Environmental Pollution 257 (2020) 113581

Contents lists available at ScienceDirect

Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol

Health risk assessment of heavy metals (Zn, Cu, Cd, Pb, As and Cr) in
wheat grain receiving repeated Zn fertilizers*
Yu-Min Liu a, Dun-Yi Liu b, Wei Zhang b, Xiu-Xiu Chen a, Qing-Yue Zhao a, Xin-Ping Chen b,
Chun-Qin Zou a, *
a
College of Resources and Environmental Science, National Academy of Agriculture Green Development, Key Laboratory of Plant-Soil Interactions, Ministry
of Education, China Agricultural University, Beijing 100193, China
b
College of Resources and Environment, Southwest University, Chongqing, 400715, China

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

Article history: Soil application of Zn fertilizer is an effective approach to improve yield and Zn accumulation in wheat
Received 2 August 2019 grain. However, it remains unclear whether repeated Zn application can result in high accumulation of
Received in revised form heavy metals (HMs) in soils and grains and thus represents a potential risk for human consumption. This
3 November 2019
study aimed to evaluate the health risk assessment of HMs in a wheat production system which had
Accepted 4 November 2019
Available online 8 November 2019
continuously received 8 years of Zn application at varying rates (0, 2.3, 5.7, 11.4, 22.7, 34.1 kg Zn ha
1).
The results showed that Zn application significantly increased the soil total Zn concentration without
affecting concentrations of As, Pb, Cd, Cu and Cr. Across Zn rates, Zn application increased grain con-
Keywords:
Heavy metals
centrations of Zn, Pb and Cd by 75%, 51% and 14%, respectively, and reduced grain As concentration by
Health risk assessment 14%. The human health risk assessment revealed that the threshold hazard quotients for the individual
Wheat grain HM were below 1, independent of Zn rates. The hazard index (HI) values at Zn rates of 11.4, 22.7 and
Zinc fertilization 34.1 kg Zn ha
1 were significantly greater than that at null Zn treatment. Furthermore, exposures to As,
Cu and Zn accounted for 97% of HI at all Zn rates. Analysis of the threshold cancer risk with Pb and As
showed that ingestion of wheat grain even from highest Zn application rate wouldn’t bring the lifetime
carcinogenic risk. In contrast, long-term Zn application significantly reduced the carcinogenic risk of As
by 9.7e26.5%. In conclusion, repeated soil applications of Zn at optimal rate (5.7 kg Zn ha
1) didn’t cause
health risk for Zn, Cu, Cd, Pb, Cr, and As, while improving productivity and grain Zn concentration of
wheat to meet human recruitment. Our study highlights the importance of appropriate Zn fertilizer
management in improving grain quality while reducing HMs risks from human consumption.
© 2019 Elsevier Ltd. All rights reserved.

1. Introduction (Cakmak, 2008). Wheat (Triticum aestivum L.) is a dominant staple

Zinc (Zn) is an important micronutrient for healthy growth and
reproduction of plants, animals, and humans (Welch and Graham,
2004). As a key component for various enzymes in the living or-
ganisms, Zn plays an important role in essential cellular functions
and metabolic pathways (Nriagu, 2011; Hafeez et al., 2013). In
plants, Zn is involved in many important biochemical pathways,
such as carbohydrate metabolism, protein synthesis, auxin meta-
bolism and pollen formation (Broadley et al., 2012). Zinc deficiency
in cereal plants can reduce crop yield and nutritional quality
*
€rg Rinklebe.
This paper has been recommended for acceptance by Dr. Jo
* Corresponding author.
E-mail address: zcq0206@cau.edu.cn (C.-Q. Zou).
cereal crop and provides 50% of daily calorie and more than 20% of
the daily Zn intake for people in the developing countries (Hotz and
Brown, 2004; Ma et al., 2008). China is the world’s largest wheat
producer and provides 18% of the global wheat grain products (Kim
and Dale, 2004). However, grain Zn concentration of wheat in China
is generally low with an average of only 23.3 mg kg
1 (Chen et al.,
2017). Agronomic biofortification (e.g. application of Zn fertilizer)
is an important and complementary approach to improve Zn
nutrition in crops (Cakmak, 2008). Compared with foliar Zn
application, soil application was more effective to sustain yield
productivity and improve Zn nutrition of crops (Cakmak and
Hoffland, 2012; Yilmaz et al., 1997). However, repeated applica-
tions of Zn fertilizer may potentially increase the heavy metals
(HMs) accumulation in soil and cereal grain and thus represents a
threat for human consumption (Jiao et al., 2012).

