Chemosphere 187 (2017) 97e105

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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Biomarker responses of rice plants growing in a potentially toxic

element polluted region: A case study in the Le'An Region
Yong Ji a, *, Jie Zhang a, b, Xiaoli Li a, Yongwen Peng a, Gaotang Cai a, c, Guiqing Gao a,
Jianqiang Wu d, Jiali Liu a, c
a
College of Water Conservancy and Ecological Engineering, Nanchang Institute of Technology, Nanchang 330099, China
b
College of Environment, Hohai University, Nanjing 210098, China
c
JiangXi Engineering Research Center of Water Engineering Safety and Resources Efficient Utilization, Nanchang 330099, China
d
Shanghai Academy of Environmental Sciences, Shanghai 200233, China

h i g h l i g h t s g r a p h i c a l a b s t r a c t

Toxic element accumulation shows

different capacity among different
stages.

Multi-biomarker responses vary
among different growing stages of
paddy.

Toxic elements induce different
biomarker response patterns.

Early stage is the most sensitive stage
for pollutant stress.

IBRs index response well with toxic
element concentrations.

a r t i c l e i n f o a b s t r a c t

Article history: Rice plants, planted and grown in the field, were chosen in this study to evaluate the potentially toxic
Received 11 June 2017 element pollution by combining pollutant analysis and a molecular biomarker response evaluation together
Received in revised form in the Le’An Region, a highly polluted area due to anthropogenic activities. Soils and crops at 18 sites clas-
18 August 2017
sified into four areas based on hydrological, geological and pollutant survey results were collected during the
Accepted 18 August 2017
Available online 25 August 2017
whole growth cycle for chemical and biological analysis. Sediment quality values and pollution indexes were
combined with statistical analyses to assess the hazard of potentially toxic elements and evaluate ecological
Handling Editor: T Cutright risks. As effective stress-related signals, chlorophyll (Chl), superoxide dismutase (SOD), peroxidase (POD),
catalase (CAT), glutathione content (GSH) and lipoperoxidation (as TBARS) were also determined during the
Keywords: rice plant growth period. The results revealed that heavy metal concentrations were significantly higher
Potentially toxic elements than corresponding background values and significantly related to those of the soils. The maximum con-
Biomarkers centration of potentially toxic elements was observed at the tillering stage, followed by the grain filling stage
Rice plants and heading stage. As biomarkers in field monitoring, a significant increase or decrease in Chl, SOD, POD, CAT,
Integrated biomarker response
GSH and TBARS in crops means a potential relationship between the indexes and pollutants. This study also
Le'An region
demonstrates that the integrated biomarker response (IBR) calculated by combining different biomarkers
could be used effectively to evaluate the pollutant-induced stress levels in different areas.
© 2017 Elsevier Ltd. All rights reserved.

* Corresponding author. No. 289 Tianxiang Dadao, Nanchang Institute of Technology, High-Tech Zone, Nanchang, 330099 Jiangxi, PR China.
E-mail addresses: jiyong@nit.edu.cn, 472388451@qq.com (Y. Ji).

http://dx.doi.org/10.1016/j.chemosphere.2017.08.092
0045-6535/© 2017 Elsevier Ltd. All rights reserved.
98 Y. Ji et al. / Chemosphere 187 (2017) 97e105

