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International Journal of Engineering & Technology IJET-IJENS Vol:14 No:05 1
144305-7171-IJET-IJENS © October 2014 IJENS I J E N S
Abstract— Phytoremediation under greenhouse condition
was investigated as an alternative to clean up an industrially-
multi-contaminated soil with both petroleum hydrocarbons and
heavy metals. The aim of this work was to study the ability of
three sunflower (Helianthus annuus) cultivars (M, H2, and P) to
absorb/degrade V, Ni, Cu, Pb, benzo(a)pyrene, and TPH (total
petroleum hydrocarbons) from multi-contaminated soil. Assays
were performed with three sunflower cultivars, suitable for
commercial biodiesel production, in pots containing 5 kg of soil
for a period of 40 days. All three varieties were able to reduce the
concentration of heavy metals, benzo(a)pyrene, and TPH in the
soil, however, the contaminants removal varied according to the
assay conditions employed. The highest removal percentage of Ni,
Pb, and Cu were obtained for H2 cultivar. On the other hand, the
highest removal percentage of V was obtained when the soil was
treated with M cultivar. TPH removal did not vary according to
the use of different sunflower cultivars. Nevertheless, the highest
removal percentage of benzo(a)pyrene was obtained with the M
cultivar. The results demonstrated the potential of the
phytoremediation technique using sunflower for the treatment of
soil multi-contaminated by heavy metals and hydrocarbon’s
petroleum.
Index Term— heavy metals, hydrocarbons, multi-
contaminated soil, phytoremediation, sunflower.
I. INTRODUCTION
The growth of industrial activity in the last century has
resulted in a strong increase in anthropogenic substances being
introduced into the environment [1]. The accidental or
intentional emission of chemical products and wastes can lead
to soil pollution, which is a significant problem in modern
economic activities, such as in transport and civil construction.
It is important to note that appropriate land use appears in the
agenda of international discussions due to the increase in
population, as well as the need for sustainable development as
a strategic safety tool. Furthermore, soil contamination is a
concern since it can negatively affect human, animals and
plants health [2].
The current world economic model is strongly tied to the
use of fossil fuels as energy source. Given that large quantities
are necessary, the increase in the number of movements,
transformations, and storage operations can also increase the
possibility of soil contamination. It is important to remember
that petroleum is chemically defined as a mixture of chemical
compounds, predominantly hydrocarbons, which also contains
heavy metals, semi-metals, and various anions from organic
material and rocks from formations and reservoirs [3]. The
presence of inorganic material in fuel mixtures and oily wastes
is also due to the use of additives, catalysts, and other
compounds. Thus, it is clear that the presence of petroleum,
petroleum derivatives, and their wastes in the soil can lead to
contamination by organic and inorganic compounds.
Considering the importance of soil, various techniques, such
as excavation, solidification, stabilization, and bioremediation,
have been used to decrease the impact of contaminants on the
soil environment [4]. Comparatively, operations based on
biological processes are considered to be more efficient, less
costly, and the least invasive. Biotechnology alternatives,
however, are strongly related to the degradation of organic
compounds in soil or their solubilization to facilitate
extraction processes [5].
Among the biotechnological alternatives, phytoremediation
is considered to be low-cost and not harmful to the physical,
chemical, and biological characteristics of soil [6]. This
technique consists of using plants to remove biota
contaminants (phytoextraction), to absorb and convert them
into non-toxic forms (phytovolatilisation) or to stabilize an
inorganic substance, transforming it into a less soluble form
(phytostabilisation) [7]. There are a number of reports that
can be found in the literature regarding the use of plants to
remove organic or inorganic compounds from soil.
Helianthus annuus (sunflower) is one of the target species
that has excellent potential as a phytoextractor since it grows
quickly, produces large amounts of biomass and is capable of
hyper accumulate heavy metals [8; 9]. Many plants have been
studied for the phytoremediation of hydrocarbons from soil
[10; 11; 12; 13; 14]. However, only Tejeda-Agredano et al.