https://doi.org/10.1016/j.envpol.2019.113581
0269-7491/© 2019 Elsevier Ltd. All rights reserved.
2 Y.-M. Liu et al. / Environmental Pollution 257 (2020) 113581
Heavy metals ingestion by human can damage the nervous,
skeletal, circulatory, enzymatic, endocrine, and immune systems
(Wang, 2013; Li et al., 2014b; Mohamed et al., 2017). Presentation of
HMs in cereal grain at even low concentrations can be toxic for
humans (Tchounwou et al., 2012). Excessive accumulation of Zn in
soil may increase crop uptake and further result in an unexpected
high level of grain Zn concentration that exceeds the consumption
safe limit (Noulas et al., 2018; Wongsasuluk et al., 2018). In addition,
Zn present in soil can influence the absorption of other HMs by
crops (Kolasinac et al., 2018). For instance, some studies showed
that Zn application reduced the toxic effect of cadmium (Cd) in
plants through decreasing its uptake by roots (Dalir et al., 2017;
Saifullah et al., 2014). Das et al. (2008) showed that soil application
of ZnSO4 reduced arsenic (As) concentration in soil and rice grain by
35.1% and 10.2%, respectively. To our knowledge, no previous
studies have evaluated the health risk of HMs in Zn-biofortified
grain of wheat. Our hypothesis is that continuous Zn fertilizer
application especially at high rate would increase the concentration
and accumulation of HMs in soils and wheat grain and thus rep-
resents a human health risk for human consumption. Human
health risk assessment (HHRS) is widely used as an effective
approach to evaluate the risk of HMs by ingestion of grains for
human health (Pandey et al., 2012; Funari and Testai, 2008). In such
an assessment, threshold hazard quotient (THQ), hazard index (HI)
and threshold cancer risk (TCR) are commonly used as indexes to
verify the non-carcinogenic and carcinogenic risks of HMs in cereal
grains on human health (USEPA, 2006; Abbasi et al., 2013).
To understand and assess the health risk of Zn-biofortified
wheat grain, HHRS method was used to evaluate the health risk
of Zn, copper (Cu), Cd, lead (Pb), As and chromium (Cr) by
consuming wheat grain which had received 8 years’ continuous Zn
application at varying rates. The specific objectives of this study
were 1) to evaluate the effects of repeated soil application of Zn
fertilizer on the concentrations of Zn, Cu, Cd, Pb, As, and Cr in wheat
grain and soil, 2) to assess the potential health risks for local con-
sumers of wheat grain that was exposed to HMs by repeated Zn
applications. This study will thus provide important information on
Zn biofortification in cereal crops in relation to human health risk.
2. Materials and methods
2.1. Site description and experiment design
The field experiment was conducted from 2009 to 2017 in
Quzhou Experiment Station (36.9 N, 115.0 E) in Hebei Province,
China. The map of the study area is provided in Fig. S1. The
experimental site has a cool to warm temperate monsoon climate.
The mean air temperature over the last 8 years was 14.2  C and
annual precipitation was 430 mm. The soil is classified as a typical
calcareous alluvial soil based on the World Reference Base classi-
fication (Chesworth et al., 2008). The initial soil had a silt loam
texture (clay 79, silt 553 and sand 368 g kg
1), pH of 8.0 (1:2.5 w/v
of soil: water), and organic matter content of 10 g kg
1. Soil organic
matter was determined by wet oxidation using a Vario Max CN
instrument (Elementar, Langenselbold, Germany). The concentra-
tions of total Zn, Cu, Cd, Pb, As and Cr in the initial soil (HNO3-HCl
digestion method) were 60.5, 21.4, 0.5, 8.5, 5.2 and 25.9 mg kg
1,
respectively.
This study investigated the winter wheat phase in a winter
wheat-summer maize rotation in which wheat was planted in early
October and harvested in early June of the following year. In each
growing season, six levels of Zn fertilizer (0, 2.3, 5.7, 11.4, 22.7, and
34.1 kg Zn ha
1) were applied to both wheat and maize at time of
planting. Each treatment had four replicates in a randomized
complete block design. The area of each plot was 75 m2. Same
wheat cultivar (Liangxing 99) was used throughout the eight years.
Before planting, Zn fertilizer as ZnSO47H2O was sprayed onto the
soil surface as an aqueous solution, following which a compound
fertilizer (N-P2O5-K2O: 15-15-15; 75 kg ha
1) was broadcasted onto
the soil surface. All fertilizers were then incorporated into the
30 cm depth using a rotary tillage machine. Additional 150 kg N
ha
1 as urea was applied at the jointing stage of wheat. Irrigations
were conducted at the before-winter, jointing, and flowering
stages. The source of irrigation water was groundwater and each
irrigation provided approximately 90 mm of water.
2.2. Sampling and analyses
2.2.1. Soil sampling and analyses
Five soil samples (0e30 cm) were collected from each plot using
a stainless-steel auger at maturity stage of wheat in the 8th year
(June 2017) after establishment of the experiment. Soil samples
from each plot were combined as one composite sample, air-dried,
ground and then sieved through a 100-mesh screen for further
analysis. A 0.25 g of dry soil was subjected to acid digestion as
described by Mico  et al. (2007) The digested solution was diluted
using the ultrapure water to a volume of 25 ml for analysis of HMs.
Concentrations of Zn, Cu, Cd, Pb, As and Cr in the solution were
determined using an inductively coupled plasma spectroscopy
(ICPAES, OPTIMA 7300 DV, PerkineElmer, USA). Certified soil ma-
terials (ISE865, Wageningen University, Netherlands) were used for
quality control.
2.2.2. Grain sampling and analyses
At maturity stage of wheat in the 8th year (June 2018), wheat
grain samples were hand-harvested by randomly collecting two
0.5-m rows from each plot. All grain samples were carefully washed
with deionized water and then dried at 60e65  C to a constant
weight. Dry samples were then grounded using a stainless-steel
grinder for HMs analysis. The samples were microwave-digested
by using a mixture acid of HNO3-H2O2 (Niemel€ a et al., 2005).
Concentrations of Zn, Cu, Cd, Pb, As and Cr in the digested solution
were then determined using an inductively coupled plasma mass
spectroscopy (ICPMS, Agilent 7700x, Agilent Technologies, USA).
Certified grain materials (IPE568, Wageningen University,
Netherlands) were used for quality control.
2.3. Calculations
2.3.1. Bioconcentration factor (BCF)
The BCF was used to assess the translation ability of HM from
soil to crop grain, calculated using Eq (1) according to Sipter et al.
(2008) and Adamoa et al. (2014).
Cgrain
BCF ¼ (1)
Csoil
Where Cgrain and Csoil were the HM concentrations in wheat grain
and soil, respectively.
2.3.2. Non-carcinogenic risk
The health risk caused by non-carcinogenic HM exposure from
the food intake was assessed using the THQ (USEPA, 2006), calcu-
lated by Eq. (2)
C  DI  EF  EDtotal
THQ ¼ (2)
RfD  BW  ATn
Where: C ¼ HM concentration in wheat grain; DI ¼ daily intake of
wheat grain (assumed 94.47 g day
1 for children and 159.9 g day
1
 Y.-M. Liu et al. / Environmental Pollution 257 (2020) 113581 3

for adults) (Zhai and Yang, 2006; Yang and Zhai, 2006);
EF ¼ exposure frequency (350 days/year); EDtotal ¼ exposure dura-
tion (6 years for children and 30 years for adults); RfD ¼ reference
dose (being 0.3, 0.04, 0.001, 0.004, 0.0003, and 1.5 mg kg
1 day
1
for Zn, Cu, Cd, Pb, As, and Cr, respectively according to USEPA,
2006); Bw ¼ average body weight (32.7 kg for children and
63.9 kg for adults) (Guo et al., 2018); ATn ¼ average time of expo-
sure to non-carcinogenic HMs (EDtotal  365 days/year).
To assess the overall non-carcinogenic effects posed by all HMs,
the THQ for each respective HM was summed (assuming additive
effects) and expressed as the HI (USEPA, 2006), according to Eq. (3)
2.4. Statistical analysis
A one-way analysis of variance (ANOVA) was performed using
SAS 8.0 (SAS Institute, Cary, NC) to determine the effect of Zn rate
on HMs concentrations in wheat grain and soil, BCF, THQ, HI and
TCR. Where the effect was significant, treatment means were
compared using Fisher’s protected least significant difference (LSD)
test at P < 0.05.
3. Results

X
i 3.1. Concentrations of HMs in soils and grains
HI ¼ THQ n (3)
n¼1 Across the six Zn application rates, the mean total concentra-
tions of Zn, Cu, Cd, Pb, As and Cr in the soil were 108.4, 21.5, 0.4, 11.0,
If the values of THQ or HI were 1, it was assumed no significant
6.3 and 21.4 mg kg
1, respectively (Table 1). The total Zn concen-
risk of non-carcinogenic effect. In contrast, values of THQ or HI
tration in soil increased with Zn rates, with the highest level of
greater than 1 indicated a greater chance of non-carcinogenic effect
185.8 mg kg
1 being observed in the treatment of 34.1 kg Zn ha
1.
(USEPA, 2006).
Concentrations of Cu, Cd, Pb, As and Cr in soil were not affected by
Zn fertilizer treatment.
The average Zn, Cu, Cd, Pb, As, and Cr concentrations in wheat
2.3.3. Carcinogenic risk
grain were 52.3 mg kg
1, 5.5 mg kg
1, 11.8 mg kg
1, 19.2 mg kg
1,
Threshold cancer risk (TCR) is a parameter used to determine
53.3 mg kg
1 and 232.6 mg kg
1, respectively (Table 2). Grain con-
the incremental probability of an individual developing cancer over
centrations of Zn, Cd and Pb significantly increased with Zn rates.
a lifetime (USEPA, 2006). The TCR of the carcinogenic HMs (Pb and
Grain As concentration was generally unaffected by Zn fertilizer
As) was calculated by Eq. (4):
except at the highest rate (34.1 kg Zn ha
1) where grain As con-
C  DI  SF  EF  EDtotal centration was significantly reduced by 26.5% than control. Grain Cr
TCR ¼ (4) concentration was significantly greater at Zn rate of 5.7 kg Zn ha
1
BW  ATc
than other levels of Zn rates. There was no treatment effect on grain
Where: ATc is the averaging time for carcinogens (70  365 days). Cu concentration.
SF was cancer slope factor, being 8.5  10
3 and 1.5 mg g
1 day
1 for The BCF values for the studied HMs decreased in the order of
Pb and As, respectively (USEPA, 2006). The C, DI, EF, EDtotal and BW Zn > Cu > Cd > Cr > As > Pb (Fig. 1). Zinc fertilizer application
were defined as above. significantly affected the BCF values of Zn and Cd. With increasing

Table 1
Total concentrations of study heavy metals in soil as affected by Zn application rates.

