Evaluacion del efecto de la fitoremediacion y el riesgo ambiental en procesos de remediacion bajo diferentes sistemas de cultivo

1. Evaluation of the phytoremediation effect and environmental risk in
remediation processes under different cultivation systems
Jie Luo a, c, *
, Shihua Qi a
, X.W. Sophie Gu b
, Jinji Wang c
, Xianming Xie c
a
China University of Geosciences, Wuhan 430074, China
b
The University of Melbourne, VIC 3010, Victoria, Australia
c
Guangdong Hydrogeology Battalion, Guangzhou 510510, China
a r t i c l e i n f o
Article history:
Received 7 July 2015
Received in revised form
18 January 2016
Accepted 18 January 2016
Available online 27 January 2016
Keywords:
Eucalyptus globulus
Chickpea
Earthworm
Environmental risk
Phytoremediation
a b s t r a c t
Remediation effectiveness and environmental risks caused by phytoremediation processes under
different cultivation systems were assessed at an electronic waste dismantling site. Non-nitrogen-fixing
Eucalyptus globulus Labill. and nitrogen-fixing Cicer arietinum (chickpea) were selected for phytor-
emediation, while Eisenia foetida and Gus gallus were chosen as impacted receptors. Chickpea mono-
culture was least effective for soil remediation, and soil cadmium under this cultivation system had the
most potential threats to the environment. The chickpea monoculture cultivation system needs 132 years
to reduce the initial soil cadmium concentration to the quality guidelines of China. The environmental
risk index of receptors was greater than in other cultivation systems. The greatest remediation effec-
tiveness occurred in the E. globulus monoculture with earthworm addition. This approach decreases the
decontamination time of cadmium in the soil by 80% compared to chickpea monoculture without
earthworms. In addition to a favorable phytoremediation effect, this cultivation system was accompanied
by a more acceptable environmental risk index value because E. globulus are evergreen trees, unpalatable
by livestock, with less litterfall production than chickpeas. E. globulus monoculture with an earthworm
addition system is especially suitable for remedying cadmium-polluted soil, as it causes the least envi-
ronmental risk and reduces the time required to decontaminate the cadmium in the soil by 30%
compared to the next most effective system. The decision of which cultivation system is more suitable for
an anthropogenically influenced site should be balanced between the capacity of the plant to remove
pollution and environmental preservation. Data from the present research have provided a new meth-
odology of efficient phytoremediation with relatively low environmental risks.
© 2016 Elsevier Ltd. All rights reserved.
1. Introduction
Since rapid economic development in developing countries is
usually coupled with industrial and mining activities, environ-
mental problems caused by continuous emission of persistent
organic pollutants and heavy metals into different media have
increased in recent years and have overwhelmed the self-
purification capacity of nature (Ullah et al., 2015). Guiyu town,
Guangdong Province, south China, is reputed to be the electronic
waste (e-waste) capital of the world for its involvement in e-waste
dismantling and the recycling business for more than 30 years
(Wong et al., 2007). Although uncontrolled open burning and
strong acid leaching of e-waste have been banned for many years in
Guiyu, soils have been polluted by metals, especially cadmium (Cd).
Heavy metals are not degradable through microbiological or
chemical processes (Mahmood et al., 2014). Thus, they generally
accumulate in the soil and then leach into groundwater under
appropriate conditions. Cd, which is highly toxic, is usually derived
from electroplating, plating, fossil fuel consumption, battery
discard and tanning (Teixeira et al., 2014).
Due to the adverse effects of Cd, a variety of methods have been
developed to treat Cd-polluted soils. Traditional physical and
chemical remediation technologies can remediate the contami-
nated land rapidly but suffer from limitations such as high ex-
penses, change in soil functions, creation of secondary
contamination and lack of suitability for treating large accident
sites containing moderate pollution (Akcil et al., 2015). Considering
* Corresponding author. China University of Geosciences, Wuhan 430074, China.
Tel.: þ86 18664739126.
E-mail address: gchero1216@hotmail.com (J. Luo).