1. Introduction Poyang Basin and has attracted extensive attention for its serious
heavy metal pollution from enormous mining activities, some of
Rice is one of the most widely cultivated crops in the world and which have operated for more than fifty years, such as the Dexing
is now grown in 113 countries worldwide. Because of this, the Copper Mine. Combined with pollutants released from lead-zinc
growth environment of paddy fields, including soil, water and miners and gold miners distributed along the River JI'Shui, one of
pollutant behaviour, has attracted much attention in recent de- the tributaries of the River Le'An, aquatic ecosystems of the lower
cades. Intensive social activities, such as mining and processing, reaches are facing an extreme challenge in survival (Chen et al.,
have caused serious impacts on soil and aquatic ecosystems by the 2016(a); Yu et al., 2016(a)). Although mining activities have been
deposition and accumulation of heavy metals through rainfall and strictly controlled over the years, high amounts of waste water and
runoff (Atibu et al., 2016; Ji et al., 2014, 2015). Hu et al. (2014), in runoff containing heavy elements are still discharged into the main
field investigations of heavy metal distribution characteristics in river waterway (Yu et al., 2016(b)). Some villages near the mining
paddy fields at different towns in northern Hunan Province, re- areas have been reported publicly for their high cancer rates,
ported that soils near mining activities were heavily polluted due to including MingKou, DaiCun and Shanglian, which were included in
long-term irrigation with contaminated waste water. Another the present study. Meanwhile, local rural residents living along the
research study, carried out in Colombia, concluded similar results main river downstream are still planting paddy crop on local
that heavy metal pollution mainly comes from anthropogenic ac- farmland and consuming it as daily food.
tivities such as mining and agricultural production and people In our survey, we found that water in the River Le'An had been
living nearby suffer higher health threats by consuming heavy pumped for agriculture irrigation, especially during the drying
metal polluted agricultural products through the daily food chain season. However, no public reports have been published on the
(Marrugo-Negrete et al., 2017). Li et al. (2014) collected literature potentially toxic element distribution in the paddy crops during the
from 2005 to 2012 regarding the heavy metal pollution for soils whole growth cycle and the pollutants' potential ecological risk
that were near mines and concluded that planted farms neigh- assessment by combining potentially toxic element concentrations
bouring mines had suffered serious pollution. and molecular biomarker response analyses together. The present
In particular, vegetation, animals and humans in agricultural study takes paddy crops as a bioindicator using an in situ approach,
zones neighbouring mining areas may be under a continuous investigates the distribution characteristics of potentially toxic el-
health risk because heavy element accumulated in soils may be ements and evaluates the ecological risk by combining a potentially
released into the environment again due to mobility and bioavail- toxic element concentration assessment and molecular biomarker
ability. A considerable concern to human health is the consumption response analyses together. The biomarker induction of chlorophyll
of agricultural products and their uptake of contaminants from soil (Chl) and antioxidant defence enzymes such as superoxide dis-
(Botsou et al., 2016; Yu et al., 2016(a)). The results from other re- mutase (SOD), peroxidase (POD), catalase (CAT), glutathione con-
searchers also suggest that daily diet is the major exposure route of tent (GSH) and lipoperoxidation (as TBARS) in a variety of farm
heavy metals (Cao et al., 2014; Yu et al., 2016(b)). Diet, inhalation plants have been widely proposed for mitigating oxidative stress in
and skin contact recommended by US documents (US a specific response to heavy metals (Aldoobie and Beltagi, 2013;
Environmental Protection Agency, 1997) have been widely used to Ashraf and Tang, 2017). The biomarkers obtained in paddy crops
evaluate heavy metal exposure in humans. Even so, Van der Oost were then used to calculate the IBR, by which a potential correla-
et al. (2003) still recommended that the risk of access to pollut- tion between the distribution of potentially toxic elements and
ants could be mitigated by combining it with the analysis of pol- their biological effects could be established.
lutants, standard assessments and biologically effect monitoring.
Calisi et al. (2013), using biological monitoring approaches com- 2. Materials and methods
bined with heavy metal analysis, selected native earthworms as
indicators and conducted research on heavy metal contaminated 2.1. Paddy crops
sites to assess the soil contaminants and evaluate the performance
of a suite of cells. Paddy crops are widely planted in flatlands throughout the Lake
As biomarkers can respond with a reliable indication of expo- Poyang Basin, and rice is consumed as a daily diet in the selected
sure to a toxicant, they have been used widely in field monitoring research areas. In the present study, “TaiYou398”, a hybrid rice sold
and assessment of environmental impacts (Vieira et al., 2017). by Jiangxi Modern Seed Industry Company and Guangdong Acad-
Unfortunately, biomarkers of indicator organisms have different emy of Agricultural Sciences, was the main breed planted around
responses in field applications and could even provide opposite the Le’An Basin. The whole growth period of “TaiYou398” is
responses in a different environment because of natural variables approximately 112 days, and it has moderate size, general growth,
and various constraints, which sometimes make it difficult to un- strong tillering and higher effective disease resistance compared
derstand the biological responses. In these circumstances, a pool of with other breeds. Based on morphological character, the growth
biomarkers is recommended to use simultaneously to fully evaluate period may also be divided into four stages: seedling, tillering,
the responses to environmental stress (Beliaeff and Burgeot, 2002). heading, and grain filling.
However, multivariate methods based on statistical analysis cannot
well describe multiple responses of a battery of biomarkers. As a 2.2. Study area
simple method, integrated biomarker responses (IBRs) combining
all biomarkers together has been calculated and displayed as radar The main tributaries receiving pollutants and the location of
plots, which make it possible to visualize the biomarker data and mining and anthropogenic social activities in the Le’An River
compare it between different sites and different exposure condi- Basin are shown in Fig. 1. Based on the characteristics of hydro-
tions. In recent years, the IBR index has been publicly reported in logical, geological and pollutant survey results (Chen et al.,
field environmental quality assessment (Zheng et al., 2013). 2016(a); Liu et al., 2013), rice plants and soils at 18 sites
Rice is one of the most important planted crops; the planted belonging to four different types of sampling areas were
areas of paddy fields account for approximately 30% of the total collected during the whole growth period for chemical and bio-
grain crops planted in China, and the rice product is close to half of logical analysis. With respect to the samples, 5 were located
the total grain output. The Le'An River Basin is in the eastern Lake around the Village Daicun and Yankou (Area A), 4 were located
 Y. Ji et al. / Chemosphere 187 (2017) 97e105 99

Fig. 1. Map showing the geographical location of the sampling sites.