[15] have related the use of sunflower to the phytoremediation
of organic contaminants.
Many wastes from the petroleum and gas sector have both
hydrocarbons and heavy metals, which can contaminate soil
Phytoremediation of Soil Multi-Contaminated
with Hydrocarbons and Heavy Metals Using
Sunflowers
Cristiane D. C. Martins1; Vitor S. Liduino
1; Fernando J. S. Oliveira
2; Eliana Flávia C. Sérvulo
1*
1Escola de Química, Universidade Federal do Rio de Janeiro – UFRJ, Av. Athos da Silveira Ramos, 149, Bloco
E Sl E-203 - Ilha do Fundão, RJ - Brasil CEP 21941-909. 2 Petróleo Brasileiro S.A. Gerência de Meio Ambiente. Av. Almirante Barroso 81, Centro, Rio de Janeiro, RJ –
20031-004.
*Corresponding author: Email: [email protected]
International Journal of Engineering & Technology IJET-IJENS Vol:14 No:05 2
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with these two classes of contaminants when the contact or
improper disposal of wastes occurs. It is also important to
emphasize that the literature does not focus on the
phytoremediation of soils co-contaminated with metals and
hydrocarbons. One of the few studies in this field was
published by Sun et al. [16], where Tagetes patula was used to
phytoremediate soil that was artificially contaminated with
benzo(a)pyrene, Cd, Cu, and Pb.
This study analyses the potential of sunflowers for the
phytoremediation of a soil collected from a contaminated area
within a petroleum refinery to remove both hydrocarbons and
heavy metals (V, Ni, Cu, Pb, and Cd). Seeds from three
different, low-cost, commercial cultivars of sunflower were
tested. The 40 days long tests were conducted in greenhouse
scale.
II. MATERIALS AND METHODS
A. Soil
Soil from a contaminated area in a Brazilian petroleum
refinery was used. Approximately 60 kg of multi-
contaminated soil was dried, sifted (<2 mm), and
homogenized before the physical, chemical, and
microbiological analyses were performed to be later used in
the phytoremediation tests.
B. Plants
Three commercial sunflower cultivars (M, H2, and P) were
tested (Dekalb Ltda, Brazil). The growth of the plants was
monitored weekly.
C. Analytical methods
The particle size distribution of the soil were determined
according to the IAC [17], and the pH of the soil was
determined in a 1:1 soil:water suspension (w w-1
) using a
digital pH meter.
The total amounts of nitrogen, phosphorous, and organic
matter were determined according to the methodologies
proposed by USEPA 351.2, USEPA 365.2 [18], and Walkley
and Black [19], respectively. The soil’s water-holding
capacity was determined using the method described by
Veihmeyer and Hendrickson [20].
The concentration of the total petroleum hydrocarbon
(TPH) was determined through the USEPA 8015C method
[18]. Ten priority polycyclic aromatic hydrocarbons (PAH)
described by the Dutch list (naphthalene, phenanthrene,
anthracene, fluoranthene, benzo(a)anthracene, chrysene,
benzo(k)fluoranthene, benzo(a)pyrene, benzo(ghi)pyrene, and
indeno(1,2,3cd)pyrene) were determined by gas
chromatography coupled with mass spectroscopy using the
methodology described by USEPA 8270D [18]. The organic
extracts were obtained using an ultrasound according to the
method described in USEPA 3550C [18].
The concentration of heavy metals in the soil was
determined by spectroscopy using the methodology described
in USEPA 6010C [18]. The digestion was performed
previously in an acidic media using a microwave in
accordance with the USEPA 3051A method [18].
The metal content in the plant biomass was determined by
the methodology described above. Previously, the plant
material was dried in an oven at 60ºC, ground in a porcelain
mill, subjected to acidic digestion, and later analyzed by
atomic absorption spectroscopy.