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Journal of Cleaner Production
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Journal of Cleaner Production 119 (2016) 25e31

2. the negative effects of those methods, phytoremediation is poten-
tially a more ecofriendly technology (Suchkova et al., 2014). Phy-
toremediation is generally accepted by the public because it has
successfully mitigated anthropogenically influenced environments
without destroying soil functions (Visoottiviseth et al., 2002). In
addition to remedying the polluted soil, phytoremediation provides
other benefits, such as soil erosion mitigation, carbon sequestra-
tion, biodiversity protection and biofuel production (Hu et al.,
2012).
There are still disadvantages of this technology; the main
obstacle is disposing of contaminated plants after remediation
(Mani et al., 2015; Pandey et al., 2015). Most published studies focus
mainly on the ability of plants to accumulate contaminants (Wei
et al., 2006) or on the effect of chelate addition to improve phy-
toextraction (Chen et al., 2003), with rare exploration of the envi-
ronmental risks caused by phytoremediation. Metals can enter food
chains and transfer to other trophic levels through bio-
magnification during phytoremediation processes, as metal-rich
plants can provide pathways for pollutants (Rathod et al., 2014).
Hence, although phytoremediation contributes to the mitigation of
contaminated soil, the resulting potential risks to the environment
must be carefully considered.
Eucalyptus globulus is considered a suitable candidate for
contaminated soil phytoremediation, although the Cd content in
this species is generally below the thresholds for Cd hyper-
accumulators (Arriagada et al., 2007). It performs in line with
known Cd hyperaccumulators because its significant biomass
production can compensate for low Cd content in plant tissues
(Fine et al., 2013). Chickpea and earthworm are well-known ni-
trogen (N) fixers. Chickpea improves the production of intercrop-
ping plants by nitrogen fixing and decontaminates heavy metals
from the soil (Huang et al., 2006). Earthworms can increase soil
nutrient status via atmospheric nitrogen fixation (Ozawa et al.,
2005), and their activity can improve soil conditions by facili-
tating water and organic matter exchange (Costello and Lamberti,
2009). Forrester et al. (2010) observed that, in direct relation with
pollutant translocation, evapotranspiration and water consumption
of Eucalyptus globules are different under various cultivation sys-
tems. Cd can enter the food chain when using edible species such as
Cicer arietinum (chickpea), Eisenia foetida (earthworm) and Gus
gallus (chicken) for soil remediation, so environmental risk during
phytoremediation is a non-negligible factor in the present study.
According to the above factors, a series of experiments without
chemical reagent addition have been designed in the present study.
The specific objectives of this study were initiated to assess (1)
the phytoremediation efficiency of each species under different
cultivation systems, (2) the role of nitrogen-fixers in promoting the
phytoremediation ability of non-nitrogen-fixing plants, and (3) the
potential environmental risks caused by Cd exposure during phy-
toremediation processes.
2. Materials and methods
A finished ecological geochemistry survey for Guiyu preceding
the present experiment showed soil in this town is a typical ferric
acrisol, slightly acidic (pH ¼ 6.4), having a CEC (cation exchange
capacity) of 12.7 cmol kgÀ1
and TOC (total organic carbon) of
42 g kgÀ1
. Heavy metals such as Cd (0.67 mg kgÀ1
), copper (Cu)
(56.2 mg kgÀ1
), mercury (Hg) (0.44 mg kgÀ1
), and lead (Pb)
(69.5 mg kgÀ1
) in this soil exceed the Chinese soil quality standard
II published by the China MEP (2008) (0.3, 50, 0.25 and 50 mg kgÀ1
for Cd, Cu, Hg and Pb, respectively). Only Cd among these elements
is further discussed because Cd was a unique element that can be
phytoremediated successfully by the chosen species without other
additional technology aids in this experiment.
Chaonan, approximately 20 km away from Guiyu, whose major
industry is fishing, was chosen as a reference site. This town was
never involved in e-waste recycling activity and the heavy metals in
its soil have significantly weaker negative impact on creatures (Li
et al., 2008).
2.1. Experimental design
In situ experiments were designed to compare the capacity of E.
globulus with different nitrogen fixers to enhance growth and Cd
uptake under different systems (Fig. 1).