Zn rate (kg Zn ha
1) Heavy metal concentration in soil (mg kg
1)

Zn Cu Cd Pb As Cr

0 61.3 ± 1.8e 21.4 ± 0.4a 0.43 ± 0.05a 10.8 ± 1.5a 5.9 ± 1.4a 21.6 ± 2.0a
2.3 69.3 ± 8.1e 21.0 ± 1.3a 0.38 ± 0.05a 10.7 ± 2.1a 5.7 ± 1.6a 20.4 ± 2.5a
5.7 85.1 ± 8.6d 21.9 ± 1.0a 0.40 ± 0.06a 11.2 ± 0.7a 6.3 ± 1.6a 22.5 ± 2.9a
11.4 101.0 ± 8.1c 21.1 ± 1.3a 0.38 ± 0.05a 10.2 ± 1.5a 6.2 ± 1.7a 21.8 ± 1.9a
22.7 147.9 ± 11.9b 22.0 ± 1.6a 0.42 ± 0.05a 11.7 ± 0.5a 7.7 ± 1.4a 21.8 ± 2.9a
34.1 185.8 ± 7.9a 21.4 ± 0.3a 0.43 ± 0.05a 11.4 ± 1.3a 6.8 ± 1.3a 20.1 ± 0.6a
Average 108.4 21.5 0.40 11.0 6.4 21.4
MACa (soil) 300 100 0.8 80 25 250

Values are means ± SD of four replicates and are not significantly different at P < 0.05 when followed by the same lowercase letters.
a
MAC is the maximum allowable concentration according to China Environmental Quality Standard for Soil (CEPA, 1995).

Table 2
Total concentrations of study heavy metals in wheat grain as affected by Zn application rates.

Zn rate (kg Zn ha
1) Heavy metal concentration in grain

Zn Cu Cd Pb As Cr

(mg kg
1) (mg kg
1)

0 32.1 ± 3.1d 5.4 ± 0.4a 10.6 ± 1.0b 13.4 ± 5.8b 60.2 ± 10.2a 242.8 ± 17.6b
2.3 43.1 ± 3.4c 5.6 ± 0.5a 10.4 ± 0.9b 12.5 ± 1.4b 58.5 ± 10.1a 232.7 ± 36.3b
5.7 47.8 ± 2.8c 5.7 ± 0.4a 10.0 ± 1.2b 18.6 ± 6.3 ab 53.8 ± 6.5 ab 309.6 ± 45.3a
11.4 58.0 ± 6.3b 5.5 ± 0.3a 12.1 ± 2.8a 24.0 ± 5.7a 52.7 ± 10.2 ab 207.8 ± 54.1b
22.7 64.1 ± 6.1a 5.6 ± 0.2a 13.7 ± 2.1a 22.2 ± 3.6a 50.2 ± 6.0 ab 216.4 ± 49.7b
34.1 68.5 ± 5.0a 5.5 ± 0.5a 13.2 ± 1.0a 24.3 ± 4.9a 44.3 ± 7.6b 186.1 ± 43.4b
Average 52.3 5.5 11.8 19.2 53.3 232.6
MACa (wheat) 50 20 100 200 500 1000

Values are means ± SD of four replicates and are not significantly different at P < 0.05 when followed by the same lowercase letters.
a
MAC is the maximum allowable concentration for cereal grain (USEPA, 2012).
4 Y.-M. Liu et al. / Environmental Pollution 257 (2020) 113581

Fig. 1. Bioconcentration factor of Zn, Cu, Cd, Pb, As and Cr affected by Zn application rates. Bars represent SD of four replicates, ** indicates a significant difference at P < 0.01 among
Zn treatments.

Table 3
Threshold hazard quotient and hazard index of heavy metals in wheat grains in exposure to the children and the adults as affected by Zn application rates.

Zn rate (kg Zn ha
1) Threshold hazard quotient in grain Hazard index

Zn Cu Cd Pb As Cr

(  10
2) (  10
3) (  10
4)

Children
0 0.30 ± 0.003d 0.37 ± 0.03a 2.93 ± 0.26b 9.29 ± 4.00b 0.56 ± 0.09a 4.48 ± 0.33b 1.26 ± 0.09b
2.3 0.40 ± 0.036c 0.39 ± 0.03a 2.90 ± 0.25b 8.65 ± 0.95b 0.54 ± 0.09a 4.30 ± 0.67b 1.37 ± 0.11 ab
5.7 0.44 ± 0.03c 0.39 ± 0.03a 2.77 ± 0.32b 12.91 ± 4.39 ab 0.50 ± 0.06 ab 5.72 ± 0.96a 1.37 ± 0.06 ab
11.4 0.54 ± 0.06b 0.38 ± 0.02a 3.56 ± 0.76a 16.65 ± 3.98a 0.49 ± 0.09 ab 3.84 ± 1.00b 1.46 ± 0.12a
22.7 0.60 ± 0.06a 0.39 ± 0.02a 3.79 ± 0.58a 15.36 ± 2.47a 0.46 ± 0.06 ab 4.00 ± 0.92b 1.47 ± 0.07a
34.1 0.63 ± 0.05a 0.38 ± 0.03a 3.66 ± 0.27a 16.82 ± 3.38a 0.41 ± 0.07b 3.44 ± 0.80b 1.49 ± 0.11a
Adults
0 0.26 ± 0.02d 0.32 ± 0.02a 2.54 ± 0.23b 8.05 ± 3.46b 0.48 ± 0.08a 3.88 ± 0.28b 1.10 ± 0.08b
2.3 0.34 ± 0.03c 0.34 ± 0.03a 2.50 ± 0.22b 7.50 ± 0.82b 0.47 ± 0.08a 3.72 ± 0.58b 1.18 ± 0.10 ab
5.7 0.38 ± 0.02c 0.34 ± 0.02a 2.40 ± 0.28b 11.18 ± 3.80 ab 0.43 ± 0.05 ab 4.95 ± 0.83a 1.19 ± 0.06 ab
11.4 0.46 ± 0.05b 0.33 ± 0.02a 3.09 ± 0.66a 14.42 ± 3.45a 0.42 ± 0.08 ab 3.32 ± 0.87b 1.26 ± 0.10a
22.7 0.51 ± 0.05a 0.33 ± 0.01a 3.29 ± 0.50a 13.30 ± 2.14a 0.40 ± 0.05 ab 3.46 ± 0.80b 1.28 ± 0.06a
34.1 0.55 ± 0.04a 0.33 ± 0.03a 3.17 ± 0.23a 14.57 ± 2.93a 0.35 ± 0.06b 2.98 ± 0.69b 1.29 ± 0.09a

Values are means ± SD of four replicates and are not significantly different at P < 0.05 when followed by the same lowercase letters.

