Environmental Pollution 132 (2004) 21e27

www.elsevier.com/locate/envpol

Phytoextraction of heavy metals by canola (Brassica

napus) and radish (Raphanus sativus) grown
on multicontaminated soil
L. Marchiol*, S. Assolari, P. Sacco, G. Zerbi
Department of Agriculture and Environmental Science, University of Udine, Via delle Science 208, 33100 Udine, Italy
Received 14 October 2003; accepted 2 April 2004

‘‘Capsule’’: As anthropogenic soil pollution is usually not limited to single pollutants, plants potentially
suited for phytoremediation should be observed in multicontaminated substrates.

Abstract

Phytoextraction can provide an effective in situ technique for removing heavy metals from polluted soils. The experiment
reported in this paper was undertaken to study the basic potential of phytoextraction of Brassica napus (canola) and Raphanus
sativus (radish) grown on a multi-metal contaminated soil in the framework of a pot-experiment. Chlorophyll contents and gas
exchanges were measured during the experiment; the heavy metal phytoextraction efficiency of canola and radish were also
determined and the phytoextraction coefficient for each metal calculated. Data indicated that both species are moderately tolerant to
heavy metals and that radish is more so than canola. These species showed relatively low phytoremediation potential of
multicontaminated soils. They could possibly be used with success in marginally polluted soils where their growth would not be
impaired and the extraction of heavy metals could be maintained at satisfying levels.
Ó 2004 Elsevier Ltd. All rights reserved.

Keywords: Soil pollution; Heavy metals; Phytoremediation; Phytoextraction

1. Introduction 1995; Chaney et al., 1997; Cunningham and Ow, 1996).

Human activities release pollutants in the environ-
ment; heavy metals, in particular, originate from indus-
trial emissions, mining activity, disposal of wastes and
fertilizer and pesticide use. Environmental restoration of
metal polluted soils by traditional physical and chemical
methods demands large investments of economic and
technological resources (Susarla et al., 2002): methods
based on plants have consequently gained a good deal of
attention.
Phytoremediation refers to the use of green plants,
soil amendments and agronomic techniques to remove,
contain or render the pollutants harmless (Salt et al.,
* Corresponding author. Fax: C39-432-558603.
E-mail address: marchiol@uniud.it (L. Marchiol).
URL: http://www.dpvta.uniud.it.
Phytoextraction refers to the use of pollutant-accumu-
lating plants that can extract and translocate contam-
inants to the harvestable parts; this option is only viable
in sites without heavy contamination.
Recently, the coupling of phytoextraction with other
soil treatments (e.g. soil washing, soil vapor extraction)
in so-called ‘‘treatment trains’’ is gaining interest; this
may be especially useful in cases where mixed conta-
minants necessitate the use of more than one technique
to effectively remediate sites (Roote, 2003).
The ideal plant for use in phytoextraction should
have the following traits: (i) ability to accumulate the
metal(s) intended to be extracted, preferably in the
aboveground parts; (ii) tolerance to high metal concen-
trations in soils; (iii) fast growth and high accumulating
biomass; (iv) easily grown as an agricultural crop and
fully harvestable.

0269-7491/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envpol.2004.04.001
22 L. Marchiol et al. / Environmental Pollution 132 (2004) 21e27