The heterogeneity of soil makes it difficult to assess phytor-
emediation efficiency of planted species for field experiments
because the success of phytoremediation is generally determined
by point-to-point evaluation instead of averages of a data matrix
from experimental sites (Han et al., 2015). To minimize the effect
of the heterogeneity of soil on phytoremediation evaluation, a
small area with a low heterogeneous soil Cd concentration was
selected according to our previous ecological and geochemical
survey. Eight contiguous anthropogenically influenced sites
(20 m  24 m) were set up, each divided into six 10 m  8 m
quadrats as replicates, and buffer trees were planted around the
perimeter of the quadrats. After large e-waste debris was removed
manually, all sites were plowed thoroughly down to the clay
basement (approximately 30 cm) and leveled homogeneously by a
rotary tiller.
The eight chosen sites were encoded as E1 to E8. E1 to E3 were
planting experiments in which earthworms were dislodged and
captured by an electrical method (Schmidt, 2001) before E. glob-
ulus and chickpeas were planted. Three-year-old E. globulus with
similar characteristics such as tree height (approximately 5.5 m)
and diameter at breast height (approximately 8 cm) were culti-
vated in E1 at a density of 2500 crops per ha in April, 2010.
Chickpeas were planted in E2 synchronously at a recommended
density of 445,000 crops per ha (Wu et al., 2008). Half of the E.
globulus and Chickpeas were planted crisscross in E3 instead of as
cultivated trees within a row in order to reduce light competition
(Forrester et al., 2005). E4, E5 and E6 categories correspond to E1,
E2 and E3, respectively, with earthworm application. For the
earthworm addition experimental sites, 10 kg of fresh earth-
worms (Costello and Lamberti, 2009) caught from similar soil
conditions were added directly to the center of every quadrat
(125 g mÀ2
). E7 was an earthworm control experiment without
plants, and E8 was a control experiment without earthworms and
plants. Sixty-three 8-month-old free-ranging chickens were
divided into 21 equal groups and placed into three of the six
quadrats (three chickens per quadrat) of every experimental site
(except E8, as there was no food for chickens). A 100-mesh nylon
net (200 cm high) was attached to the buffer trees to prevent
chickens moving between quadrats. The experimental designs are
summarized in Table 1.
E1 is E. globules in the monoculture with chicken, E2 is chickpea
in the monoculture with chicken, E3 is E. globules and chickpea
mixed cultivation with chicken, E4 is E. globules in the monoculture
with chicken and earthworm, E5 is chickpea in the monoculture
with chicken and earthworm, E6 is E. globules and chickpea mixed
cultivation with chicken and earthworm, E7 is the unplanted site
with earthworm and chicken and E8 is the bare control.
Each 10 m  8 m quadrat was further divided into four equal-
area sampling sites (5 m  4 m). Five subsamples of topsoil
(20 cm) were collected at each corner and the center of the
established sampling site by a stainless steel sampler and then
combined into one composite sample. Four collected composite
samples for every quadrat were air dried and sieved through a 2.0-
mm mesh for analysis.
J. Luo et al. / Journal of Cleaner Production 119 (2016) 25e3126

3. In late August, five E. globulus were harvested randomly in each
quadrat by felling at 30 cm above the ground. Half of the chickpeas
were harvested by felling at 15 cm above the ground. The harvested
plants were separated into roots and shoots, washed thoroughly
with deionized water three times to remove adhered soil particles
and oven dried at 80 C until reaching a constant weight. Weighed
samples were ground into powder in a stainless steel mill for
analysis.
Earthworms were seized by an electrical method in four of the
six quadrats at each experimental site and those in the other two
quadrats were caught by a hand sorting technique that is
considered to provide realistic species representation estimates
(one with chicken addition and one without). All data were
presented on a centiare basis for comparison. After starving for
72 h to clean the soil in the guts, earthworms were washed with
distilled water to remove soil particles and then oven dried at
90 C for 8 h.
Although Cd concentrations are different among various or-
gans, different edible portions were not considered separately
because predators consume their prey whole. Chickens were
numbered and slaughtered. Edible portions such as muscle tissue,
internal organs, fat and cartilage were sampled from each chicken
on a proportional weight basis. Materials of each edible portion
were blended thoroughly and 20 g of homogenized samples were
dried at 140 C for 4 h and incinerated in a muffle furnace at
600 C for 12 h.
Earthworm and chicken samples collected from an uncontam-
inated site were processed in the same way as background values
for comparison.