Zn rate from 0 to 34.1 kg ha
1, the BCF value of Zn increased firstly
and then decreased, while the BCF value of Cd continued to
increase.
3.2. Human health risk assessment
3.2.1. Non-carcinogenic risk of Zn, Cu, Cd, Pb, As and Cr
The THQ values of all studied HMs were below 1, with values
being generally greater for children than for adults (Table 3). The
mean THQ values of Zn, Cu, Cd, Pb, As and Cr for children were 0.48,
0.38, 3.3  10
2, 1.3  10
2, 0.49 and 4.3  10
4, respectively. For
adults, the mean THQ values of Zn, Cu, Cd, Pb, As and Cr were 0.42,
0.33, 2.8  10
2, 1.2  10
2, 0.43 and 3.7  10
4, respectively. The
THQ values of the studied HMs in wheat grain for both children and
adults were in order of As > Zn > Cu > Cr > Cd > Pb. With increasing
Zn rates, the THQ values of Zn, Cd, and Pb of children and adults
significantly increased, whereas that of As decreased and Cu was
unchanged. The HI values ranged between 1.26 and 1.49 for chil-
dren and between 1.10 and 1.29 for adults. The HI values for Zn rates
of 11.4, 22.7, and 34.1 kg Zn ha
1 were significantly greater than that
for the unfertilized control.
The THQ of Zn, Cu and As contributed most to HI, representing
nearly 97% in all Zn rate treatments for both children and adults
(Fig. 2). The total contribution of Cd, Pb and Cr to HI was less than
3%. The percentage contribution of Zn to HI increased and that of As
decreased in response to increasing Zn rates.
 Y.-M. Liu et al. / Environmental Pollution 257 (2020) 113581
Fig. 2. Percentage contributions of the studied heavy metals in wheat grain to the hazard index (HI) with the consumption by children (a) and adults (b) under different Zn rates.
Table 4
Threshold cancer risk in wheat grains in exposure to the children and the adults as affected by Zn application rates.
Zn rate (kg Zn ha
1) Threshold cancer risk
Pb (  10
8)
Children
0 2.71 ± 1.17b
2.3 2.52 ± 0.27b
5.7 3.76 ± 1.28 ab
11.4 4.85 ± 1.16a
22.7 4.48 ± 0.72a
34.1 4.90 ± 0.99a
Values are means ± SD of four replicates and are not significantly different at P < 0.05 when followed by the same lowercase letters.
3.2.2. Carcinogenic risk of Pb and As
The TCR values of Pb and As were generally greater for adults
than for children (Table 4). The TCR value of Pb ranged from
2.71  10
8 to 4.90  10
8 for children and from 11.73  10
8 to
21.23  10
8 for adults. The mean TCR of As was 1.90  10
5 for
children and 8.22  10
5 for adults. Continues Zn application
increased the TCR values of Pb for both children and adults, with
the increasing being more obvious at higher Zn rates. In contrast,
the TCR value of As was generally not affected by levels of Zn ap-
plications except that the highest application (34 kg Zn ha
1)
significantly reduced TCR value by 26.5% compared to the control.
4. Discussions
4.1. Concentrations of HMs in soil and wheat grain as affected by
repeated Zn application
Availability of the HMs in cropland soil is affected by nutrient
management practices such as fertilizer application (Lv et al., 2015).
Repeated Zn fertilizer application over 8 years at rates from 2.3 to
34.1 kg Zn ha
1 had resulted in a total input of 36.8e545.6 kg Zn
ha
1 into the studied soil. Soil total Zn concentration was 1.2e3.2
times greater for the Zn-fertilized treatments than for the control.
The maximal soil total Zn concentration of 186 mg kg
1 for the
highest Zn rate was 2.4 times of the background Zn concentration
in soil of North China Plain (Zhang et al., 2014). Different from Zn,
the Cu concentration in soil was similar to the corresponding
background concentration and Cd, Pb, As and Cr concentration
were lower than the background value (Zhang et al., 2014). Soil
total concentrations of Zn, Cu, Cd, Pb, As and Cr were within the
corresponding Grade II criterion of the Environmental Quality
Standard for Soils of China (Table 1, CEPA, 1995), indicating that
5
As (  10
5)
Adults Children Adults
11.73 ± 5.05b 2.14 ± 0.37a 9.28 ± 1.58a
10.92 ± 1.20b 2.08 ± 0.36a 9.03 ± 1.56a
16.29 ± 5.54 ab 1.92 ± 0.23 ab 8.31 ± 1.01 ab
21.01 ± 5.02a 1.88 ± 0.37 ab 8.12 ± 1.58 ab
19.39 ± 3.13a 1.79 ± 0.21 ab 7.74 ± 0.93 ab
21.23 ± 4.27a 1.58 ± 0.27b 6.83 ± 1.14b
repeated Zn fertilizer application even at high rates did not result in
obvious HMs pollution for the croplands on North China Plain.
According to the standards of USEPA (2012), the maximum
allowable concentrations (MAC) of Zn, Cu, Cd, Pb, As and Cr in cereal
grain are 50, 20, 0.1, 0.2, 0.5 and 1.0 mg kg
1, respectively (Table 2).
The HMs concentrations in crop grains can frequently exceed the
safe standard resulted from the agricultural activity in farmland.
For example, ten years’ organic fertilizer application on a typical
Hapludalf soil had increased Cr and Zn concentrations in wheat
grain up to levels exceeding the MAC (Couto et al., 2018). Waste-
water irrigation had increased Cd concentration in wheat grain
beyond the recommended dietary limit (Asgari and Cornelis, 2015).
In this study, application of Zn fertilizer at 2.3e34.1 kg Zn ha
1 for
eight years had increased grain Zn concentration by 34%e113%
compared with control. When Zn rate was at or greater than 11.4 kg
Zn ha
1, grain Zn concentration exceeded the USEPA standard value
(50 mg kg
1), indicating that the long-term Zn fertilizer application
at high rates could bring a direct health risk on consumers. How-
ever, in a biofortification view, Zn concentration in wheat grain
should reach at least 40 mg kg
1 to satisfy human requirement
(Cakmak et al., 2010). Therefore, application of Zn fertilizer at
optimal rate (5.7 kg Zn ha
1), can not only ensure food safety, but
also ensure the adequate Zn nutrition for human consumption.
Different from Zn, concentrations of other HMs in wheat grain
including Cd, Pb, Cu, As, Cr were less than the standard values. The
relative low concentrations of Cu, Cd, Pb, As and Cr in wheat grain
were mainly associated to the low concentration and availability of
these HMs in soil. This result is consistent with a previous study on
a calcareous soil in Northwest China (Yang et al., 2019). Different
from its effect on soil HMs availability, Zn application either
increased or decreased HMs concentrations in wheat grain. For
example, Zn application decreased grain concentration of As
6 Y.-M. Liu et al. / Environmental Pollution 257 (2020) 113581
whereas increased that of Cd and Pb. Plants have similar uptake and
transport mechanisms for Zn and some other non-essential toxic
HMs. Therefore, Zn can exert an antagonistic effect with other HMs
for completing the uptake sites in crop roots (Sarwar et al., 2015;