among the Village Minkou, Lanken and Liufang (Area B), 4 were grade) and 2 ml HClO4 (excellent grade) were added for digestion
located among the Village Zhengjia, Hanjia and Weijia (Area C), on a hot plate. In this study, the contents of potentially toxic
and 5 were obtained from locations ranging from the Village elements Cu, Zn, Pb, Cd Cr and As in soils, plants and grains were
Yanhu to Shanliang (Area D). The site (CK) at a higher elevation determined by inductively coupled plasma-atomic emission
and irrigated directly from the Dongfanghong Reservoir in Area C spectrometry (ICP-AES, Thermo 6300). Standard mother liquor
was selected as a control site used for comparison due to its clean ranging from 0 to 40 mg L1 was used to draw the standard curve
water resource and less social activities. with a correlation coefficient value greater than 0.999 prior to
sampling analysis. Reference materials purchased from the Chi-
2.3. Plants and soil sampling nese Academy of Geological Sciences were used to guarantee the
accuracy.
In June 2015, the Plum Blossom Method was used to sample the
paddy field surface soils from 5 to 20 cm under the positioning
coordination of a GPS (Garmi eTrex 30). At each sampling site, 2.5. Biomarker assays and IBR calculation
approximately 1 kg of mixed samples was collected and marked.
From June to September 2015, whole rice plants were also collected After being cut into small pieces to a size less than 1 mm, 0.3 g of
at the tillering, heading and grain filling stage. More than 5 rice leaves were fully ground with ethanol, quartz sand and calcium
plants were collected at each sampling point and stored in a carbonate until the samples turned white. Then, the liquid mixture
portable refrigerator (FYL-YS-18A, Fuyilian Beijing), which was was transferred into a 25 ml volumetric flask after filtering. The
supplied car power and kept at a constant temperature of 25  C. chlorophyll (Chl) content of the final transparent sample was
After transportation to the lab, these samples were rinsed with tap measured at a wave length of 652 nm. Kits provided by Nanjing
water and distilled water, dried by filter paper, cut separately into Jiancheng Biological Technology Co. Ltd. were used to determine
edible parts by stainless steel scissors, and then separately packed the soluble protein content and enzyme activities, including SOD,
into polyethylene plastic bags, numbered and labelled and then POD and CAT, and GSH and TBARS contents in the leaves of the rice
placed in the refrigerator at 40  C. plants. The Kit used the xanthine oxidase method (hydroxylamine
method) to determine the SOD activity (U mg1 protein). POD ac-
2.4. Chemical analysis tivity (U mg1 protein) was measured at 420 nm by catalysing the
corresponding substrate. CAT activity (U mg1 protein) was
After moistening with ultrapure water, 0.1 g freeze dried soil measured at 405 nm at which a light yellow complex could be
samples placed in a vial were digested on the hot plate by adding produced by reaction between the hydrogen peroxide and ammo-
4 ml pure HNO3 (excellent grade) and 1 ml HClO4 (excellent nium molybdate. GSH content (mg g1 protein) was quantitatively
grade), subsequently. To use acid reflux, a small glass funnel was determined using colorimetric assay based on the reaction with
laid on the vial. When the resultant yellow brown smoke mercaptan to compounds to produce a yellow compound. The
emptied and cooled, the left transparent digestion was filtrated, TBARS (nmol mg1 protein) in the lipid degradation product could
filled up to 50 ml in a volumetric flask and placed in a refriger- be condensed with thiobarbituric acid (TBA) to form a red product
ator until final laboratory analysis. The analysis of heavy metal with a maximum absorption peak of 532 nm. The method
contents of the rice plants and grains was similar. 0.1 g of crushed mentioned in reference by Beliaeff and Burgeot (2002) was applied
rice plants and grains were placed in a flask and wetted with a in the present study to combine all the measured biomarker re-
few drops of ultrapure water, and then 10 ml HNO3 (excellent sponses into IBR.
100 Y. Ji et al. / Chemosphere 187 (2017) 97e105

2.6. Pollution assessment model

The pollution degree of potentially toxic elements in the soils

was fully evaluated by the pollution load index (PLI), which was
mentioned by Tomlinson et al. (1980) and has been used widely as a
criterion for pollutants. Recommended by the USEPA (US