For phytoextraction to be practical we need to couple Data on the biomass growth of these species and the
a high biomass and a very high metal accumulation in basic carbon dioxide assimilation parameters are re-
the plant tissues (Chaney et al., 1998). Unfortunately, no ported. To test the phytoextraction potential of these
wild plants or crops have yet been identified that com- species, the heavy metal concentrations in the plant
bine these properties (Li et al., 2003). parts were determined and the bioaccumulation coef-
Most studies on phytoextraction have been based on ficients calculated for each metal.
hydroponic experiments, which have increased the un-
derstanding of metal uptake by hyperaccumulator, ac-
cumulator and tolerant plants (Brown et al., 1995;
2. Materials and methods
Pollard and Baker, 1996; Salt et al., 1997; Haag-Kerwer
et al., 1999; Wenzel et al., 1999; Lasat, 2000; Zhao et al.,
The contaminated soil used in the experiment was
2000). This type of experiment has also been important
sampled in a farming area near Carpiano (Milan, Italy);
for determining the uptake efficiency and metal toler-
the pollution source had been identified as bad quality
ance of potential phytoremediation species.
irrigation water being used for more than a century.
Anthropogenic soil pollution is usually not limited to
Previous experiments to screen the most tolerant vari-
a single pollutant; several metals may be present at high
eties among different accessions of B. napus and R.
concentration in soils. A limited amount of research has
sativus showed that B. napus cv Kabel and R. sativus cv
been dedicated to describing the behavior of accumu-
Rimbo were the most resistant cultivars among those
lating plants in these conditions even if this is the most
tested, so these were used for the experiment.
frequent case in soil restoration. These studies involve
Preliminary tests on polluted soil indicated that both
serious difficulties as plant heavy metal uptake is sub-
canola (B. napus) and radish (R. sativus) performed
jected to the antagonistic, additive and synergetic effects
poorly when sown in pots containing this soil; 2 weeks
that heavy metals exert on one another (Grifferty and
after germination the seedlings of both species appeared
Barrington, 2000; Baker, 2000).
stunted as compared to controls grown on a standard
However, some interesting papers have recently been
soil, and their leaves were a purple color with necrotic
published dealing with in situ phytoextraction experi-
areas. After a few days most of them died.
ments and multicontaminated soil. Keller et al. (2003)
A milder substrate was prepared by mixing this soil
pointed out the importance of choosing plant species
with sand in a 1:1 (w/w) proportion. An uncontaminated
according to depth and heterogeneity of localisation of
agricultural soil (Typic Udifluvent) collected from the
the pollution. Perronn et et al. (2003) reporting the dis-
Experimental Farm of Udine University was used to fill
tribution of Cd and Zn in the hyperaccumulator Tlaspi
control pots. Physical and chemical characteristics of
caerulescens in different growth stages, showed that the
control soil and polluted substrate are reported in Table 1.
concentration of the metals varied according to the
Seeds of B. napus cv Kabel and R. sativus cv Rimbo
organ and plant age; Zn concentration in shoots de-
were sown in 2 l plastic pots containing the control soil
creased with time while Cd concentration remained con-
and the soilesand mixture. To prevent emergence fail-
stant, despite an increase in biomass. Also, in old plants,
ures, more seeds were sown in each pot than the number
the Cd concentration was higher in the youngest leaves.
The development of large-scale phytoextraction tech- Table 1
niques could consider crop species as bioaccumulators Physical and chemical characteristics of control soil and polluted
of heavy metals; in fact, some of them can accumulate substrate
heavy metals while producing high biomass in response Parameter Control Polluted
to established agricultural management (Ebbs and substrate
Kochian, 1997). Particle size Sand 36.8 78.0
This approach has been followed by Huang et al. distribution (%) Silt 35.5 17.4
(1997) and Burke et al. (2000) on maize, by Shahandeh Clay 27.6 4.6
and Hossner (2000) on sunflower, and by Linger et al. pH 7.3 6.2
(2002) on hemp; fast growing trees such as Salix spp. Organic C g kg
1 17 40
(Punshon and Dickinson, 1999; Mottier et al., 2000; Total N g kg
1 30 3.7
Extr. P P2O5 Olsen (mg kg
1) 40 78
Klang-Westin and Perttu, 2002) and Populus spp. EC dS m
1 0.13 0.48
(Shannon et al., 1999; Banũelos et al., 1999; Vose CEC cmol(C) kg
1 17.2 14.6
et al., 2000) have also been tested.
Cd mg kg
1 0.90 38.6
The experiment reported in this paper was under- Cr mg kg
1 18.2 165
taken to study the behavior of two Brassica speciesd Cu mg kg
1 37.9 286
Brassica napus and Raphanus sativusdgrown in pots Ni mg kg
1 19.6 46.9
on a soil containing Cd, Cr, Cu, Ni, Pb and Zn collected Pb mg kg
1 37.9 884
Zn mg kg
1 66.1 6685
from a contaminated field site.
 L. Marchiol et al. / Environmental Pollution 132 (2004) 21e27