2.2. Chemical analysis
The prepared dry powder of soil, plant, earthworm and chicken
samples were digested using a di-acidic mixture of concentrated
nitric acid (HNO3) and hydrochloric acid (HCl) (3:1, v/v). A 1-mL
solution was diluted with 3% HNO3 in a 10-mL colorimetric tube.
Total Cd was determined by inductively coupled plasma mass
spectrometry (ICP-MS). Quality assurance tests were performed by
comparison with certified reference material GBW10012 for plants,
GBW07410 for soil and GBW10018 for earthworms and chickens.
Limits of detection for Cd were 0.02, 0.03 and 0.02 mg/kg in soil,
plant and animal organ samples, respectively. The recovery rates of
Cd for soil, plant and animal organ samples were 94 ± 7%, 94 ± 5%
and 97 ± 3%, respectively.
2.3. Data analysis
The bioconcentration factor (BCF) and translocation factor (TF)
were used to evaluate Cd uptake and translocation (Wu et al., 2015).
BCF ¼ Cd content in shoot or root/Cd content in soil
TF ¼ Cd content in shoot/Cd content in root
The total Cd extraction by plant (TE) (Niazi et al., 2012) was
calculated as
TE ¼ Cd content in plant  dry biomass of plant.
The environmental risk index (ERI) was calculated by dividing
the Cd content in earthworms or chickens caught from each
experimental site by the Cd content in the corresponding creature
collected from the clean location (Rodriguez-Ruiz et al., 2015; Song
et al., 2015).
Fig. 1. Map of the experiment sites.
Table 1
Experimental designs.
E1 E2 E3 E4 E5 E6 E7 E8
Eucalyptus globulus þ e þ þ e þ e e
Chickpea e þ þ e þ þ e e
Earthworm e e e þ þ þ þ e
Chicken þ þ þ þ þ þ þ e
J. Luo et al. / Journal of Cleaner Production 119 (2016) 25e31 27

4. Experimental data were processed using two-way analysis of
variance (ANOVA) with STATISTICA 6.0 software, using cultivation
conditions, nitrogen fixers and experimental sites as fixed factors.
All reported data are the means ± SD of six replicates (three rep-
licates for chickens). The statistical significance of the differences
between experimental sites was analyzed by Duncan's Multiple
Range Tests at the level of p 0.05.
3. Results and discussion
The results are presented and discussed in the following order:
physical signs of plants under different cultivation systems,
biomass production of plants and receptors and characteristics of
Cd in different media, and the success of phytoremediation and
possible environmental risks during the remediation process.
3.1. Physical signs of plants
E. globulus grew well in all cultivation systems without visible
phytotoxicity caused by high Cd content in the soil. This result
corresponds well with the finding of Gomes et al. (2012), who
observed that Cd at 5.5 mmol mÀ3
only depressed 10% of E. globulus
production. That level was much higher than the Cd concentration
in this study, which varied within a relatively small range from 0.58
to 0.82 mg kgÀ1
, with a mean value of 0.68 mg kgÀ1
.
The leaves of chickpeas slightly crinkled, showing symptoms of
early senescence in monoculture cultivation regardless of the
presence of earthworms. The more obvious phytotoxic signs in
chickpeas under mixture cultivation may be due to competition for
nutrients and water from E. globules. This result corresponds well
with the results of Forrester et al. (2005), who reported E. globules
inhibited the growth of Acacia mearnsii under a mixture cultivation
system. Crops showed necrosis from blade tips to petioles and
stipules and subsequently to the entire lamina. The health status of
the plants under different cultivation systems are summarized in
Table 2.
3.2. Biomass production of plants and animals
E. globulus produced the greatest roots and shoots dry biomass
in mixture cultivation with earthworm addition (E6), followed by
mixture cultivation without earthworms (E3) and monoculture
with earthworms (E4). E. globulus monoculture without earth-
worms (E1) produced the lowest roots and shoots dry biomass. E.
globulus in E3 produced less roots but more shoots dry biomass
than in E4. In both experiments, similar total dry biomass was
produced (Table 3).