Environmental Protection Agency, 1989), a human health risk
assessment model combining Hazard Quotient (HQ) and Hazard
Index (HI) has been used to evaluate the element risk of grains to
adults. In this study, HQ and HI can be calculated by the following
equation:

Ci *IR*ED*EF
ADD ¼ (1)
BW*AT*365
ADD is the daily oral dose of potentially toxic elements (i) (mg
kg1 d1); Ci is the content of metal in grain (mg kg1); IR is daily
appetite for rural adults and has a value 0.389 kg per person per day
(Tu et al., 2015); ED is the exposure period (US Environmental
Protection Agency, 1997); EF represents the exposure frequency
and has a value of 350 d a1; BW represents the average weight of
adults and has a value of 62.7 kg; AT is the life expectation and has a
value of 70 y; and 365 is the days in a year.

ADD
HQ ¼ (2)
TDI

where TDI refers to the tolerable daily intake which can be deter-
mined by the reference (CCME, 1998; Sample et al., 1996).
X
HIn ¼ HQj (3)

where HIn is equal to the sum of the HQ of all potentially toxic el-
ements and is used to estimate the final risk for multiple elements.

2.7. Statistical analysis Fig. 2. Pollutants distribution at biomonitoring sites for soils (A), rice plants at tillering
stage (B), heading stage (C) and grain filling stage (D).

Analysis of the correlation between the total amount of heavy

metal elements in soil and rice at P < 0.05 levels was determined Cd and Pb were recorded at S17, and the highest value of Cr was
using IBM SPSS Statistics 19.0. ArcGIS 10.0 was used to assess the observed at S18.
distribution characteristics of potentially toxic elements by spatial Accumulation degree of potentially toxic elements in the whole
interpolation. The change tendency of the pollutants was calculated rice plants is presented in Fig. 2 (B)for the tillering stage, Fig. 2 (C)
using Microsoft Excel 2010. Adobe Photoshop CS 5 was selected to for the heading stage and Fig. 2 (D) for the grain filling stage. In
enhance the quality of the photographs. general, the trend of potentially toxic element concentrations, such
as Cu, Cd and As, decreased from the tillering stage to the heading
3. Results stage, and then increased from the heading stage to the grain filling
stage. Based on the values of the different stages, the accumulation
3.1. Toxic element distribution in soils and rice plants level of potentially toxic elements had a decreasing trend as fol-
lows: the tillering stage > grain filling stage > heading stage. The
The concentrations of potentially toxic elements determined at contents of potentially toxic elements in grains are listed in
the 18 sampling sites in the four areas are shown in Fig. 2. As shown Fig. 2(E). The content of potentially toxic elements among soils,
in Fig. 2(A), sites that were near the confluence of the River Jishui plants and grains is highly related, and the correlation between
and River Shuangxi and located on lower flats that flooded potentially toxic elements in organs is significant, which may be
frequently during the rainy season had higher pollutant contents. related to the absorption, transportation and enrichment process of
Contents of Cu ranged from 33.2 to 1122.7 mg kg1; Zn ranged from potentially toxic elements in plants. Compared with the values in
58.3 to 403.4 mg kg1; Pb ranged from 28.3 to 114.4 mg kg1; Cd the rice plants, the elements in grains were at a lower level. Con-
ranged from 0.21 to 11.2 mg kg1; Cr ranged from 39.4 to tents of Cu ranged from 10.64 to 26.47 mg kg1; Zn ranged from
294.4 mg kg1 and As ranged from 39.4 to 264.3 mg kg1. In 14.28 to 40.86 mg kg1; Pb ranged from 0.38 to 1.89 mg kg1; Cd
particular, sites S1 and S2 in the neighbourhood of Village Daicun in ranged from 0.15 to 0.37 mg kg1; Cr ranged from 1.3 to
area A had slightly higher concentrations of Cu, Cd and Cr. Sites S6, 7.42 mg kg1 and As ranged from 0.85 to 3.5 mg kg1.
S8 and S9 in area B were determined to have the highest pollution
levels for Cu, Zn, Cr and As based on mean values. Although sam-
pling sites in area C were on higher terrain that was not flooded 3.2. Biomarker responses
frequently during the rainy season, values of pollutants in these
areas were still slightly higher than those in the background soils of The responses of Chl, SOD, POD, CAT, GSH and TBARS are pre-
the Jiangxi Province (Gong et al., 2006). The maximum values of Cu, sented in Fig. 3. Taking the whole growth cycle into consideration,
 Y. Ji et al. / Chemosphere 187 (2017) 97e105 101