of plants required for the experiment; after the first pair
of true leaves appeared seedlings were thinned to 10
plants per pot.
These plants were grown for 60 days on a laboratory
bench lit by lamps which gave 500 mmols m
2 s
1 of
photosynthetically active radiation (PAR) at the plant
top with a 12:12 h photoperiod; the pots were rotated
randomly daily to equalize their light exposure.
Ambient temperature was maintained at 22 G 2 (C.
Each pot was watered twice a week during the first
month and then every two days, alternating distilled
water and a modified, diluted (1:5) Hoagland solution
(Brown et al., 1995) to supply mineral nutrients.
2.1. Gas exchange measurements
Gas exchange measurements were performed on both
treated and control plants at the end of the growing
cycle. Light saturation response curves and C assimila-
tion vs. CO2 mesophyll concentration (Aeci) curves
were determined sampling a leaf area of 6 cm2 on 5
leaves per treatment. Data on CO2 assimilation (A),
transpiration (T ) and internal CO2 concentration (ci)
were collected using a Li-6400 (LI-COR) photosynthesis
system. During measurements, leaf temperature was
maintained at 22 G 0.2 (C, dew point temperature in
the cuvette at 12 G 0.5 (C and 360 ppm of CO2. The
light response curves were obtained by varying light
intensity from 0 to 2000 mmol m
2 s
1 using a 6400-02
LED (Li-Cor) light source; Aeci response curves were
obtained by changing the CO2 entering the cuvette from
0 to 1200 ml l
1.
2.2. Further observations
After the gas exchange measurements an index of
chlorophyll content was measured on the same fully
expanded leaves using a Minolta SPAD-502 chlorophyll
meter.
At the end of the experiment, the total leaf areas in
control and treated plants were measured using a Li-Cor
3000 Leaf Area Meter (Li-Cor). Finally, the leaves, stems
and roots of 5 plants per treatment were collected, taking
particular care with the root apparatus; fractions were
fresh weighed and oven-dried to determine their dry
matter. The plant fractions from treated and control pots,
plus soil samples from the same pots, were subjected to
microwave-assisted total digestion (US EPA, 1994).
Total Cd, Cr, Cu, Ni, Pb and Zn contents in the plant
and soil samples were determined by inductively coupled
plasma emission spectrometry (Perkin Elmer 3000).
2.3. Data analysis
The trial was arranged in a randomized design with
two treatments (control and treated); each treatment
23
was replicated four times for statistical purposes. During
gas exchange measurements data were logged continu-
ously and recorded when a steady state of readings was
reached (coefficient of variability 1%) at different levels
of irradiance and CO2 concentration in the leaf
chamber. The water use efficiency (WUE) was calculated
as the ratio of net carbon dioxide uptake to transpira-
tion (Sinclair et al., 1984). The Matthews and Boyer’s
method (1984) was used to quantify the chloroplast and
stomatal limitations for photosynthesis.
Two indicators were calculated to evaluate plants for
phytoextraction purposes. The Tolerance Index (TI),
which is the ratio between a variable measured in treated
plants and that same variable measured in control plants
expressed as a percentage, was calculated considering
the plant biomass (Wilkins, 1978). The Translocation
Factor (TF ¼ Cshoots =Croots ) was calculated from the
compartment concentrations of heavy metals to evaluate
plants for phytoextraction purposes and in particular
the plant’s ability to translocate heavy metals from roots
to the harvestable aerial part (Mattina et al., 2003).
Analysis of variance and the StudenteNewmanne
Keuls test (P ¼ 0:05) were used to compare treatment
means. Data are presented as means with standard error.
3. Results
3.1. Plant growth
During the experiment no obvious symptoms of
metal toxicity were observed even if plant growth on the
polluted soil resulted lower than the controls and the
leaves of treated plants were paler green in both B. napus
and R. sativus. This evidence was confirmed by the
indirect measurements of leaf chlorophyll content by
means of the SPAD measurements. The species differ in
this parameter even in the control, however the soil
pollution significantly affected the Brassica species in
different ways, with R. sativus appearing to be more
sensitive than B. napus (Table 2).
The dry weight of shoot biomass of B. napus and R.
sativus was affected by the contaminated substrate; on
average, the metal caused a reduction of about 30% of
aboveground biomass.
The percent dry matter content of the aboveground
biomass did not differ between the two species, but
treated B. napus had 47% less DM than its control,
whereas in R. sativus only a 32.5% reduction was
recorded. Table 2 also reports data on total plant
biomass; the contribution of the root fraction is not
substantial and ANOVA generally gave the same results
as those described for the shoots and confirmed that R.
sativus is more tolerant than B. napus (35.8% and 45.8%
less DM production than the respective controls).
24 L. Marchiol et al. / Environmental Pollution 132 (2004) 21e27