Chickpeas produced less dry biomass in all experimental sites
compared to their production in clean soil (Soltani and Sinclair,
2011), as they are sensitive to contaminants (Gupta et al., 2006).
Chickpeas produced the least dry biomass in mixture cultivation
with earthworm addition (E6), followed by mixture cultivation
without earthworms (E3) and the greatest dry biomass in mono-
culture cultivation without earthworms (E2).
Nitrogen fixers can improve the growth of non-nitrogen-fixing
plants (Forrester et al., 2010) and intraspecific competition was
decreased in mixture cultivation due to the development of canopy
stratification (Hubbard et al., 2004). Therefore, E. globulus under
mixture cultivation systems produced more biomass. Chickpeas
can fix 30 kg of nitrogen per ha from the atmosphere in the area
having a similar planting density with the monoculture chickpea
cultivation systems in the present study (Zhang et al., 2011).
Chickpeas cannot fix the same amount of nitrogen under mixture
cultivation systems due to low planting density and the competi-
tive environment in this study. Earthworms can also increase soil
nitrogen concentration, either directly via metabolite excretion and
carcass decomposition, or indirectly via soil structure changing,
oxidation increase, organic substance fragmentation and nitrifying
bacteria promotion (Ozawa et al., 2005). Parkin and Berry (1999)
reported that earthworms can fix 6.9 kg of nitrogen per ha at a
density of 50 earthworm (approximately 25 g fresh weigh) per
square meter. Thus, they may produce more nitrogen (approxi-
mately 34.5 kg per ha) than chickpeas in the present study. Previous
research has concluded that competition was more intense among
different plant species relative to plant, animal and microorganisms
(Nie et al., 2010; Yu et al., 2005). These results support the present
observation that compared with chickpea, earthworms help E.
globulus produce more biomass (Table 3).
Earthworms in experimental sites without chickens had
significantly higher fresh weight per square meter than those with
chickens regardless of whether the earthworms had been removed
beforehand. Earthworms in chickpea monoculture and mixture
cultivation systems had more fresh biomass than those in E. glob-
ulus monoculture, indicating that chickpeas improve earthworm
fresh biomass more effectively than E. globulus. E. globulus may
compete with earthworms for nutrients such as N, calcium (Ca),
magnesium (Mg) and organic matter (Mboukou-Kimbatsaa et al.,
2007). Chickpeas facilitated the growth of earthworms in two
ways, namely, providing organics to earthworms directly and
reducing earthworm consumption by chickens through offering an
alternative food source.
The body weight of chickens at the end of the experiment fol-
lowed the descending order of E6 E5 E7 E4 E2 E3 E1.
Chickens in experimental sites with earthworm addition were
significantly heavier than those in sites without earthworms. E.
globulus promoted the growth of chickens less effectively than
Chickpeas because E. globulus are unpalatable to chickens, and
evergreen trees produce less plant debris for earthworms than
deciduous trees such as chickpea.
3.3. Cd concentrations in different media
The soil Cd concentrations were not significantly different in all
experimental sites due to the short duration of the experiment
(Table 4).
The Cd content of E. globulus was higher when a nitrogen fixer
existed. Root BCF values of E. globulus ranged from 4.6 to 12.8
(Table 5). The maximum value occurred in E4, followed by E6 and
E3. The trend of shoot BCF values of E. globulus were similar to root
BCF values, i.e., E4 E6 E3. All BCF values of E. globulus except
shoot BCF in E1 were greater than one, which means the plant is
suitable for Cd phytoremediation (Kim and Lee, 2010). Root BCF
values of chickpeas were greater than one, ranging from 3.8 to 7.7
and shoot BCF values were lower than one.
The TF values in all experimental sites were lower than one; the
TF values of E. globulus ranged from 0.13 to 0.15, which were similar
in E1, E3 and E6 and significantly higher in E4. TF values of chick-
peas ranged from 0.06 to 0.11, which were significantly lower than
the values of E. globulus.