Khan et al., 2014; Qaswar et al., 2017). However, results from the
literature on the interaction between Zn and Cd, Pb uptake in plants
are not always inconsistent. As many previous publications re-
ported, Zn application decreased Cd and Pb in grain of wheat
(Qaswar et al., 2017). However, less publication reported Zn appli-
cation increasing effects on the Cd concentrations in plants (Smilde
et al., 1992; Chen et al., 2019). Some studies reported that appli-
cation of Zn fertilizer could increase HMs (i.e. Cd and Pb) availability
in soils due to the contaminations from the commercial fertilizer
(Mortvedt, 1996). However, this is not the case in the current study
as we used analytic purity reagent of ZnSO4 which does not contain
other HMs as contaminants. In our experiment, Zn application
especially at higher rates over 11.4 kg Zn ha
1 (total soil Zn con-
centration > 100 mg kg
1) significantly increased Cd and Pb con-
centration in grain of wheat, which may be associated with their
increasing availability in soil. Smilde et al. (1992) reported that
higher Zn application (total soil Zn concentration > 150 mg Zn
kg
1) increased available CaCl2-extractble Cd concentration on a
loam soil with a pot experiment, and thereby increased Cd uptake
in maize plants. From these results published, we can see the Zn
application effects on Cd and Pb in crop are highly dependent on
soil types, crop species and cultivars, background levels of Cd, Pb
and Zn in the growth medium, and Zn application rate. In addition,
the increases in grain Cd and Pb concentrations had resulted in an
increase in health risk with consumption of wheat grain. In
contrast, the reduction of grain As concentration in response to Zn
fertilization was likely due to the precipitation/fixation of As on
ZnSO4 (Das et al., 2008; Craw and Chappell, 2000).
The BCF value is a key parameter to evaluate human exposure to
HMs from soil to wheat grain. Among the studied HMs, Zn had the
highest BCF value, which was consistent with previous reports
(Abbasi et al., 2013; Boussen et al., 2013; Liu et al., 2017a). This
indicated that Zn was more easily transferred from soil to grain
than other studied HMs. The low BCF values for HMs of Cu, Cd, Pb,
As and Cr were likely attributed to their low availability in soil due
to the high soil pH (8.0) and CaCO3 content (4.5%) (Cakmak, 2008).
The mean BCF values of Cu, Cd, Pb, As and Cr were similar to those
reported by Lei et al. (2015) and Yang et al. (2019). However, with
increasing Zn rates, the BCF value of Zn initially increased and then
decreased. When soil Zn level was low, the ability of plant to
exclude Zn from the root symplasm was weak, resulting in an in-
crease of net Zn uptake (Santa-Marıa and Cogliatti, 1998). At high
soil Zn concentration close to the toxic level, plant Zn uptake can be
controlled by root-induced changes in the rhizosphere such as
changes in pH and exudation of organic acids (Verbruggen et al.,
2009; Martinez and Motto, 2000; Xiao et al., 2017). Christie et al.
(2004) also indicated that at the excess Zn supply, arbuscular my-
corrhiza could bind with Zn to depress the translocation to plant
roots. Therefore, the BCF values of HMs are strongly dependent on
the interaction between plant and soil (Kabata-Pendias, 2011). In
this study, Zn fertilizer application did not affect the BCF values of
Cu, Pb, Cr, and As but significantly decreased the BCF of Cd, which
was agreed with Kolasinac et al. (2018).
4.2. Human health risk assessment of HMs in grain of wheat as
affected by repeated Zn application
Health risk assessment is an effective method to evaluate the
effect of an individual HM on human health based on HM con-
centration, human body weight and the quantity of food consumed
(Mahmood et al., 2014). In China, wheat is consumed at a rate of
159.9 g day
1 for adult, whereas the consumption rate may be
much greater in other countries (Asgari and Cornelis, 2015; Rizwan
et al., 2017). In the current study, the THQ and HI values were
higher for children than adults, which was consistent with previous
reports (Guo et al., 2018; Couto et al., 2018; Doabi et al., 2018). This
means that children are more at risk for health than adults due to
the HMs intake from wheat grain in this study. Still, for both chil-
dren and adults, the THQ values of selective HMs in grain were all
less than 1, indicating that the wheat grains in our study, including
those from the repeated high Zn rate treatments, are safe for hu-
man consumption. However, special attention should be paid on
certain high THQ value for some HMs. For example, the THQ value
of Zn was affected mostly by Zn fertilizer rate and eight years of
repeated application of Zn at high level (34.1 kg Zn ha
1) had
resulted in the maximum THQ value of 0.63 for children. Although
this value is still in safe range, ingesting the wheat grain with such
high value for a long period time presents a potential threat for
human health (USEPA, 1997). Therefore, controlling grain Zn at an
optimal level is necessary to reduce its risk for human consump-
tion. It is worth noting that the risk of As in wheat grain decreased
with increasing Zn application rate, indicating that Zn fertilizer
application is an effective way to reduce the risk of As exposure to
Zn-biofortified wheat. Although the THQs of Cd and Pb increased
with Zn application rate, the values were still less than 0.1 without
causing any risk. The HI, as the sum of THQ for the tested HMs, was
1.40 for children and 1.22 for adults, which were higher than values
from previous studies in the main grain production regions of China
(Zhang et al., 2018; Yang et al., 2019). The reason might be related to
the differences in wheat cultivars and environmental conditions.
The non-carcinogenic HI greater than 1 in this study demonstrated
that a long-time ingestion of wheat grain in this area would result
in a potential non-carcinogenic risk, which could be explained by
two factors. First, in this study, we evaluated the health risk of
consumption of whole wheat grain instead of wheat flour. Previous
studies showed that the presentation for most HMs in wheat grain
is in the bran and can be reduced during the flour processing
(Ajiboye et al., 2015). Li et al. (2014a) reported that the Zn con-
centration in wheat flour was only 30% of that in the whole grain.
Second, an accurate health risk assessment should base on the
bioavailability, rather than the total amount of HM (Oomen et al.,
2002). Factors inhibiting the bioavailability of HMs such as phytic
acid content in grain should be taken into account in the future