Table 2
Index of chlorophyll content (SPAD), dry weight of shoots and total plant biomass, dry matter (%) of shoots and total plant biomass and tolerance
index (TI) of B. napus cv Kabel and R. sativus cv. Rimbo
B. napus R. sativus ANOVA
Ctrl Trea Ctrl Trea Treatment Species
SPAD 36.9 G 0.98y 26.4 G 0.91 33.5 G 1.68 20.4 G 1.76 ***z ***
DW shoots 0.49 G 0.04 0.34 G 0.04 0.55 G .007 0.38 G 0.08 * ns
( g plant
1)
Shoots DM % 15.4 G 0.53 8.14 G 0.45 14.1 G 0.75 9.52 G 0.55 *** ns
DW total biomass 0.57 G 0.05 0.38 G 0.04 0.61 G 0.07 0.43 G 0.09 * ns
( g plant
1)
Total biomass DM % 15.9 G 0.55 8.62 G 0.46 15.4 G 0.77 9.88 G 0.56 *** ns
TI 67:8 G 6:5 76:5 G 23 ns
yStandard error; ***zP ! 0:01, **P ! 0:05, *P ! 0:1, ns: not significant.

The TI of B. napus and R. sativus is respectively 67.8
and 76.5; on an average the contaminated soil caused
about 30% reduction in plant biomass.
3.2. Gas exchanges
There were differences in the light response curves of
the tested species (Fig. 1). The lower slope of the linear
portion of the curve in treated B. napus as compared to
the control curve of the same species should be under-
lined; a difference in the efficiency of light utilization by
B. napus grown on contaminated soil can be inferred.
In R. sativus the light utilization efficiency in control
and treated plants appeared to be similar and the differ-
ences between the two curves are limited to the satura-
14
Control
12 Treated
10
tion values (the A value of treated plants was reduced by
40% in both species).
The WUE curves are reported in Fig. 2. Treated
plants of B. napus had a much lower WUE than the
controls; no differences were found in R. sativus.
When leaf carbon assimilation of the different treat-
ments was analyzed under increasing CO2 rates, typical
response curves were obtained (Fig. 3); even at high
internal CO2 concentration the C assimilation of plants
grown on contaminated soil was always lower than
controls. Graphical analysis of the Aeci curves (Mat-
thews and Boyer, 1984) reveals different behavior of the
species. In fact, the stomatal limitation at ambient CO2
to photosynthesis in R. sativus is higher than in B. napus
6
Control
5 Treated
4
3

8 2
6 1
4 B.napus
0
( µmol co2 m-2 s-1 )

WUE

2
B.napus
0
Control
5
A

Treated
Control
12 Treated 4
10
3
8
2
6
1
4
R.sativus
2 0
R.sativus
0 -1
0 200 400 600 800 1000
-2
0 200 400 600 800 1000 PAR
PAR ( µmol m-2 s-1 )
( µmol m-2 s-1 )
Fig. 2. Water use efficiency (WUE) in response to light saturation for
Fig. 1. Light response curves for control and treated plants of B. napus control and treated plants of B. napus and R. sativus. Vertical bars for
and R. sativus. Vertical bars for each point represent the standard error each point represent the standard error for five replicates. (PAR =
for five replicates. (PAR = Photosynthetically Active Radiation.) Photosynthetically Active Radiation.)
 L. Marchiol et al. / Environmental Pollution 132 (2004) 21e27 25

Species
16
Control
14 Treated

ns
ns
ns
ns
ns
*
12

Treatment
10

ANOVA
Chloroplast
8

***

***
***

***
6

**

**
4 Stomatal
( µmol co2 m-2 s-1 )

Trea (mg kg
1)

