of roots, with values of
4.70.8, 4.20.7, and 4.41.0 g for primary, secondary
and tertiary treated wastewater, respectively. The
irrigation with treated wastewater seems to increase the

Primary treated wastewater was obtained from the

sewage treatment plant of Heraklion (180,000 p.e.),
Crete, Greece. This primary wastewater was further
treated using a compact packed bed biofilter
(Advantex-AX20, Orenco) to produce the secondary
treated wastewater. Tertiary wastewater was obtained
by treating the effluent of the packed bed filter using a
sand filter and a chlorination process. The

Table 2 Concentrations of individual PAH and PAHs (sum of ten PAHs) in the soils at the end of experiment
PAH

Application of TW (control)

Application of PTW

Application of STW

Application of TTW

Mean

SD

Mean

SD

Mean

SD

Mean

SD
0.48

Fl

0.40

0.34

1.16

0.08

0.44

0.28

0.36

Phe

12.46

0.96

20.42

5.58

16.58

1.1

15.68

0.96

Ant

0.04

0.04

0.26

0.32

0.04

0.04

0.04

0.02

Flu

14.20

0.56

15.60

2.62

14.28

0.62

14.38

0.38

Pyr

30.64

2.44

32.38

0.04

31.02

2.68

30.82

2.34

Baa

0.18

0.12

1.02

0.78

0.22

0.1

0.16

0.22

Chr

0.42

0.12

0.9

0.44

0.54

0.28

0.56

0.38

Bbf

0.42

0.16

1.76

1.34

0.48

0.24

0.4

0.24

Bkf

n.d

0.15

0.13

n.d

n.d

Bap

2.24

0.44

3.90

1.60

2.34

0.62

2.46

1.02

10 PAHs

61.02

5.18

77.72

13.28

65.94

5.96

64.86

6.04

SD standard deviation, n.d. not detected; unit: g/kg

Water Air Soil Pollut (2013) 225:1791

3.2 PAHs
Table 2 shows the individual PAH concentrations in the
soils irrigated with the different types of water and
wastewater at the end of the experiment. The soil used
for the experiment was found to be contaminated with
elevated PAH concentrations. PAH levels have been
previously reported in soils from urban sites mainly
due to traffic (Papageorgopoulou et al. 1999). Nadal
et al. (2004) found a median concentration of 37
27 g/kg in unpolluted sites from Tarragona County,
Spain.
The most abundant PAHs investigated were
phenanthrene, fluoranthene and pyrene. These three
compounds accounted for 93 % of the total examined
PAHs. Comparing the results of the present work with
other studies into PAH distributions in anthropogenically
contaminated soils, it is apparent that the trends are very
similar. Generally, the medium-molecular-weight
PAHs (e.g., fluoranthene and pyrene) are present in
the greatest quantities (Nadal et al. 2004; Morillo
et al. 2007). Total PAH concentrations were found
to be slightly higher for soils irrigated with secondary
and tertiary wastewater compared with the control
soil. However, significantly higher concentrations of
PAHs were observed in the soil irrigated with
primary wastewater. As expected, soil contamination
was correlated with the PAH levels in the irrigation
water or wastewater.
The concentrations of PAHs in radish roots at the end
of the experiment showed substantial variation, from
non-detectable to 124.06 g/kg (Fig. 2). Radish roots
were enriched with low and medium molecular weight
PAHs such as Fl, Phe, Flu and Pyr. Slightly higher
values for total PAHs were observed in the radish roots
irrigated with primary treated wastewater. The results
indicated that the PAH concentrations in the roots were
correlated with the soil concentrations (high PAH
concentration in the soil resulting in high PAH
concentration in the root).
Cai et al. (2008) examined the accumulation of PAHs
in R. sativus after sewage sludge and compost
application. They found that at a concentration of
107 g/kg of total PAHs in sewage sludge amended
soil, the accumulation of total PAHs in radish roots

160

PAHs concentration (g/kg)

dry mass production compared with tap water; however,

one-way ANOVA analysis (Tukey test) showed no
significant difference.