The success of phytoremediation depends on the metal forms,
nutrient availability and physics-chemical properties of soil,
Table 2
Health status of the plants.
E1 E2 E3 E4 E5 E6
E. globulus Grow
well
e Grow
well
Grow
well
e Grow well
Chickpea e Early
senescence
Leaf tip
necrosis
e Early
senescence
Petiole and
stipule necrosis
J. Luo et al. / Journal of Cleaner Production 119 (2016) 25e3128

5. biomass production of the chosen plant species and response of
plants to contaminants (Kuppens et al., 2015). The nutrient and
metal absorption ability of plants is a key factor influencing their
biomass, physiology and remediation effectiveness (Guo et al.,
2002). Water use efficiency plays a vital role in extracting nutri-
ents and metals because such materials can be dissolved in water
and then assimilated by plants via water absorption (Licht and
Isebrands, 2005). Nitrogen-fixing plants enhanced water con-
sumption of E. globules and, consequently, nutrient accumulation
and biomass production (Forrester et al., 2010; Lou et al., 2013).
Therefore, the root and shoot Cd concentration of E. globulus
increased in mixture cultivation (E3) compared to E. globulus
monoculture (E1), which may be explained by high growth rate,
nutrient use efficiency and water consumption.
BCF and TF values of E. globulus were higher in E. globulus
monoculture with earthworm application (E4) than in mixture
cultivation without earthworms (E3), indicating that earthworms
can more effectively facilitate the uptake and transport capability of
Cd from the sediment by the corresponding planted crops than
nitrogen-fixing plants such as chickpeas in present study. The trend
of BCF and TF values of E. globules was consistent with their biomass
production under the different cultivation systems mentioned
above, which concurs with the finding of Yu et al. (2005) that
earthworms can improve bioavailability and mobility of Cd because
they can change the physical and chemical characteristics of soil,
including organic materials, pH, redox potential (Eh) and moisture.
Ll et al. (2012) concluded that earthworms had more positive ef-
fects on regulating the soil ecosystem than maize and Glomus
intraradices. Interestingly, although the BCF value of E. globulus in
mixture cultivation with earthworms (E6) was higher than in
mixture cultivation without earthworms (E3), it was lower than in
E. globulus monoculture with earthworms (E4). The interaction
effect of two nitrogen fixers may not simply be the superposition of
their individual effects and their synthetic action may be more
complex. This complexity remains to be elucidated in the future.
The Cd contents of earthworms were generally higher when
chickpeas existed, elucidating that earthworms' metal uptake
ability depends on food source. In support of this result, Du et al.
(2014) and Mo et al. (2012) concluded that Cd concentrations of
earthworms increased significantly in the presence of edible plant
species, and Manna et al. (1997) reported the synergistic effect that
earthworms with maize stover and chickpea straw accelerated
residue breakdown. The Cd concentrations of chickens were
significantly higher in experimental sites with earthworms (E4 to
E7) than in soils without earthworms (E1 to E3), and the values
have approached or even exceeded the maximum toxicity
threshold of 0.5 mg/kg permitted by Chinese legislation for live-
stock offal. Chickpeas deteriorated the Cd contamination of chicken
more obviously than E. globulus regardless of the presence of
earthworms because chickens were more inclined to grazing on
them. This study corresponds well with the findings of Guruge et al.
(2005) and Zhuang et al. (2009) regarding biomagnification of
organic and inorganic pollutants in the food chain of domestic
Table 3
Biomass production of plants and creatures.