2

4029 G 1993
59.5 G 15.2
62 G 31.7

51.6 G 8.41
B.napus

563 G 151

407 G 199
0
A

Control
14
Treated
12 Chloroplast
10

Ctrl (mg kg
1)

8

0.45 G 0.09
5.73 G 0.53

8.25 G 0.45
19.4 G 4.01
38.5 G 9.39
35.8 G 4.5
R. sativus
6 Stomatal
4
2
0 R.sativus

Trea (mg kg
1)

-2
0 200 400 600 800 1000 1200

23.5 G 3.87
82.5 G 14.9
414 G 69.8

472 G 104
5983 G 879
45.7 G 7.9
Ci
( µmol Co2 mol-1 )

Fig. 3. Aeci curves for control and treated plants of B. napus and
R. sativus. Vertical bars for each point represent the standard error for

Ctrl (mg kg
1)

five replicates. A, B and Al, Bl indicate carbon dioxide assimilation

0.91 G 0.43
5.77 G 0.89
32.1 G 2.67
5.87 G 0.59
13.6 G 0.92
57.1 G 7.56
at saturating CO2 for control and treated plants, of B. napus and

B. napus
R. sativus respectively; the limitation of photosynthesis at ambient CO2

Roots
(mL L
1) due to inhibition of chloroplast activity in control and treated
plants is indicated by C, D and C l , D l . The limitation of
Concentration of heavy metals in the shoots and roots of B. napus cv. Kabel and R. sativus cv. Rimbo

photosynthesis at ambient CO2 (mL L
1) due to stomatal closure in

Species
treated leaves of B. napus and R. sativus is indicated by the reduction in

***
**

**
ns
ns

ns
photosynthesis from D to E and Dl to E l, respectively, where the
diffuse resistance in the leaves results in an internal CO2 concentration
Treatment
ANOVA

less than ambient.

***z

***
***

***
(
28% and
20% respectively). An inhibition of
*

chloroplast activity in response to the treatment was *

Trea (mg kg
1)

stronger in B. napus (Fig. 3).

18.4 G 1.06
2.46 G 0.55
28.7 G 6.42
7.13 G 0.62

3371 G 299
16 G 6.3

yStandard error; ***zP ! 0:01, **P ! 0:05, *P ! 0:1, ns: not significant.
3.3. Heavy metals in plant tissues

Table 3 reports the metal content in the shoots and

Ctrl (mg kg
1)
0.19 G 0.03
0.28 G 0.04
1.91 G 0.05
0.84 G 0.04
1.88 G 0.92
26.9 G 1.23

roots of B. napus and R. sativus.

R. sativus

As expected, the concentration of heavy metals

measured in the tissues of plants grown on polluted soil
substrate was higher than in the controls. This increase
was often 10-fold or more and in the case of zinc it was
Trea (mg kg
1)
12.6 G 1.11
0.77 G 0.05
23.6 G 0.05
4.12 G 0.33
5.48 G 0.51
1305 G 0.14

about two orders of magnitude. A statistically signifi-

cant difference between species was evidenced in the
phytoextraction of heavy metals: radish is more efficient
than canola in concentrating metals in the shoots, with
a significantly higher concentration of Cd, Ni and Pb
Ctrl. (mg kg
1)
0.61 G 0.31y

(Table 3). This was confirmed observing the metal

0.76 G 0.37
4.56 G 1.41
0.86 G 0.31
1.30 G 0.37
27.6 G 1.77

uptake in the root system only in the case of Cd.

B. napus
Shoots

The translocation factor (TF) calculated for B. napus

and R. sativus is reported in Table 4; the values indicate
an appreciable efficiency in translocating Zn and Cd,
Element
Table 3

whereas a little amount of Pb and Cr reached the shoots.

Cd

Cu

Zn
Pb
Cr

Ni

Intermediate values have been obtained for Cu and Ni.