a a a
a
a
b b
b
b a
a

b b

a a
a b
a aa a
b a b
b b b
b b
b b

b
b
a b
b a b
b
b aa
a b
b b
b b
b
a a

Fig. 3. Responses of Chl, SOD, POD, CAT, GSH and TBARS measured in the leaves of Paddy plants at different sampling areas at three stages. The lower case a and b indicate values
that are significantly different from background values (a indicating P < 0.05, b indicating P < 0.01).

the biomarker responses detected at the CK site show a slowly significantly reduced at all sites at the tillering stage, and the
decreasing trend from the tillering stage to the grain filling stage. In greatest reduction in SOD activities was observed at area B. Un-
particular, the enzymatic activities of the polluted sampling sites do fortunately, SOD activities were not significantly different from the
change significantly during the whole growth cycle compared with CK site at the heading and grain filling stages. Although there are no
the CK site. Among these enzymatic responses, Chl contents at all significant differences in CAT activities between the tillering and
sites were significantly reduced and the greatest inhibition of Chl heading stage at the CK site, CAT showed a trend of decreasing at
contents was observed at sites in area B and area D. Furthermore, a the grain filling stage. Compared with the CK site, CAT activities in
significant reduction was also found at sites in area A at the tillering other cases significantly increased. The maximum values of CAT
and heading stages. In addition to area C, SOD activities were also activities were observed at the tillering stage and decreased along
102 Y. Ji et al. / Chemosphere 187 (2017) 97e105

the whole growth cycle. Similar to CAT activities, POD activities at 4. Discussion
all the sampling sites significantly induced at different stages. From
the tillering to the grain filling stage, there was a decreasing trend In general, waste water containing high concentrations of
in POD activities. The maximum induction was observed at areas A potentially toxic elements coming from upstream, tributaries and
and C. GSH content was significantly induced at all sampling sites. social activities can cause more accumulation and serious pollution
The maximum values of GSH content were observed at the tillering in downstream areas. The Jishui River carrying large amounts of
stage, and the largest induction was measured at areas A and C. A waste water from Dexin City has resulted in higher potentially toxic
significant increase in TBARS content was observed at all sampling element accumulation around S1 and S2, which are just down-
areas, especially at the heading and grain filling stages. An increase stream of the tributary. Sites in area B have been polluted under a
in TBARS content reached a peak in Areas B and D, where the mixed assemblage of pollutants coming from upstream and passing
potentially toxic element concentrations were also measured as through area A and the Shuangxi River, which is one of tributaries
maximum values. receiving numerous pollutants from surrounding nonferrous pro-
cessing factories and enterprises in townships. S7 and S9 recorded
3.3. Integrated biomarker responses the maximum concentration for Zn and As, respectively. Areas
around S17 and S18 were flooded frequently historically and are
As shown in Fig. 4, the integrated biomarker responses more easily affected by social activities focused in the township.
combining Chl, SOD, POD, CAT, GSH and TBARS at three different Moreover, landscape elevation has a negative effect on potentially
growing stages are represented as a star plot. In Fig. 4, the radius toxic element accumulation. Comparing to S1 and S2 in the flood-
coordinates are IBR values for Fig. 4(A), (B) and (C) and potentially plain area, concentrations of potentially toxic elements in S3 and S4
toxic element concentrations for Fig. 4(D), and the capital letters A, are slightly lower due to their higher elevation. A similar result
B and C refer to the sampling areas. IBR values visualized in a star could also be concluded from S10 and S11 in area C, and S14 and S15
plot can make the comparison between different sites more in area D, all of which have potentially toxic element pollution
acceptable. Fig. 4(D) is a star plot to represent the distribution levels much lower than in the other stations that are flooded
characteristics of potentially toxic elements which are expressed in frequently during the rainy season.
mg kg1. In general, the biomarker responses presented by IBR Compared with the background values mentioned in the refer-
values do show a large range variation at different growing stages ence (Gong et al., 2006), values of potentially toxic elements in CK
and in different sampling areas. The contents of potentially toxic are similar, except As, which is almost four times greater than the
elements also exhibit obvious spatial changes that are consistent background values. This is probably due to the natural background
with the IBR values. In the tillering stage, biomarker responses in ores of the soils. By dividing the mean potentially toxic element
Area A and Area C show greater induction for CAT, POD and GSH. As concentrations of the studied areas with the corresponding back-
previously mentioned, the IBR shown in Fig. 4(A) well reflect the ground values (Gong et al., 2006), potentially toxic elements Cu, Zn,
comprehensive response to stress of potentially toxic elements. Pb, Cd, Cr and As are 17.49, 3.34, 1.99, 25.31, 3.62 and 10.67 times
Similarly, biomarker responses in Area B and Area D at the heading the background values, respectively. The pollution degree of
stage and grain filling stage show higher TBARS contents and lower potentially toxic elements has a decreasing order as follows:
activities of Chl. The IBR values displayed in the two stages, as Cd > Cu > As > Cr > Zn > Pb. Performed by the method mentioned
shown in Fig. 4(B) and , (C) accurately reflect the comprehensive in reference (Tomlinson et al., 1980), values of CF, PLI, PLIzone and
response degree. PLIarea are listed in Table 1. Based on the criterion as well as back-
ground ores, only three sites, including the control site, S10 and S11,
could be considered as unpolluted. This is probably due to the
higher elevation of both S10 and S11 which are near the CK site.
However, the remaining 15 sites are in a very strong pollution
range. Area B is highly polluted with a PLIzone value of 10.20.
As heavy metals are not essential elements of paddy crops, their