Page 5 of 8, 1791
Fl
Ph

Ant
Flu

Pyr
B(a)An

B(a)Py

120

80

40

0
TW

TTW

STW

PTW

Irrigation type
Fig. 2 The mean concentrations of PAHs detected in radish roots
(Raphanus sativus) on a dry weight basis. TW tap water, TTW
tertiary treated wastewater, STW secondary treated wastewater,
PTW primary treated wastewater

was 104 g/kg. The results observed in this experiment

show a slightly higher accumulation of PAHs in radish
roots (107124 g/kg) compared with the concentration
of PAHs in the soil (6177 g/kg). The maximum limits
for PAHs in foods vary from country to country and
many countries have not established acceptable limits in
either soil or vegetables. Current EU legislation (2005)
sets maximum allowed concentrations for BaP in
various food products (not including vegetables) in the
110 g/kg wet weight range.
Table 3 Root Concentration factors of PAHs in the treated
radishes
PAHs

Irrigation type
Tap
water

Primary
treated
wastewater

Secondary
treated
wastewater

Tertiary
treated
wastewater

Fl

9.3

3.2

8.3

9.8

Phe

2.6

2.3

2.3

2.1

Ant

24.4

2.6

17.1

16.1

Flu

1.4

1.3

1.4

1.4

Pyr

1.7

1.6

1.6

1.5

Baa

1.5

0.2

15.7

22.7

Chr

<0.1

<0.1

<0.1

<0.1

Bbf

<0.1

<0.1

<0.1

<0.1

Bkf

<0.1

Bap

1.5

0.8

1.3

1.3

10PAHs

1.9

1.6

1.8

1.7

1791, Page 6 of 8
Table 4 Heavy metal and macronutrient concentrations in the soils
at the end of experiment (mean
values and standard deviations)

Water Air Soil Pollut (2013) 225:1791

Parameter

Application of
TW (control)

n.d. not detected

Application
of STW

Application
of TTW

Cr (mg/kg)

80.47.3

84.11.9

76.57.4

80.17.2

Cu (mg/kg)

26.72.3

25.99.3

27.09.6

24.83.1

Ni (mg/kg)

74.77.0

71.25.6

74.87.4

75.79.4

Zn (mg/kg)

32.35.2

31.68.3

26.33.0

35.48.2

B (mg/kg)

5.11.5

7.61.6

6.31.5

5.81.6

Mg (g/kg)

11.71.0

11.82.9

11.31.2

10.82.1

Ca (g/kg)

142.713.6

146.64.9

143.113.6

141.612.1

K (g/kg)

1.50.1

1.60.3

1.60.4

The root concentration factors (RCFs) expressed as

the ratio of PAH concentration in the mass root (dry
weight) to the residual concentration in the soil are
presented in Table 3. These factors are often used to
determine contaminant concentrations in plants because
soil-to-plant transfer is one of the major pathways for
pollutants to enter the food chain (Khan and Cao 2011).
It was found that the accumulations in radish roots were
higher for low-molecular-weight PAHs. Wang et al.
(2011) examined PAH concentrations in roots and
shoots of six vegetables from wastewater irrigated areas
in China, and found the highest PAH concentration in
radish roots and higher RCFs for acenaphtene, fluorene
and phenanthrene. In general, the RCFs depend on the
type of vegetable, PAH concentration in soil and PAH
solubility, as well as the physicochemical properties of
the soil. The RCF values found in this study were higher
than those previously reported (Cai et al. 2008; Khan
and Cao 2011; Wang et al. 2011). In the containers the
soil was watered to almost 100 % water holding
capacity, and as a result the mass transfer
(bioavailabilty) issues that would be present in open

Table 5 Root dry matter heavy

metal and macro-nutrient content
at the end of experiment (mean
values and standard deviations)