Eucalyptus globulus kg/plant (dry
weight)
Chickpea g/plant (dry
weight)
Earthworm g/m2
(fresh weight) Chicken g/chicken (fresh weight)
Root Shoot Root Shoot Chicken No chicken Earthworm No earthworm
E1 1.31 ± 0.27a 2.90 ± 0.42a e e 4.7 ± 2.3a 9.7 ± 3.1a e 1087(6) ±116a
E2 e e 6.3 ± 1.8a 17.1 ± 3.2a 5.3 ± 1.7a 12.9 ± 4.5a e 1264(7) ±201b
E3 1.72 ± 0.45b 3.78 ± 0.53b 4.9 ± 1.2b 14.2 ± 4.1b 6.6 ± 2.6a 11.4 ± 2.8a e 1259(7) ±158b
E4 1.89 ± 0.22b 3.54 ± 0.29b e e 20.9 ± 6.4b 192.8 ± 38.6b 1297(6) ±111a e
E5 e e 5.2 ± 1.6b 14.9 ± 2.1b 37.2 ± 4.8c 224.3 ± 42.1c 1429(9) ±261b e
E6 2.07 ± 0.56c 4.52 ± 1.03c 3.7 ± 1.2c 13.6 ± 4.2b 34.5 ± 7.2c 201.4 ± 16.8b 1514(8) ±181b e
E7 e e e e 9.8 ± 3.1d 216.8 ± 30.5c 1327(8) ±128a e
E8 6.9 ± 1.4a 8.7 ± 3.2a
The numbers in parentheses refer to the chicken alive at the end of the experiment. Numerical values followed by different letters in the same column were significantly
different at p 0.05.
Table 4
Cd concentrations of plants, earthworms and chickens.
Soil mg/kg Eucalyptus globulus mg/kg Chickpea mg/kg Earthworm mg/kg Chicken mg/kg
Root Shoot Root Shoot Chicken No chicken Earthworm No earthworm
E1 0.74 ± 0.16a 3.37 ± 0.28a 0.46 ± 0.21a e e 3.84 ± 1.04a 4.27 ± 1.29a e 0.22(6) ±0.07a
E2 0.69 ± 0.07a e e 2.59 ± 1.02a 0.07 ± 0.05a 5.52 ± 1.32b 5.47 ± 2.31b e 0.37(7) ±0.11b
E3 0.62 ± 0.08a 5.21 ± 2.03b 0.77 ± 0.29b 4.79 ± 0.58b 0.29 ± 0.07b 5.02 ± 0.62b 5.19 ± 1.92b e 0.31(7) ±0.09b
E4 0.58 ± 0.12a 7.42 ± 1.16c 1.49 ± 0.56c e e 4.53 ± 1.89b 3.91 ± 0.86a 0.44(6) ±0.09a e
E5 0.67 ± 0.04a e e 4.53 ± 0.76b 0.52 ± 0.07c 8.43 ± 0.92c 5.80 ± 0.97b 0.57(9) ±0.21b e
E6 0.64 ± 0.10a 6.89 ± 1.81c 0.93 ± 0.09b 4.42 ± 1.21b 0.40 ± 0.12d 5.41 ± 1.43b 3.66 ± 1.82a 0.52(8) ±0.16b e
E7 0.70 ± 0.08a e e e e 3.12 ± 2.12a 2.48 ± 0.56c 0.39(8) ±0.12a e
E8 0.82 ± 0.21b 2.57 ± 1.16d 2.03 ± 1.09c
Numerical values followed by different letters in the same column were significantly different at p 0.05.
Table 5
Phytoremediation potential and environmental risk.
Eucalyptus globulus Chickpea ERI
RBCF SBCF TF TE RBCF SBCF TF TE Chicken Earthworm
E1 4.6a 0.6a 0.1a 5.7a e e e e 2.00a 2.75a
E2 e e e e 3.8a 0.1a 0.03a 0.02a 2.71a 4.63b
E3 8.4b 1.2b 0.1a 11.9b 7.7b 0.5b 0.06b 0.03a 2.51a 3.88a
E4 12.8c 2.6c 0.2a 19.3c e e e e 2.08a 5.50b
E5 e e e e 6.8b 0.8c 0.11c 0.03a 3.50b 7.13c
E6 10.8c 1.5b 0.1a 18.5c 6.9b 0.6b 0.09b 0.02a 2.23a 6.50c
E7 e e e e e e e e 1.38a 4.88b
E8 e e e e e e e e 1.13a e
Numerical values followed by different letters in the same column were significantly
different at p 0.05. RBCF means BCF of root and SBCF means BCF of shoot.
J. Luo et al. / Journal of Cleaner Production 119 (2016) 25e31 29

6. animals. Chickens with higher Cd contents having a generally
higher (not significantly) survival rate indicated that chicken
mortality was controlled by famine rather than pollution.