(White and Broadley, 2009). Therefore, the HI based on the tested
HMs concentrations in this study could be over-estimated. Heavy
metals of Zn, Cu and As contributed most to HI for different receptor
population, which was consistent with Huang et al. (2008). The HI
value significantly increased with Zn application rates, indicating
that Zn fertilizer can potentially bring the non-carcinogenic risk.
However, the HI values at Zn rates of 2.3 and 5.7 kg Zn ha
1 were
not different from control, suggesting that wheat grain at an
optimal Zn application is safe for consumption without causing any
non-carcinogenic risk.
Arsenic and Pb are classified as carcinogenic to humans through
oral intake (IARC, 2009). Therefore, calculating the carcinogenic risk
of exposure of Zn biofortified wheat in the dietary intake pathway
is necessary. The acceptable range of carcinogenic risk of HM in
wheat grain was 1  10
6 - 1  10
4 (Abbasi et al., 2013). The TCR
values of Pb and As in this study were all within the safe range,
indicating that the Zn-biofortified wheat had no lifetime carcino-
genic risk for both children and adults. Being consistent with pre-
vious studies (Lei et al., 2015; Yang et al., 2019), the TCR was greater
for As than for Pb. Although the carcinogenic risk of Pb increased
with increasing Zn rates, the TCR value of Pb in wheat grain was less
than the critical value of 1  10
6. The highest TCR value of As was
observed for control, which was close to the MAC of 1  10
4 and
 Y.-M. Liu et al. / Environmental Pollution 257 (2020) 113581
greater than that of wheat grown in non-mining areas (Ahmed
et al., 2015; Guo et al., 2018). Therefore, results of this study have
raised the attention for the potential carcinogenic risk of As in this
region. Our results also clearly showed that Zn fertilizer application
at appropriate rate is an effective strategy to reduce such risk.
4.3. Zn fertilizer management in the Zn biofortified wheat
production
Zinc application through soil and foliar is an effective approach
to realize the Zn biofortification of wheat (Rengel et al., 1999;
Cakmak, 2008). We confirmed the benefit of soil Zn application as it
not only increased grain yield (Fig. S2), but also improved grain Zn
nutrition in the current study. Our previous study had indicated
that the optimal rates of Zn fertilizer (5.7e11.4 kg Zn ha
1) could
synergistically achieve high yield (8e14% increase than control)
(Liu et al., 2017b) and high Zn nutrition quality (33e46% in grain Zn
concentration than control) (Liu et al., 2017c). Meantime, it did not
cause the decrease of other nutrients such as Fe and Mn (Liu et al.,
2017c). In the current study, we further quantitatively evaluated the
effect of Zn fertilizer application on the health risk of human in
exposure to HMs with ingestion of the Zn-biofortified wheat grains.
After 8 years’ repeated application of fertilizer at rate of 5.7 kg Zn
ha
1, grain Zn concentration has achieved the biofortification
target, without exceeding the human safety limit (40e50 mg kg
1).
Moreover, Zn application at the optimal rate of 5.7 kg Zn ha
1 did
not result in any risk for non-carcinogenic metals and reduced 11%
of the carcinogenic risk for As (8.7  10
6). Therefore, soil Zn
application at 5.7 kg Zn ha
1 can achieve Zn nutrition target for
human consumption, without causing any risks of Zn, Pb, Cu, Cd, Cr,
and As for population with diet based on the wheat grain in this
area.
5. Conclusions
Results from this long-term experiment provided a better un-
derstanding of the effect of continuously repeated application of Zn
fertilizer on HMs accumulations in soil and cereal grain, and further
on the risk assessment of human health. Application of Zn fertilizer
at appropriate levels can achieve the Zn biofortification target for
wheat grain without causing soil HMs pollution. Repeated Zn
application at high rates caused grain Zn concentration to exceed
MAC while increased the non-carcinogenic risk of Zn, Cu, Cd, Pb, As
and Cr. Zinc fertilizer had the potential to decrease carcinogenic risk
of As. We concluded that 5.7 kg Zn ha
1 is an optimal rate for
croplands on North China Plain, which can improve grain Zn
nutrition without causing any non-carcinogenic risks. Results of our
study thus provided valuable advices on the biofortification
through soil Zn application from human health risk perspective.
Overuse of Zn fertilizer for wheat production can likely result in
health risk due to the consumption of wheat grain and cause po-
tential threat to soil environment. Results of the current study will
help farmers to improve Zn fertilizer management in crop pro-
duction in the future.
Acknowledgements
This research was funded by the 973 Project (2015CB150402),
the National Natural Science Foundation of China (31672240), and
the Innovative Group Grant of National Natural Science Foundation
of China (31421092).
Appendix A. Supplementary data
Supplementary data related to this article can be found at
7
https://doi.org/10.1016/j.envpol.2019.113581.
References
Abbasi, A.M., Iqbal, J., Khan, M.A., Shah, M.H., 2013. Health risk assessment and
multivariate apportionment of trace metals in wild leafy vegetables from Lesser
Himalayas, Pakistan. Ecotoxicol. Environ. Saf. 92, 237e244.
Adamoa, P., Iavazzo, P., Albanese, S., Agrelli, D., Vivo, B.D., Lima, A., 2014. Bioavail-
ability and soil-to-plant transfer factors as indicators of potentially toxic
element contamination in agricultural soils. Sci. Total Environ. 500e501, 11e22.
Ahmed, M.K., Shaheen, N., Islam, M.S., Habibullah-Al-Mamun, M., Islam, S.,
Banu, C.P., 2015. Trace elements in two staple cereals (rice and wheat) and
associated health risk implications in Bangladesh. Environ. Monit. Assess. 187,
326.
Ajiboye, B., Cakmak, I., Paterson, D., Jonge, M.D.D., Howard, D.L., Stacey, S.P.,
Torun, A.A., Aydin, N., McLaughlin, M.J., 2015. X-ray fluorescence microscopy of
zinc localization in wheat grains biofortified through foliar zinc applications at
different growth stages under field conditions. Plant Soil 392 (1e2), 357e370.
Asgari, K., Cornelis, W.M., 2015. Heavy metal accumulation in soils and grains, and
health risks associated with use of treated municipal wastewater in subsurface
drip irrigation. Environ. Monit. Assess. 187, 410.
Boussen, S., Soubrand, M., Bril, H., Ouerfelli, K., Abdeljaouad, S., 2013. Transfer of
lead, zinc and cadmium from mine tailings to wheat (Triticum aestivum) in
carbonated Mediterranean (Northern Tunisia) soils. Geoderma 192, 227e236.
Broadley, M., Brown, P., Cakmak, I., Rengel, Z., Zhao, F., 2012. Function of nutrients:
micronutrients. In: Marschner, P. (Ed.), Marschner’s Mineral Nutrition of Higher
Plants. Academic Press, San Diego, pp. 191e248.
Cakmak, I., 2008. Enrichment of cereal grains with zinc: agronomic or genetic
biofortification? Plant Soil 302, 1e17.
Cakmak, I., Hoffland, E., 2012. Zinc for the improvement of crop production and
human health. Plant Soil 361, 1e2.
Cakmak, I., Pfeiffer, W.H., McClafferty, B., 2010. Review: biofortification of durum
wheat with zinc and iron. Cereal Chem. 87, 10e20.
CEPA, 1995. Environmental Quality Standard for Soils (GB15618-1995). Chinese
Environmental Protection Administration.
Chen, S., Xin, Z., Lu, Q., Zheng, F., He, B., 2019. Effects of exogenous zinc on cadmium
uptake and transport in rice. J. Agro Environ. Sci. 38 (10), 2270e2277 (In
Chinese).
Chen, X., Zhang, Y., Tong, Y., Xue, Y., Liu, D., Zhang, W., Deng, Y., Meng, Q., Yue, S.,
Yan, P., Cui, Z., Shi, X., Guo, S., Sun, Y., Ye, Y., Wang, Z., Jia, L., Ma, W., He, M.,
Zhang, X., Kou, C., Li, Y., Tan, D., Cakmak, I., Zhang, F., Zou, C., 2017. Harvesting
more grain zinc of wheat for human health. Sci. Rep. UK. 7, 7016.
Chesworth, W., Camps Arbestain, M., Macías, F., Spaargaren, O., Spaargaren, O.,
Mualem, Y., Morel-Seytoux, H.J., Horwath, W.R., Almendros, G., Chesworth, W.,
Grossl, P.R., Sparks, D.L., Spaargaren, O., Fairbridge, R.W., Singer, A., Eswaran, H.,
Micheli, E., 2008. Classification of soils: world reference base (WRB) for soil
resources. Encycl. Soil Sci. 120e122.
Christie, P., Li, X., Chen, B., 2004. Arbuscular mycorrhiza can depress translocation of
zinc to shoots of host plants in soils moderately polluted with zinc. Plant Soil
261, 209e217.
Couto, R.D.R., Faversani, J., Ceretta, C.A., Ferreira, P.A.A., Marchezan, C., Basso
Facco, D., Garlet, L.P., Silva, J.S., Comin, J.J., Bizzi, C.A., Flores, E.M.M., Brunetto, G.,
2018. Health risk assessment and soil and plant heavy metal and bromine
contents in field plots after ten years of organic and mineral fertilization.
Ecotoxicol. Environ. Saf. 153, 142e150.
Craw, D., Chappell, R., 2000. Metal redistribution in historic mine wastes, Coro-
mondal Peninsula, New Zealand. J. Geol. Geophys. 43, 187e189.
Dalir, N., Tandy, S., Gramlich, A., Khoshgoftarmanesh, A., Schulin, R., 2017. Effects of
nickel on zinc uptake and translocation in two wheat cultivars differing in zinc
efficiency. Environ. Exp. Bot. 134, 96e101.
Das, D.K., Sur, P., Das, K., 2008. Mobilisation of arsenic in soils and in rice (Oryza
sativa L.) plants affected by organic matter and zinc application in irrigation
water contaminated with arsenic. Plant Soil Environ. 54, 30e37.
Doabi, S.A., Karami, M., Afyuni, M., Yeganeh, M., 2018. Pollution and health risk
assessment of heavy metals in agricultural soil, atmospheric dust and major
food crops in Kermanshah province, Iran. Ecotoxicol. Environ. Saf. 163, 153e164.
Funari, E., Testai, E., 2008. Human health risk assessment related to cyanotoxins
exposure. Crit. Rev. Toxicol. 38, 97e125.
Guo, G., Lei, M., Wang, Y., Song, B., Yang, J., 2018. Accumulation of As, Cd, and Pb in
sixteen wheat cultivars grown in contaminated soils and associated health risk
assessment. Int. J. Environ. Res. Public Health 15, 2601.
Hafeez, B., Khanif, Y.M., Saleem, M., 2013. Role of zinc in plant nutrition- a review.
Am. J. Exp. Agric. 3 (2), 374e391.
Hotz, C., Brown, K.H., 2004. Assessment of the risk of zinc deficiency in populations
and options for its control. Food Nutr. Bull. 25, 94e204.
Huang, M., Zhou, S., Sun, B., Zhao, Q., 2008. Heavy metals in wheat grain: assess-
ment of potential health risk for inhabitants in Kunshan, China. Sci. Total En-
viron. 405, 54e61.
International Agency for Research on Cancer, 2009. Complete List of Agents eval-
uated and their classification. URL: http://monographs.iarc.fr/ENG/
Classification/index.php.
Jiao, W., Chen, W., Chang, A.C., Page, A.L., 2012. Environmental risks of trace ele-
ments associated with long-term phosphate fertilizers applications: a review.
Environ. Pollut. 168, 44e53.
8 Y.-M. Liu et al. / Environmental Pollution 257 (2020) 113581
Kabata-Pendias, A., 2011. Trace Elements in Soils and Plants, fourth ed. CRC Press
Taylor and Francis Group, Boca Raton, FL, p. 552.
Khan, M.A., Castro-Guerrero, N., Mendoza-Cozatl, D.G., 2014. Moving toward a
precise nutrition: preferential loading of seeds with essential nutrients over
non-essential toxic elements. Front. Plant Sci. 5, 51.
Kim, S., Dale, B.E., 2004. Global potential bioethanol production from wasted crops
and crop residues. Biomass Bioenergy 26 (4), 361e375.
Kolasinac, S.M., Leki c, S.S., Golijan, J., Petrovi
c, T., Todorovic, G., Kosti  2018.
c, A.Z.,
Bioaccumulation process and health risk assessment of toxic elements in to-
mato fruit grown under Zn nutrition treatment. Environ. Monit. Assess. 190, 9.
Lei, L., Liang, D., Yu, D., Chen, Y., Song, W., Li, J., 2015. Human health risk assessment
of heavy metals in the irrigated area of Jinghui, Shaanxi, China, in terms of
wheat flour consumption. Environ. Monit. Assess. 187, 647.
Li, M., Wang, S., Tian, X., Zhao, J., Li, H., Guo, C., Chen, Y., Zhao, A., 2014a. Zn dis-
tribution and bioavailability in whole grain and grain fractions of winter wheat
as affected by applications of soil N and foliar Zn combined with N or P. J. Cereal
Sci. 61, 26e32.
Li, Z., Ma, Z., van der Kuijp, T.J., Yuan, Z., Huang, L., 2014b. A review of soil heavy
metal pollution from mines in China: pollution and health risk assessment. Sci.
Total Environ. 468e469, 843e853.
Liu, B., Ai, S., Zhang, W., Huang, D., Zhang, Y., 2017a. Assessment of the bioavail-
ability, bioaccessibility and transfer of heavy metals in the soil-grain-human
systems near a mining and smelting area in NW China. Sci. Total Environ.
609, 822e829.
Liu, D., Zhang, W., Pang, L., Zhang, Y., Wang, X., Liu, Y., Chen, X., Zhang, F., Zou, C.,
2017b. Effects of zinc application rate and zinc distribution relative to root
distribution on grain yield and grain Zn concentration in wheat. Plant Soil 411,
167e178.
Liu, D., Liu, Y., Zhang, W., Chen, X., Zou, C., 2017c. Agronomic approach of zinc
biofortification can increase zinc bioavailability in wheat flour and thereby
reduce zinc deficiency in humans. Nutrients 9, 465.
Lv, J., Liu, Y., Zhang, Z., Zhou, R., Zhu, Y., 2015. Distinguishing anthropogenic and
natural sources of trace elements in soils undergoing recent 10-year rapid ur-
banization: a case of Donggang, Eastern China. Environ. Sci. Pollut. Res. Int. 22
(14), 10539.
Ma, G., Jin, Y., Li, Y., Zhai, F., Kok, F.J., Jacobsen, E., Yang, X., 2008. Iron and zinc