Fig. 4. Star plots of four selected areas for Integrated Biomarkers Responses at Tillering
stage (A), Heading stage (B), Grain Fillering stage (C) and distribution of potentially
toxic elements in soils (D). Fig. 5. Values of Hazardous Index for grains in different sampling areas.
 Y. Ji et al. / Chemosphere 187 (2017) 97e105 103

Table 1
Pollution load indexes and status in the study area.

Area Site Contamination Factor PLI Degree PLI (zone) PLI (area)

Cu Zn Pb Cd Cr As

A S1 27.08 3.27 1.68 28.63 2.94 14.43 7.51 III 4.42 5.44
S2 23.34 2.96 1.35 21.30 2.67 9.22 6.04 III
S3 6.42 1.75 1.07 14.41 1.43 6.60 3.43 III
S4 4.31 2.65 1.69 9.22 1.09 6.86 3.32 III
S5 4.08 2.10 1.22 11.67 1.33 7.20 3.25 III
B S6 37.89 4.57 3.06 38.33 5.55 13.42 10.72 III 10.20
S7 42.64 7.42 2.80 43.52 4.41 19.55 12.22 III
S8 24.23 3.84 2.33 30.19 5.13 20.92 9.43 III
S9 12.59 4.43 2.25 27.33 6.00 22.10 8.77 III
Control CK 1.08 0.86 0.79 0.94 0.87 3.32 1.12 I
C S10 1.35 1.06 0.74 0.72 0.92 3.54 1.17 I 2.43
S11 1.37 0.97 1.12 0.80 1.53 2.50 1.29 I
S12 13.03 1.94 1.91 20.07 1.90 9.09 5.06 III
S13 12.68 1.54 1.66 18.70 1.71 8.72 4.57 III
D S14 7.59 3.91 1.03 13.89 4.09 14.42 5.41 III 8.04
S15 11.71 2.93 2.35 39.00 5.90 3.91 6.46 III
S16 9.98 3.59 2.47 37.59 4.92 5.83 6.76 III
S17 47.14 5.79 4.20 53.52 6.16 11.53 12.78 III
S18 27.44 5.47 2.93 46.74 7.53 12.30 11.13 III

absorption is a passive procedure and the main driving force is significant positive correlation among each other, indicating these
transpiration (Salt et al., 1995). The stem of a paddy serves as the might come from a similar source and are effected by the same
main transpiration channel and transports elements from the soil social activities. In particular, there were significant correlations for
to different organs (Liu et al., 2003; Salt et al., 1995). The main the same elements between the soils and rice grains, the correla-
reason for the accumulation pattern of heavy metals in different tion coefficients were 0.991 for Cu, 0.888 for Zn, 0.973 for Pb, 0.952
growth stages is that the concentration and activity of heavy metals for Cd, 0.957 for Cr and 0.970 for As, respectively. All these indicate
in the soil are higher at the tillering stage with smaller leaves, that the heavy metal pollution in the soils has affected the contents
which will cause less dry matter accumulation at this stage. During of potentially toxic elements in the grains, resulting in a higher
the growth period, the dry matter will reach a maximum with a accumulation level in the final products. In summary, the poten-
higher generation rate than the heavy metals, which will then tially toxic element contents in the rice plants and grains could be
decrease with the relative unit weight. Elements in grains mainly affected by the growth rate of dry matter accumulation at each
come from transportation from other organs, and the absorption of stage as well as the concentration of potentially toxic elements in
ions is theoretically the least (Chen et al., 2016(b); Xu et al., 2006). paddy soils.
Taking the growing stage of rice into consideration, the heavy metal Hazard Quotient (HQ) and Hazard Index (HI) mentioned by the
ions in the paddy field will change from an active state to a non- USEPA (US Environmental Protection Agency, 1989) for the four
active state through a series of chemical effects, resulting in a sampling areas are shown in Fig. 5. Based on the evaluation crite-
decrease in the total amount of heavy metal ions that can be rion, the potentially toxic element risk in grains can be classified
absorbed by the rice. Theoretically, the more paddy dry matter that into three grades (class 0: HI  1.0, indicating no apparent health
accumulates, the less heavy metals are absorbed. risk; class 1: HI > 1.0, great risks to human health; class 2: HI > 10,
Based on the enrichment coefficient of potentially toxic ele- chronic toxicity). Based on the calculation results of HI, values of HI
ments performed by dividing concentration between soils and for all sampling sites fall into class 1, indicating potential health
grains, the enrichment degree of the six elements decreases in the risks for humans. In addition, sampling sites in areas B and D nearly
sequence of Zn > Cd > Cu > Cr > Pb > As. A Pearson correlation fall into class 2, which means people who consume grains produced
analysis was also used to analyse the contents of potentially toxic in these areas are facing even higher health risks. The degree of
elements in soil and rice using SPSS 19.0. As shown in Table 2, both health risk in grains yielded the following ranking: D > B > A > C.
potentially toxic elements in soil and rice grains indicated Taking different elements into consideration, HQ for the six

Table 2
Pearson correlation analysis between the contents of potentially toxic elements in soil and rice grains.