3.4. Phytoremediation effectiveness and environmental risks
TE (total metal extraction by plant) values were used widely to
estimate the time required to reduce pollutants in soil to their safe
thresholds (Beames et al., 2015; Harris et al., 2009; Niazi et al.,
2012; Wei et al., 2006). It is worth noting that the time required
for remediation can only be predicted roughly through this
method, although the method has been adopted generally. The TE
values of E. globulus were 5.7, 11.9, 19.3 and 18.5 mg per plant in E1,
E3, E4 and E6 and the TE values of chickpeas were 0.018, 0.028,
0.031 and 0.022 mg per plant in E2, E3, E5 and E6, respectively. TE
values were assumed to remain constant. To estimate the quantity
of Cd to be removed, pollution was assumed to occur only in the top
20 cm soil, which gives a total soil mass of 2700 t haÀ1
(Ferric
Acrisols density of 1.35 g cmÀ3
).
It would require 72, 132, 49, 21, 72 and 37 years for E. globulus
monoculture (E1), chickpea monoculture (E2), mixture cultivation
without earthworms (E3), E. globulus monoculture with earth-
worms (E4), chickpea monoculture with earthworms (E5) and
mixture cultivation with earthworms (E6) to reduce the initial
average soil Cd content (0.68 mg/kg) to safe thresholds. Biomass
production and Cd accumulation and transportation demonstrated
that animal N-fixers assist phytoremediation better than plant N
fixers, at least in the present study.
The average Cd content of earthworms and chickens from clean
locations were 2.03 and 0.08 mg/kg, and the ERI values of earth-
worms ranged from 1.13 to 3.50. These values did not significantly
vary among experimental sites except in E5. ERI values of earth-
worms were generally higher when chickpea existed due to bio-
magnification. Rodriguez-Ruiz et al. (2015) observed a similar
phenomenon that environmental risk of Eisenia fetida from a
chronically polluted site was significant in the present of Lactuca
sativa and Dictyostelium discoideum. The ERI values of chickens
ranged from 2.75 to 7.15, and the presence of earthworms made
chicken ERI values significantly higher. ERI values of chickens were
generally greater than the corresponding values of earthworms.
Moreno-Jimenez et al. (2011) assessed the risk affecting potential
receptors in an abandoned mine in Spain and found earthworms
influenced terrestrial vertebrates activity in the lower positions of
the food chain; Song et al. (2015) concluded that chironomids and
flutter earthworm can transfer Cu, zinc (Zn), Pb and Cd to higher
trophic levels and trigger various ecological risks. Combining pre-
vious and present studies, these observations suggest that slight
variations in the bottom of the trophic chain may lead to large
differences in the upper part of the trophic chain.
4. Conclusion
The phytoremediation effectiveness of plants and potential
environmental risks during remediation processes in different
cultivation systems have been assessed. Nitrogen fixers, including
chickpeas and earthworms, can enhance biomass production and
Cd accumulation of E. globulus significantly. E. globulus monoculture
with an earthworm addition system had the best phytoremediation
effect which would reduce the time required to decontaminate the
pollution in soil by 30% compared to the next most effective system.
Earthworms can more effectively promote the uptake capability of
Cd in the soil by the corresponding planted crops than chickpeas
but the presence of them also exacerbated ERI values of corre-
sponding chickens in a process known as biomagnification. The
decision of which cultivation system is more suitable for an
anthropogenically influenced site should be balanced between the
capacity of the plant to remove pollution and environmental
preservation. The conclusions of this study provide an efficient
phytoremediation method with low environmental risks. Addi-
tional studies as to the exposure pathways of each receptor and as
to the cost and economic benefit of different cultivation systems are
warranted in the future.
Compliance with ethical standards
All applicable international, national, and/or institutional
guidelines for the care and use of animals were followed. All pro-
cedures performed in studies involving animals were in accordance
with the ethical standards of the institution or practice at which the
studies were conducted.
Informed consent was obtained from all individual participants
included in the study.
Acknowledgments
We gratefully acknowledge the financial support from the
Department of Finance of Guangdong Province ([2007]137).
Thanks are also given to our colleagues for their support.
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