The total heterotrophic bacteria (THB) and total fungi (TF)
in the soil were determined using the plate counting and the
pour-plate methods [21] in nutrient agar and Sabouraud agar,
respectively. The quantification of hydrocarbon-degrading
microorganisms (HDM) was performed through the Most
Probable Number technique in Bushnell Haas mineral media
with the addition of a drop of light Arabian oil as the only
carbon source [22].
All of the analytical results are reported as the average of
three replicas for all the microbiological, physical, and
chemical assays.
D. Phytoremediation tests
The experiments were conducted in greenhouse scale
(Figure 1). The seeds were germinated directly in the multi-
contaminated soil. The pots used in the tests had a surface area
of 600 cm2. Five kilograms of soil were added to each pot, and
equidistant furrows were made in the soil, with four seeds
being deposited into each hole. When at least one of the
seedlings in each hole reached a height of 10 cm, the plants
were thinned to one plant per hole.
Fig. 1. Greenhouse’s views.
The phytoremediation tests were conducted for 40 days,
during which the soil moisture was maintained at
approximately 80% of its water-holding capacity.
Each soil sampling procedure consisted of taking five
individual samples at equidistant points in each pot to obtain a
representative sample. Individual samples of 50 g of soil each
were mixed to obtain a single sample of 250 g. For each test,
the sampling procedure was performed in triplicate.
At the end of 40 days, the heights of the plants were
measured and the dry weight of the plant biomass was
determined with an analytical balance. The plant drying
process was performed in an oven at 50ºC for three days. All
of the results from the phytoremediation tests were expressed
as the average of three replicas.
The results consider the losses from the biotic and abiotic
control tests. The control tests were performed under the same
conditions, where the abiotic control was made without
International Journal of Engineering & Technology IJET-IJENS Vol:14 No:05 3
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planting sunflowers, and the biotic control, the soil in pots
were treated with an AgNO3 solution (10% w w-1
).
E. Statistical analysis
A two-way analysis of variance (ANOVA) was used in
order to understand the effect of the different sunflower
cultivars in the phytoremediation process. Comparisons of
means were done using the Tukey’s HSD test (p≤0.05). All
statistical analyses were performed using STATISTICA
software v. 7.0 (STATSOFT, USA).
III. RESULTS AND DISCUSSION
A. Soil
Table 1 shows the results of the physical, chemical, and
microbiological characteristics of the multi-contaminated soil
used in this study. The soil was sandy-silty with a neutral pH
in water. The water-holding capacity was low, which is
typical for tropical soils with low clay content. Soil
phosphorous and, especially, N can serve as nutritional
sources for the biota.
Lead, copper, nickel, and TPH were present in the soil at
concentration values above the intervention limit set by local
legislation for industrial used soils with low clay content. The
local legislation uses the same intervention values listed in the
Dutch Legislation [23]. The sum of the concentration of the
10 PAHs listed by the Dutch legislation (naphthalene,
phenanthrene, anthracene, fluoranthene, benzo(a)anthracene,
chrysene, benzo(k)fluoranthene, benzo(a)pyrene,
benzo(ghi)pyrene and ideno(1,2,3cd)pyrene) were found to be
below the intervention values according to the current local
legislation.
Copper was the only metal present at a concentration higher
than the intervention value listed in federal legislation [24].
Benzo(a)pyrene was near the limit of the intervention
concentration (3.5 mg kg-1
), and the other nine monitored
PAHs exhibited concentrations below the limit of detection of
the samples, which was 0.33 mg kg-1
. Therefore, the dataset
justifies the treatment of this soil.
The amount of hydrocarbon-degrading microorganisms
(HDM) was similar to the total number of heterotrophic
bacteria. This fact corroborates the data of increased
concentration of petroleum hydrocarbons and PAHs in the
soil, which can be used as a carbon source by these
microorganisms. The natural selection of the native microbiota
is probably due to the area contamination history by oily
wastes.