Application
of PTW

1.60.6

field soils were partly reduced. It is reported that

hydroponic systems remove mass transfer limitations,
resulting in higher heavy metal accumulation in plants,
and a similar process could have occurred in the pots
with fully wetted soil (January et al. 2008).
3.3 Macro- and Microelements
There was no significant effect on macro- and micro
nutrients or heavy metal content in soils irrigated with
the three different qualities of treated wastewater and tap
water (Table 4). The Ca and Mg concentration in
wastewaters was approximately double (120 and
50 mg/l, respectively) that of tap water (60.5 and
19.1 mg/l, respectively) while no differences was found
in soil content. Kalavrouziotis et al. (2008) found that
Ca concentration decreased and Mg concentration
increased in soil irrigated with treated wastewater in
comparison with soil irrigated with tap water. In that
case, the wastewater:water concentration ratio was
slightly lower for Ca (90 mg/l in wastewater and
49 mg/l in tap water) and significantly higher (21 mg/l

Parameter

Application of
TW (control)

Application
of PTW

Application
of STW

Application
of TTW

Cr (mg/kg)

n.d

n.d

n.d

n.d

Cu (mg/kg)

25.18.7

23.55.6

26.84.6

23.94.2

Ni (mg/kg)

0.30.2

0.30.2

0.40.3

0.40.3

Zn (mg/kg)

19.28.3

18.77.5

17.45.0

17.39.2

B (mg/kg)

23.87.7

26.211.4

20.75.2

22.81.8

Mg (g/kg)

1.50.3

1.40.3

1.60.3

1.80.2

Ca (g/kg)

5.81.4

5.40.8

6.51.0

7.42.2

K (g/kg)

28.38.2

26.91.4

26.42.2

31.71.2

Water Air Soil Pollut (2013) 225:1791

in wastewater and 4.2 mg/l in tap water) for Mg. Heavy

metal content was not significantly different for the
different treatments. The treated wastewaters used for
the experiment were not polluted with heavy metals, as
shown in Table 1, with the exception of Zn. The results
show that the other characteristics of water and
wastewater (pH, EC, organic content) did not affect
the heavy metal concentration in the irrigated soils.
Boron concentration in soil irrigated with primary
treated wastewater was 7.71.6 mg/kg, while the soil
irrigated with tap water had a boron concentration of 5.1
1.5 mg/kg.
The data presented in Table 5 show the comparative
macro- and micro-nutrients and heavy metal
composition of roots irrigated with three different
qualities of wastewater and tap water. There were no
significant differences in the inorganic composition of
radishes. The calculated RCFs for the examined heavy
metals were similar for every treatment used. Higher
RCFs were observed for Cu (0.95) and Zn (0.58), while
Ni and Cr did not tranfer from the soil to the roots. The
ratio of metals between soil and roots (RCFs) may be
affected by several factors, such as the type of heavy
metal, soil, temperature, pH, organic matter and plant
species (Antoniadis and Alloway 2001; Kachenko and
Singh 2006; Kalavrouziotis et al. 2012).

4 Conclusions
The effect of irrigation with different types of treated
wastewater on organic (PAHs) and inorganic (nutrients
and heavy metals) pollutants content in a soilradish
system was examined during this study. No significant
effect on the macro and micro-nutrient status of the
edible part of radishes was observed for any type of
application. On the other hand, results show that PAHs
were taken up by radish from soils. PAH compounds
have been reported as highly carcinogenic and
mutagenic, thus their presence in food is of great
concern and intakes should be as low as reasonably
practicable. Application of primary treated wastewater
could lead to accumulation of PAHs in soil and radish
roots. The concentration of total PAHs in the roots was
positively correlated with the concentration of total
PAHs in the soil. The most abundant PAHs were
phenanthrene, fluoranthene and pyrene, both in the soil
and in radish roots. Comparing the calculated
bioaccumulation factors with those estimated in the past

Page 7 of 8, 1791

for other vegetables, it was concluded that radish has a

higher potential health risk as a result of contamination
by PAHs.
Acknowledgments This research was co-funded by the LIFE
project ENV/GR/00551.

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