26 L. Marchiol et al. / Environmental Pollution 132 (2004) 21e27
Table 4
Translocation factor [TF ¼ ðCaerial =Croot Þ ! 100] calculated
B. napus cv. Kabel and R. sativus cv. Rimbo
Element TF
B. napus
Cd 0.53
Cr 0.09
Cu 0.57
Ni 0.09
Pb 0.01
Zn 0.22
4. Discussion
Heavy metal accumulator species belonging to the
Brassica family (B. napus, Brassica juncea and R. sativus)
have been suggested for phytoremediation (Kumar et al.,
1995; Ebbs et al., 1997; Ebbs and Kochian, 1997). Interest
in these crops has risen recently with the phytoremedia-
tion potential of transgenic canola plants (Nie et al.,
2002).
In the chain of events necessary for plant growth,
CO2 assimilation stands at the beginning as the primary
conversion of light energy to chemical energy stored in
organic substances; moreover, heavy metal uptake and
translocation from the root zone to stems and leaves of
plants are driven by transpiration, the water status of
the plant tissues and soil moisture. Therefore, gas ex-
change parameters, as considered in our experiment, in
addition to biomass data and metal concentration in the
plant fractions, provide insights for the selection of
suitable plant species for phytoremediation.
The importance of these parameters has been con-
firmed by a recent paper by Angle et al. (2003) dealing
with the effects of soil moisture on phytoextraction of Ni
and Zn by some hyperaccumulators. It was demonstrat-
ed that metal hyperaccumulation is not associated with
drought tolerance mechanisms in plants as stated by
Severne (1974). Greater metal uptake was observed at
higher soil moisture values; also, plants produce greater
biomass at higher soil moisture levels that further
enhances the amount of metals extracted from soil.
From this point of view it could be interesting to study
the WUE and phytoextraction of heavy metals, and the
relationships between water transpiration and metal
translocation via the rooteshoot pathway.
In the perspective of phytoremediation our data
suggest that radish respond to the stress by lowering the
transpiration without any effects on the uptake of the
metals. These aspects will have strong implications in
the management of large scale projects of phytoreme-
diation and therefore they need more research.
The negative effects of a mix of contaminating metals
on the physiology (assimilation and growth) of plants in
the presence of metals has been verified in our experi-
ment; it is known that heavy metals affect both light and
dark reactions of photosynthesis (Krupa and Baszyn-
for sky, 1995). This was, in part, explained by Kupper et al.
(1999) who observed that the in vivo substitution of Mg
by Hg, Cu, Cd, Ni, Zn and Pb in chlorophyll leads to
R. sativus a breakdown in photosynthesis. Actually, the concom-
0.31 itant presence of metals had a severe influence on plant
0.04 growth and also on the phytoextraction efficiency, prob-
0.51 ably due to the additive effects on plant metabolism.
0.14
0.04
The data obtained in our experiment confirmed that
0.84 both cultivars are partially tolerant to heavy metals and
that radish is more tolerant than canola. In our con-
ditions these species showed only a low phytoremedia-
tion potential for soils contaminated by many metals.
Our results do not isolate single or cumulate negative
effects of metals on plant growth and phytoextraction
efficiency, but canola and radish succeeded in surviving
and demonstrated a simultaneous accumulation of sev-
eral metals. It could thus be realistically hypothesized
that they could perform better in the case of light soil
pollution. On the other hand, Lasat (2000) and, more
recently Li et al. (2003), have pointed out the role of soil
fertility in the future of phytoremediation, increasing the
potential of induced phytoextraction.
Besides fertilization, research needs to be developed
on optimizing crop management practices that can max-
imize the cleanup potential of remediative plants, such
as (i) sowing practices, (ii) seedbed conditions, (iii)
irrigation, (iv) harvesting schedule and methods, (v)
crop rotations and crop cycle duration (Lasat, 2002).
The effects on metal uptake of all these aspects must
be tested in the field under different site-specific con-
ditions and on multicontaminated soil.
Acknowledgements
This trial was supported by the Italian Ministry of
Education, Universities and Research. The authors are
grateful to Patrizia Zaccheo, University of Milan (Italy)
for providing the polluted soil and its chemical
characterization. We would also like to thank Liviana
Leita and Paolo Cantone, respectively, director and lab
technician of the Institute of Plant NutritiondGorizia
(Italy) for the ICP/ES analysis on plant material.
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