The population of fungi found in the soil sample was
elevated prior to the phytoremediation treatment, which also
contributes to the degradation of organic compounds.
According to the lab-scale study published by Atagana et al.
[25], fungi showed the potential to degrade high-molecular-
weight polycyclic aromatic hydrocarbons and other
recalcitrant organic compounds due to their complex
enzymatic systems with the ability to synthesize and excrete
various extracellular enzymes.
Table I
Physical, chemical, and microbiological composition of multi-contaminated
soil
Soil Properties Measurements
Clay (<0.002 mm), % 2
Silt (0.002-0.5 mm), % 33
Sand (0.05-2 mm), % 52
pH (in H2O) 6.81
Water-holding capacity, % 30
Total phosphorus, g kg-1
0.2
Total nitrogen, g kg-1
5.2
Organic matter, g kg-1
129
Cadmium, mg Cd g-1
3.2
Lead, mg Pb g-1
528.1
Copper, mg Cu g-1
684.5
Nickel, mg Ni g-1
109.3
Vanadium, mg V g-1
180.2
10 priority PAH, mg kg-1
5.1
Benzo(a)pyrene, mg kg-1
2.97
TPH, mg kg-1
8.890
THB, CFU g-1
1.8 x 107
TF, CFU g-1
8.7 x 104
HDM, MPN g-1
1.2 x 107
THB: total heterotrophic bacteria; TF: total fungi; HDM:
hydrocarbon-degrading microorganisms.
In the chromatographs of organic extracts from the soils
(Figure 2), compounds were detected mostly as an unresolved
complex mixture (UCM). No linear hydrocarbons, pristane, or
phytane were detected. The absence of pristane and phytane
and the increase in the baseline indicate the weathering of the
oily wastes, increasing their recalcitrance and, consequently,
increasing the difficulty for biotreatment. Weathering refers to
the result of chemical, biological, and physical processes on
the waste that can affect the type of compounds that remain in
the soil. In addition to the described factors, the elevated TPH
concentration makes phytoremediation even more difficult.
Fig. 2. Chromatograph of the total petroleum hydrocarbons present in the
multi-contaminated soil sample from the petroleum refinery.
B. Phytoremediation of multi-contaminated soil
Over the 40 days, all of the cultivated plants showed similar
average heights, measuring approximately 28 3 cm. The
development of the plant height was linear during this time
period. All of the plants had the same number of leaves and
flowers. After the 40 days, the total dry weights of the plants
obtained were 3.08, 4.06, and 2.60 g for the H2, P, and M
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biomasses, respectively. These results were consistent with
those reported in the literature for the treatment of soil
contaminated with different lead concentrations [26].
Results of microbiological analysis of the soil after 40 days
of phytoremediation with sunflowers are represented in Table
2. The results indicate that the presence of the sunflowers was
beneficial to microbial activity, particularly for the fungal
population. Fungi can be related to the release of chemical
substances and enzymes by the plant roots, which favor
microbial growth [27; 28].
The percentage of heavy metals removed from the soil by
the three sunflower cultivars is shown in Figure 3. The largest
percentage of vanadium removal from the soil (39.8%) was
achieved by seeding cultivar P. The removal of nickel and
cadmium was approximately 46 and 100%, respectively, for
the three tested cultivars. The greatest percentage removals of
lead (55.8%) and copper (73.3%) were observed when the H2
cultivar was used.
Usman and Mohamed [29] studied different conditions for
soil phytoremediation by sunflowers for 60 days. They
observed that the Zn removal varied between 2 and 4%, Cu
removal varied between 1.6 and 3%, Cd removal varied
between 2.6 and 4%, and Pb removal varied between 0.4 and
1.3%. The study published by Chen et al. (2004) showed
removals of Pb, Cu, Zn, and Cd of 4, 16, 13, and 23%,
respectively, after 53 days of phytoremediation by sunflowers.
Thus, the promising results for the three tested Brazilian
cultivars were confirmed.