Cu Zn Pb Cd Cr As GCu GZn GPb GCd GCr GAs

Cu 1 .832** .829** .837** .639** .596** .991** .758** .848** .728** .577** .651**
Zn 1 .797** .846** .793** .723** .824** .888** .776** .752** .736** .776**
Pb 1 .926** .806** .451 .802** .644** .973** .872** .790** .524*
Cd 1 .873** .503* .802** .661** .886** .952** .872** .574*
Cr 1 .562* .598** .525* .765** .879** .957** .647**
As 1 .576** .570* .387 .368 .410 .970**
GCu 1 .776** .829** .704** .545* .642**
GZn 1 .671** .556* .487* .642**
GPb 1 .831** .737** .474*
GCd 1 .925** .481*
GCr 1 .520*
GAs 1

Note: ** Significant level of correlation 0.01 (bilateral), * Significant level of correlation 0.05 (bilateral), G means potentially toxic elements in Grains.
104 Y. Ji et al. / Chemosphere 187 (2017) 97e105

selected potentially toxic elements in the present research areas responses was used to display the bio responses and make a
decreases in the following sequence: Cr > As > Cu > Pb > Cd > Zn. comparison among different areas. Overall, the most contaminated
The top three elements for the HI are Cr, As and Cu. sites were observed with larger biomarker responses. The com-
Chl is one of the most important pigments related to photo- parison results based on the IBR values reveal that Areas B and D,
synthesis. The content of chlorophyll can determine the growth and seriously polluted by potentially toxic elements, are a more
accumulation of dry matter. Published papers have indicated that stressful place for paddy growth. There is a good visual relationship
heavy metals can affect chlorophyll content, which could be between the potentially toxic element gradient observed in soils
induced by lower concentrations of heavy metals and inhibited by and the IBR variation at different growing stages. Hou et al. (2016)
higher concentrations of heavy metals. As shown in Fig. 4(A), used the integrated biomarker responses index combined by the
chlorophyll content in this research was significantly inhibited at activities of CAT, SOD, glutathione reductase (GR), and glutathione
different stages, especially in Areas B and D which were seriously peroxidase (GSH-Px) to evaluate the toxicity of ZnO Nps on fluvial
polluted by potentially toxic elements. Large amounts of hydrogen biofilm. The IBR index proved to be a useful evaluation system for
peroxide would be produced during the peroxidation process un- ZnO NPs toxicity evaluation. Beaumelle et al. (2017), calculating the
der the threat of heavy metals, and these materials can enter the IBR in three subcellular fractions of earthworms (Aporrectodea
chloroplast through the wax and attack the chloroplast pigment caliginosa) after 21 days exposed to field contaminated soils,
protein complex (Bazzaz et al., 2010). In addition, the accumulation revealed good accordance between IBR values and potentially toxic
of heavy metals could damage the structure of chloroplast and element levels. To evaluate the pollution degree and toxicity in Lake
reduce the content of soluble proteins and activities of Mg2þ- Tai, Lu et al. (2010) and Ji et al. (2010) conducted an active bio-
ATPase (Ebbs and Uchil, 2008). monitoring examination using IBR index combined with biotrans-
In normal situations, production and elimination of active oxy- formation enzymes 7-ethoxyresorufin-O-deethylase (EROD), GSH,
gen can maintain a balance until a change of the outer environ- CAT, TBARS and revealed good agreement between IBR values and
ment, which can cause accumulation of radical and membrane lipid organic pollutant concentrations.
peroxidation. In this case, plant antioxidases, including SOD, POD
and CAT, and non-enzymatic substances, such as GSH, can work 5. Conclusions
together to reduce the effect caused by extra active oxygen. Under
the threat of heavy metals, plants can use a stress protective This research completed an integrated investigation on poten-
response to protect health and suffer depression during metabolic tially toxic element pollution and biomarker responses of rice
processes (Zhang et al., 2006). As shown in Fig. 4(B), (C)(D) and (E), plants in different growing stages in the Le’An Region. The results
not including the tillering stage, the responses of SOD activities indicate that social activities have caused a serious effect on the
were not obviously different from the CK site. However, CAT and distribution of pollutants and resultant higher health risks for
POD were significantly induced for all sampling sites, and the consuming rice. Transpiration can directly affect the pattern of
maximum values were observed at the heavily polluted sites. All accumulation of potentially toxic elements at different stages and
these indicate that heavy metals had caused a start-up of the lead to less accumulation of pollutants in grains. This research also
antioxidase protection system. In areas B and D with a higher demonstrated that the early stage of a paddy was the most sensitive
concentration of potentially toxic elements, active oxygen can period for heavy metal stress. Potentially toxic element analysis
accumulate at a higher level, which causes the damage of mem- combined with IBR evaluation describes a good relationship be-
brane structure and leads to a reduction in enzymatic activities. The tween biomarkers and pollutants for different polluted sites.
present research also observed the trend of enzymatic activities
including SOD, CAT and POD with a following decreasing sequence: Acknowledgments
tillering stage > heading stage > grain filling stage. This is mainly
because the antioxidant system enzymes are affected by two fac- This study was supported by Jiangxi Provincial Education
tors in the process of growth; one is the extrinsic factors of heavy Department (GJJ161094), National Natural Science Foundation of
metals, and the other is the internal metabolic process of plant China (51469017; 51579127; 51679141), Jiangxi Provincial
senescence. This research also revealed that the early stage of a Technology Department (20142BBF60012; 20142BAB213024) and
paddy was the most sensitive period for heavy metal stress. With Open Foundation of JiangXi Engineering Research Center of Water
the growth process, the resistance of the paddy to potentially toxic Engineering Safety and Resources Efficient Utilization (OF201608).
elements increased, and a series of antioxidant enzymes activities
gradually decreased. References
The experimental results show that GSH and MDA contents
could increase gradually along with an increase in potentially toxic Aldoobie, F.N., Beltagi, S.M., 2013. Physiological, biochemical and molecular re-
sponses of common bean (Phaseolus vulgaris L.) plants to heavy metals stress.
element concentrations in different growth periods. GSH can keep Afr. J. Biotechnol. 12 (29), 4614e4622.
the membrane protein structure stable, remove extra ROS induced Ashraf, U., Tang, X., 2017. Yield and quality responses, plant metabolism and metal
by potentially toxic elements and effectively reduce the reactive distribution pattern in aromatic rice under lead (Pb) toxicity. Chemosphere 176,
141e155.
oxygen substances stress. GSH can also provide material for the Atibu, E.K., Devarajan, N., Laffite, A., Giuliani, G., Salumu, J.A., Muteb, R.C.,
synthesis of peptides, thereby reducing the accumulation of Mulaji, C.K., Otamonga, J.P., Elongo, V., 2016. Assessment of trace metal and rare
potentially toxic elements. Accumulation of MDA showed that the earth elements contamination in rivers around abandoned and active mine
areas. The case of Lubumbashi River and Tshamilemba Canal, Katanga. Democr.
rice plant was stressed by free radicals, which caused peroxidation Repub. Congo. Chem. Erde-Geochem 76 (3), 353e362.
of cell membranes in the leaves of the paddy. Bazzaz, F.A., Rolfe, G.L., Carlson, R.W., 2010. Effect of Cd on photosynthesis and
As different stressors target different biomarkers, using a pool of transpiration of excised leaves of Corn and Sunflower. Physiol. Plant. 32 (4),
373e376.
available biomarkers can fully take advantage of biomarkers to
Beaumelle, L., Hedde, M., Vandenbulcke, F., Lamy, I., 2017. Relationships between
assess the effect of outer pollutants and provide a more reliable metal compartmentalization and biomarkers in earthworms exposed to field-
interpretation of exotoxicological examinations. In this study, contaminated soils. Environ. Pollut. 224, 185e194.
antioxidase and oxidative stress parameters of phases I and II were Beliaeff, B., Burgeot, T., 2002. Integrated biomarker response: a useful tool for
ecological risk assessment. Environ. Toxicol. Chem. 21 (6), 1316e1322.
chosen to provide early signals of potentially toxic elements in a Botsou, F., Sungur, A., Kelepertzis, E., Soylak, M., 2016. Insights into the chemical
paddy. The star radar plot drawing by the integrated biomarker partitioning of trace metals in roadside and off-road agricultural soils along two
 Y. Ji et al. / Chemosphere 187 (2017) 97e105 105