deficiencies in China: what is a feasible and cost-effective strategy? Public
Health Nutr. 11 (06), 632e638.
Mahmood, A., Malik, R.N., Li, J., Zhang, G., 2014. Human health risk assessment and
dietary intake of organochlorine pesticides through air, soil and food crops
(wheat and rice) along two tributaries of river Chenab, Pakistan. Food Chem.
Toxicol. 71, 17e25.
Martinez, C., Motto, H., 2000. Solubility of lead, zinc and copper added to mineral
soils. Environ. Pollut. 107 (1), 153e158.
Mico , C., Peris, M., Recatala, L., Sa
nchez, J., 2007. Baseline values for heavy metals in
agricultural soils in an European Mediterranean region. Sci. Total Environ. 378,
13e17.
Mohamed, B.A., Ellis, N., Kim, C.S., Bi, X.T., 2017. The role of tailored biochar in
increasing plant growth, and reducing bioavailability, phytotoxicity, and uptake
of heavy metals in contaminated soil. Environ. Pollut. 230, 329e338.
Mortvedt, J.J., 1996. Heavy metal contaminants in inorganic and organic fertilizers.
Fert. Res. 43, 55e61.
Niemela €, M., Kola, H., Per€
am€ aki, P., Piispanen, J., Poikolainen, J., 2005. Comparison of
microwave-assisted digestion methods and selection of internal standards for
the determination of Rh, Pd and Pt in dust samples by ICP-MS. Microchim. Acta
150, 211e217.
Noulas, C., Tziouvalekas, M., Karyotis, T., 2018. Zinc in soils, water and food crops.
J. Trace Elem. Med. Biol. 49, 252e260.
Nriagu, J., 2011. Zinc Deficiency in Human Health. Encyclopedia of Environmental
Health, pp. 789e800.
Oomen, A.G., Hack, A., Minekus, M., Zeijdner, E., Comelis, C., Sxhoeters, G.,
Schoeters, G., Verstrraete, W., Van de Wiele, T., Wragg, J., Rompelberg, C.J.M.,
Sips, A.J.A.M., Van Wijnen, J.H., 2002. Comparison of five in vitro digestion
models to study the bioaccessibility of soil contaminants. Environ. Sci. Technol.
36, 3326.
Pandey, R., Shubhashish, K., Pandey, J., 2012. Dietary intake of pollutant aerosols via
vegetables influenced by atmosphericde position and wastewater irrigation.
Ecotoxicol. Environ. Saf. 76, 200e208.
Qaswar, M., Hussain, S., Rengel, Z., 2017. Zinc fertilisation increases grain zinc and
reduces grain lead and cadmium concentrations more in zinc-biofortified than
standard wheat cultivar. Sci. Total Environ. 605e606, 454e460.
Rengel, Z., Batten, G.D., Crowley, D.E., 1999. Agronomic approaches for improving
the micronutrient density in edible portions of field crops. Field Crop. Res. 60,
27e40.
Rizwan, M., Ali, S., Hussain, A., Ali, Q., Shakoor, M.B., Zia-ur-Rehman, M., Farid, M.,
Asma, M., 2017. Effect of zinc-lysine on growth, yield and cadmium uptake in
wheat (Triticum aestivum L.) and health risk assessment. Chemosphere 187,
35e42.
Saifullah, Sarwar, N., Bibi, S., Ahmad, M., Ok, Y.S., 2014. Effectiveness of zinc
application to minimize cadmium toxicity and accumulation in wheat (Triticum
aestivum L.). Environ. Earth Sci. 71, 1663e1672.
Santa-Marıa, G.E., Cogliatti, D.H., 1998. The regulation of zinc uptake in wheat
plants. Plant Sci. 137, 1e12.
Sarwar, N., Ishaq, W., Farid, G., Shaheen, M.R., Imran, M., Geng, M., Hussain, S., 2015.
Zinc-cadmium interactions: impact on wheat physiology and mineral acquisi-
tion. Ecotoxicol. Environ. Saf. 122, 528e536.
Sipter, E., Rozsa, E., Gruiz, K., Ta
trai, E., Morvai, V., 2008. Site-specific risk assess-
ment in contaminated vegetable gardens. Chemosphere 71, 1301e1307.
Smilde, K.W., Luit, B.V., Driel, W.V., 1992. The extraction by soil and absorption by
plants of applied zinc and cadmium. Plant Soil 143, 233e238.
Tchounwou, P.B., Yedjou, C.G., Patlolla, A.K., Sutton, D.J., 2012. Heavy metals toxicity
and the environment. Mol. Clin. Environ. Toxicol. 101, 133e164.
USEPA, 1997. Environmental Protection Agency. Exposure Factors Handbook -
General Factors, vol. I. Office of Research and Development. National Center for
Environmental Assessment. Unites States Environmental Protection Agency,
Washington, DC. EPA/600/P-95/002Fa.
USEPA, 2006. Environmental Protection Agency, Region 3, Risk-Based Concentration
Table: Technical Background Information. Unites States Environmental Protec-
tion Agency, Washington, DC.
USEPA, 2012. Environmental protection agency, region 9, preliminary remediation
goals. http://www.epa.gov/region9/superfund/prg/.
Verbruggen, N., Hermans, C., Schat, H., 2009. Molecular mechanisms of metal
hyperaccumulation in plants. New Phytol. 181 (4), 759e776.
Wang, W.X., 2013. Dietary toxicity of metals in aquatic animals: recent studies and
perspectives. Chin. Sci. Bull. 58, 203e213.
Welch, R.M., Graham, R.D., 2004. Breeding for micronutrients in staple food crops
from a human nutrition perspective. J. Exp. Bot. 55, 353e364.
́
White, P.J., Broadley, M.R., 2009. Biofortification of crops with seven mineral ele-
ments often lacking in human dietsdiron, zinc, copper, calcium, magnesium,
selenium and iodine. New Phytol. 182, 49e84.
Wongsasuluk, P., Chotpantarat, S., Siriwong, W., Robson, M., 2018. Using hair and
fingernails in binary logistic regression for bio-monitoring of heavy metals/
metalloid in groundwater in intensively agricultural areas, Thailand. Environ.
Res. 162, 106e118.
Xiao, L., Guan, D., Peart, M.R., Chen, Y., Li, Q., Dai, J., 2017. The influence of
bioavailable heavy metals and microbial parameters of soil on the metal
accumulation in rice grain. Chemosphere 185, 868e878.
Yang, W., Wang, D., Wang, M., Zhou, F., Huang, J., Xue, M., Dinh, Q.T., Liang, D., 2019.
Heavy metals and associated health risk of wheat grain in a traditional culti-
vation area of Baoji, Shaanxi, China. Environ. Monit. Assess. 191, 428.
Yang, X.G., Zhai, F.Y., 2006. A Survey on the Chinese National Health and Nutrient III:
the Resident Constitution and Nutrition in 2002. Peoples Medial Publishing
House, Beijing, China, pp. 55e58.
Yilmaz, A., Ekiz, H., Torun, B., Gultekin, I., Karanlik, S., Bagci, S.A., Cakmak, I., 1997.
Effect of different zinc application methods on grain yield and zinc concen-
tration in wheat cultivars grown on zinc-deficient calcareous soils. J. Plant Nutr.
20 (4e5), 461e471.
Zhai, F.Y., Yang, X.G., 2006. A Survey on the Chinese National Health and Nutrition
II: the National Diet and Nutrition in 2002. Peoples Medial Publishing House,
Beijing, China, pp. 145e146.
Zhang, Y., Yin, C., Cao, S., Cheng, L., Wu, G., Guo, J., 2018. Heavy metal accumulation
and health risk assessment in soil-wheat system under different nitrogen
levels. Sci. Total Environ. 622e623, 1499e1508.
Zhang, X., Zhang, X., Zhong, T., Jiang, H., 2014. Spatial distribution and accumulation
of heavy metal in arable land soil of China. Environ. Sci. 35, 692e703 (In
Chinese).