Fig. 3. Removal of V, Ni, Pb, Cd and Cu by the sunflower cultivars (H2, M
and P) in multi-contaminated soil from an industrial area. Values are means ±
SE of three replicates. Different letters indicate significant differences among
the means of different treatments (p<0.05).
The concentrations of heavy metals in the plant biomass
after 40 days of testing are shown in Table 3. There is no
significant difference between the values for vanadium and
cadmium found in the biomasses of the three cultivars. The
greatest concentrations of lead and copper were found in the
biomass of cultivar H2. However, the highest concentration of
nickel was found in the biomass from cultivar M.
c
a
c
a
b b
a a
a
a
a a
b
a
b
0
20
40
60
80
100
120
V Ni Pb Cd Cu
Re
mo
val (
%)
M H2 P
Table II
Soil microbiology after 40 days of sunflower phytoremediation
Cultivar THB (CFU g-1
) HDM (MPN g-1
) TF(CFU g-1
)
H2 5.0 ± 0.2 x 108a 2.0 ± 0.1 x 10
8a 8.6 ± 0.3 x 10
6a
M 2.4 ± 0.1 x 108b 7.8 ± 0.3 x 10
7b 7.1 ± 0.2 x 10
6b
P 1.7 ± 0.1 x 108c 6.9 ± 0.2 x 10
7b 4.3 ± 0.1 x 10
6c
Biotic control 7.2 ± 0.3 x 107 9.5 ± 0.4 x 10
7 7.8 ± 0.3 x 10
4
THB: total heterotrophic bacteria; TF: total fungi; HDM: hydrocarbon-degrading microorganisms; CFU: colony
forming unit; MPN: most probable number. Values are means ± SE of three replicates. Different letters indicate
significant differences among the means of different treatments (p<0.05).
Table III
Heavy metal concentration in plant biomass after 40 days of phytoremediation
Metals (mg kg-1
) Sunflower cultivar
H2 P M
V 0.50 ± 0.01a 0.50 ± 0.01a 0.50 ± 0.01a
Ni 2.61 ± 0.08b 1.72 ± 0.07c 3.43 ± 0.1a
Pb 2.65 ± 0.05a 0.54 ± 0.01c 1.74 ± 0.05b
Cd 0.051 ± 0.010a 0.051 ± 0.010a 0.052± 0.010a
Cu 21 ± 1.5a 14± 1.0b 16± 1.0b
Values are means ± SE of three replicates. Different letters indicate significant differences among the means of
different treatments (p<0.05).
International Journal of Engineering & Technology IJET-IJENS Vol:14 No:05 5
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Sung et al. [6] studied the accumulation of copper, nickel, and
chromium in various plants, including sunflowers, in
phytoremediation assays with steel tailings. The authors
found concentrations of Cu, Ni, and Cr in the plant tissues
varying between 25-50, 25-80, and 50-80 mg kg-1
,
respectively. Turgut et al. [7] evaluated the accumulation of
Cr, Ni, and Cd in two different cultivars of sunflower, teddy
bear and dwarf sunspot. The two cultivars showed
concentrations of Cr, Ni, and Cd in their plant tissues of
approximately 190; 6.5 and 120 mg kg-1
, respectively. Thus,
it was inferred that the accumulation of metals in plant tissues
is heterogeneous, corroborating the quantitative images of
metals in plant tissues published by Becker et al. [31].
Table 4 shows the removal of benzo(a)pyrene after 40 days
of phytoremediation of multi-contaminated soil by sunflower
cultivars. Benzo(a)pyrene is a high molecular weight PAH,
with a recognized persistence in the soil and half-life values
varying between months to many years [32; 33]. The
metabolites originating from benzo(a)pyrene are considered to
be highly carcinogenic and mutagenic and are classified as
carcinogenic group 1 by IARC [34].
Table IV
Removal of benzo(a)pyrene by sunflower cultivars (H2, M and P).