major highways in Attica's region, Greece. Ecotox. Environ. Safe 132, 101e110. Lu, G.H., Ji, Y., Zhang, H.Z., Wu, H., Qin, J., Wang, C., 2010. Active biomonitoring of
Calisi, A., Zaccarelli, N., Lionetto, M.G., Schettino, T., 2013. Integrated biomarker complex pollution in Taihu Lake with Carassius Auratus. Chemosphere 79 (5),
analysis in the earthworm Lumbricus terrestris : application to the monitoring 588e594.
of soil heavy metal pollution. Chemosphere 90 (11), 2637e2644. Marrugo-Negrete, J., Pinedo-Hern andez, J., Díez, S., 2017. Assessment of heavy metal
Cao, S., Duan, X., Zhao, X., Ma, J., Dong, T., Huang, N., Sun, C., He, B., Wei, F., 2014. pollution, spatial distribution and origin in agricultural soils along the Sinú
Health risks from the exposure of children to As, Se, Pb and other heavy metals River Basin, Colombia. Environ. Res. 154, 380e388.
near the largest coking plant in China. Sci. Total. Environ. 472, 1001e1009. Salt, D.E., Prince, R.C., Pickering, I.J., Raskin, I., 1995. Mechinisms of cadmium
CCME, 1998. Protocol for the Derivation of Canadian Tissue Residue Guidelines for mobility and accumulation in Idian mustard. Plant Physiol. 109 (4), 1427e1433.
the Protection of Wildlife that Consume Aquatic Biota. Canadian Council of Sample, B.E., Opresko, D.M., Suter, G.W.I., 1996. Toxicological Benchmarks for
Ministers of the Environment, Winnipeg. Wildlife: 1996 Revision. Risk Assessment Program Health Sciences Research
Chen, H., Chen, R., Teng, Y., Jin, W., 2016(a). Contamination characteristics, ecolog- Division Oak Ridge. Tennessee.
ical risk and source identification of trace metals in sediments of the Le'An River Tomlinson, D.L., Wilson, J.G., Hariis, C.R., Jeffrey, D.W., 1980. Problems in the
(China). Ecotox. Environ. Safe 125, 85e92. assessment of heavy metal levels in estuaries and the formation of a pollution
Chen, Q., Mu, X., Chen, F., Yuan, L., Mi, G., 2016(b). Dynamic change of mineral index. Helgol. Meeresun 33 (1e4), 566e575.
nutrient content in different plant organs during the grain filling stage in maize Tu, J.F., Liu, L.Y., Luo, Q., Chen, H.Z., Wu, Y.Q., Chen, W.W., Song, Y.K., Huang, W.,
grown under contrasting nitrogen supply. Eur. J. Agron. 80, 137e153. Yao, Q.H., 2015. Contents and health risk assessment of Cadmium in milled rice
Ebbs, S., Uchil, S., 2008. Cadmium and zinc induced chlorosis in Indian mustard in Fujiang Province. J. Agro-Environ. Sci. 34 (4), 695e701 (in Chinese).
[Brassica juncea, (L.) Czern] involves preferential loss of chlorophyll b. Photo- US Environmental Protection Agency, 1997. Exposure Factors Handbook. EPA 600/P-
synthetica 46 (1), 49e55. 95/002Fa (Update to Exposure Factors Handbook (EPA/600/8-89/043)). United
Gong, X.F., Chen, C.L., Zhou, W.B., Jian, M.F., Zhang, Z.H., 2006. Assessment on heavy States Environmental Protection Agency, Washington, D.C.
metal pollution in the sediment of Poyang Lake. Environ. Sci. 27, 732e736 (in US Environmental Protection Agency, 1989. Risk Assessment Guidance for Super-
Chinese). fund, Volume I: Human Health Evaluation Manual (Part a). EPA/540/1e89/002.
Hu, X.F., Jiang, Y., Shu, Y., Hu, X., Liu, L., Luo, F., 2014. Effects of mining wastewater United States Environmental Protection Agency, Washington, D.C.
discharges on heavy metal pollution and soil enzyme activity of the paddy Van der Oost, R., Beyer, J., Vermeulen, N.P.E., 2003. Fish bioaccumulation and bio-
fields. J. Geochem. Explor 147 (B), 139e150. markers in environmental risk assessment: a review. Environ. Toxicol. Phar 13
Hou, J., You, G., Xu, Y., Wang, C., Wang, P., Miao, L., Dai, S., Lv, B., Yang, Y., 2016. (2), 57e149.
Antioxidant enzyme activities as biomarkers of fluvial biofilm to ZnO NPs Vieira, C.E.D., Costa, P.G., Cabrera, L.C., Primel, E.G., Fillmann, G., Bianchini, A.,
ecotoxicity and the Integrated Biomarker Responses (IBR) assessment. Ecotox. Martinez, C.B.D.R., 2017. A comparative approach using biomarkers in feral and
Environ. Safe 133, 10e17. caged Neotropical fish: implications for biomonitoring freshwater ecosystems
Ji, Y., Lu, G., Wang, C., Song, W., Wu, H., 2010. Fish transplantation and stress-related in agricultural areas. Sci. Total. Environ. 586, 598e609.
biomarkers as useful tools for assessing water quality. J. Environ. Sci. 22 (11), Xu, J., Yang, L., Wang, Z., Dong, G., Huang, J., Wang, Y., 2006. Toxicity of copper on
1826e1832. rice growth and accumulation of copper in rice grain in copper contaminated
Ji, Y., Zhang, J., Huang, X., Bai, C., Chen, X., 2014. Investigation and assessment of soil. Chemosphere 62 (4), 602e607.
heavy metals in surface sediments of Ganjiang River, China. J. Environ. Biol. 35 Yu, Y., Hui, W., Qi, L., Wang, B., Yan, Z., Ding, A., 2016(a). Exposure risk of rural
(6), 1173e1179. residents to copper in the Le'An River Basin, Jiangxi Province, China. Sci. Total.
Ji, Y., Zhang, J., Li, R., Pan, B., Zhang, L., Chen, X., 2015. Distribution and partitioning Environ. 548e549, 402e407.
of heavy metals in sediments of the Xinjiang river in Poyang Lake Region, China. Yu, Y., Li, Q., Wang, H., Wang, B., Lu, Q., Yan, Z., Ding, A., 2016(b). Exposure risk of
Environ. Prog. Sustain 34 (3), 713e723. young population to lead: a case study in Le'An River Basin in Jiangxi Province,
Li, Z., Ma, Z., Kuijp, T.J.V.D., Yuan, Z., Huang, L., 2014. A review of soil heavy metal China. Environ. Pollut. 209, 140e146.
pollution from mines in China: pollution and health risk assessment. Sci. Total. Zhang, X., Wang, D., Chu, K., Yang, C., Mou, R., Chen, M., Zhu, Z., He, Q., Liao, X., 2006.
Environ. 468e469, 843e853. Changes of Sod activity and MDA content in rice exposed to Cd stress as affected
Liu, G., Tao, L., Liu, X., Hou, J., Wang, A., Li, R., 2013. Heavy metal speciation and by genotype. Chin. J. Rice. Sci. 20 (2), 194e198 (in Chinese).
pollution of agricultural soils along Jishui River in non-ferrous metal mine area Zheng, Q., Feng, M., Dai, Y., 2013. Comparative antioxidant responses in liver of
in Jiangxi Province, China. J. Geochem. Explor 132 (3), 156e163. Carassius auratus, exposed to phthalates: an integrated biomarker approach.
Liu, J., Li, K., Xu, J., Zhang, Z., Ma, T., Lu, X., Yang, J., Zhu, Q., 2003. Lead toxicity, Environ. Toxicol. Phar 36 (3), 741e749.
uptake, and translocation in different rice cultivars. Plant. Sci. 165 (4), 793e802.