Initial H2 M P
Concentration
(mg kg-1
soil)
2.97 2.01 ± 0.2 1.55 ± 0.1 2.42 ± 0.2
Removal (%) - 32.1 ± 3.2b 47.7 ± 4.2a 18.3 ± 2.0c
Values are means ± SE of three replicates. Different letters
indicate significant differences among the means of different
treatments (p<0.05).
The largest percentage reductions of benzo(a)pyrene in the
soil were obtained through the cultivation of the M and H2
cultivars, considering the losses in the biotic and abiotic
control reactors. Liste and Alexander [35] performed
phytoremediation tests over 56 days with the Avena sativa and
Brassica napus var. radicola grains, resulting in a reduction in
pyrene of 55.4 and 73.5%, respectively, 18% of which were a
result of the microbial activity and the abiotic losses (control
tests). Through the growth of the herbs Anethumgraveolens,
Capsicum annuum, and Raphanussativus, the same authors
found decreases in the concentration of pyrene varying
between 51.2 and 66.9% after 28 days of phytoremediation, of
which only 6.7% corresponded to losses in the control tests.
Comparing the results now presented in the literature, the
applicability of sunflower cultivars is verified for the removal
of a high-molecular-weight PAH.
Phytoremediation was also investigated for the removal of
TPH after 40 days of sunflower growth on multi-contaminated
soil. In this period, there was a reduction of 10 ± 2% of the
TPH in the soil, regardless of the sunflower cultivar used;
these values were calculated considering the losses in the
control reactors. The petroleum hydrocarbons are a class of
organic compounds in which aromatic compounds, PAH,
alkanes, and other substances that show various
physicochemical properties are included. An increase in the
size of the carbon chain leads to a reduction in the solubility of
the compound and an increase in the partition coefficient in
octanol-water (Kow) and, consequently, a reduction in the
availability of the contaminant to the plants [36].
Siciliano and Germida [37] reported a reduction of
approximately 21% in the concentration of TPH in
phytoremediation tests using forage species. However, these
results were achieved only after 20 months of treatment for
soil contaminated only with hydrocarbons. More recently,
Moreira et al. [38] observed a decrease of 12% in the
concentration of TPH in mangrove sediment also
contaminated with only petroleum hydrocarbons, after 90 days
of phytoremediation with Rizophora mangle L. Thus, the
results in the present study are promising and innovative, as
they demonstrate the concomitant removal of hydrocarbons
and heavy metals in real soil from an industrial area.
Considering Dutch regulations to be among the most
restrictive in the world, the seeding of H2 and M cultivars
provided a reduction in the concentration of heavy metals that
allowed the soil to be classified as rehabilitated for use in an
industrial area (with reference to these parameters). However,
there was insufficient removal of TPH to consider the soil
rehabilitated for use in industrial areas in relation to all of the
analyzed parameters. Nevertheless, considering the low cost
and the short time required, the results prove that the treatment
is promising for the treatment of this type of multi-
contaminated soil.
IV. CONCLUSION
The three tested sunflower cultivars presented the same
growth and biomass under multi-contaminated and non-
contaminated soils. All of them were also capable of removing
benzo(a)pyrene, TPH, and metals from multi-contaminated
soil. The amount of contaminants removed from soil varied
according to the sunflower cultivar used. Sunflower H2, P and
M can hyperaccumulate Cu, Pb, Ni and V from multi-
contaminated soil. Nevertheless, Cd absorption was lower than
other metals. After the treatment with the H2 and M cultivars,
the soil was considered rehabilitated for use in an industrial
area, according to Brazilian federal legislation. Considering
the costs and time required, the phytoremediation with
sunflower proved to be an efficient, convenient, low-cost
process for the treatment of soils contaminated with organic
and inorganic compounds.
ACKNOWLEDGMENTS
The authors thank the Brazilian National Petroleum Agency
(ANP) and Petrobras for providing financial support.
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