Accepted Manuscript

Title: The influence of biochar type on long-term stabilization

for Cd and Cu in contaminated paddy soils

Author: Hongying Li Xinxin Ye Zhigang Geng Hongjian

Zhou Xisheng Guo Yunxia Zhang Huijun Zhao Guozhong
Wang

PII: S0304-3894(15)30175-8
DOI: http://dx.doi.org/doi:10.1016/j.jhazmat.2015.10.048
Reference: HAZMAT 17193

To appear in: Journal of Hazardous Materials

Received date: 24-6-2015

Revised date: 23-9-2015
Accepted date: 22-10-2015

Please cite this article as: Hongying Li, Xinxin Ye, Zhigang Geng, Hongjian Zhou,
Xisheng Guo, Yunxia Zhang, Huijun Zhao, Guozhong Wang, The influence of biochar
type on long-term stabilization for Cd and Cu in contaminated paddy soils, Journal of
Hazardous Materials http://dx.doi.org/10.1016/j.jhazmat.2015.10.048

This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.
 The influence of biochar type on long-term stabilization for
Cd and Cu in contaminated paddy soils

Hongying Li, a,b Xinxin Ye, *,a Zhigang Geng, a Hongjian Zhou, a Xisheng Guo, b
Yunxia Zhang, a Huijun Zhao, a,c Guozhong Wang,*,a
a
Key Laboratory of Materials Physics, Centre for Environmental and Energy
Nanomaterials, Anhui Key Laboratory of Nanomaterials and Nanotechnology,
Institute of Solid State Physics, Chinese Academy of Sciences
b
Institute of Soil and Fertilizer, Anhui Academy of Agricultural Sciences, Hefei
230031, China
c
Centre for Clean Environment and Energy, Gold Coast Campus, Griffith University,
Queensland 4222, Australia

Corresponding authors at: Key Laboratory of Materials Physics, Centre for
Environmental and Energy Nanomaterials, Anhui Key Laboratory of Nanomaterials
and Nanotechnology, Institute of Solid State Physics, Chinese Academy of Sciences,
Hefei 230031, P.R. China. Fax: +86 0551 6559 1434; Tel: +86 0551 6559 5616; E-mail:
xxye@issp.ac.cn (X.X. Ye), gzhwang@issp.ac.cn (G.Z. Wang).

Highlights
 Biochar species can affect the long-term stabilization of Cd and Cu in acid soils.
 Corn straw biochar consistently reduces the bioavailability of Cd and Cu.
 Aging process of biochar influences the immobilization stability of Cd and Cu.
 More oxygen-containing groups can form on corn straw biochar in acid
condition.

Abstract Long-term effect of biochar on PTEs (potential toxic elements)

immobilization depends upon biochar own property and its aging process in soil. To
understand the role of biachar type on PTEs stabilization, two types of biochar, corn-
straw-derived biochar (CB) and hardwood-derived biochar (HB), were compared for
their efficacy in achieving a stable decrease in the bio-availability of Cd and Cu in soils.
The 3-year pot-culture experiment showed that HB reduced the concentration of CaCl2-
extractable Cd and Cu by 57.9% and 63.8% in soil, and Cd and Cu uptake by 63.6%
and 56.3% in rice tissue respectively, in the first year, whereas these values increased
in the next two years. On the other hand, CB decreased these values steadily year by
year. At the end of the three years, CB at 5% level had lowered the levels of CaCl2-
extractable Cd and Cu by 53.6% and 66.8%, respectively. These variations between CB
and HB were due to the differences in the way the two types of biochar age in the soil.
The aging process was simulated in the laboratory, and the XPS results showed that the
oxidization of the biochars introduced more oxygen-containing groups (especially
carboxyl) on the surface of CB than HB, leading to a correspondingly greater number
of oxygenated binding sites for Cd and Cu in the case of CB. The content of lignin was
the major factor resulting in the variation of oxidation degree in two biochars. These
results suggest that it is important to select the right kind of biochar to stably decrease
the bio-availability of potential toxic elements (Cd and Cu) in contaminated soils.
Key words: Rice, Soil remediation, Biochar, Acid soil, Bio-availability

1. Introduction
PTEs often coexist in contaminated soils and can accumulate in the human body
through the soil–crop–food chain [1–3]. Compared with other remediation technology
such as mechanical separation, chemical washing, electrokinetic remediation, the in-
situ immobilization of PTEs in contaminated soils is an effective and low-cost
remediation technology for decreasing the mobility and bio-availability of such
elements [4], and amending the contaminated soils with biochar has been proved
particular effect to immobilize PTEs in the soils and decrease the uptake of PTEs by
plants [5–7]. However, the long-term effects of different biochars (wood and crop
residues, for example) on the immobilization of PTEs appear to be inconsistent [8,9]:
biochar derived from wheat straw reduced the Cd and Pb contents of rice significantly
in a three-year field trial [8] whereas biochar derived from hardwoods increased the
uptake of the same metals in ryegrass in the last two harvests compared to that in the
first harvest [9]. Therefore, the effects of biochar on immobilizing PTEs in
contaminated soils need to be investigated in greater detail.
The extent to which biochar can immobilize PTEs stably is related to the bio-
geochemical processes the biochar is subjected to in soils [10]. Particularly, the surface
properties of biochar are affected by the physical, chemical, and biological processes
that result in the aging of biochar [11]. Biochars undergo a slow but measurable in-situ
oxidation in soils, which may lead to the formation of carboxylic, carbonylic, phenolic,
and other oxygen-containing functional groups on the surface of aged biochar [12,13],
and such aging process is faster in acidic soils [14]. Aging is thus an important process
that influences the surface properties of biochar in soil, which, in turn, affect the
absorption capacity of the biochar and the extent to which it can immobilize PTEs in
the long term. For example, the capacity of biochar to adsorb metals increased with the
increase of acid concentration in the simulation process of biochar activation [15].
Uchimiya et al. [16] reported that in soils contaminated with Cu, Zn and Pb, biochars
oxidized by HNO3 were rich in carboxyl functional groups and immobilized PTEs for
significantly greater stability than unoxidized biochars.
Oxidation is considered to be a major contribution to the aging of biochar in soils
[17], and the surface properties and oxidation ability of biochar through acid treatment
varies a great deal depending on the source from which the biochar is derived [18].
Some studies showed that the CEC and specific area were the important factors
influencing the adsorption of metal ions [19,20]. The CEC of biochar in previously
published data were quite variable, ranging from 34 cmol kg−1 [21] to 304 cmol kg−1
[22], and could also increased after the activation [23]. The research found that the
specific area of biochar derived from different feedstock had slight variations from 2
m2 g–1 to 6 m2 g–1, depending on the pyrolysis temperature [19]. Moreover, biochars are
not commonly treated or further activated before they are added to soils, and the
feedstocks are most of biological residues. Thus, depending on their source, biochars
differ in the physicochemical properties of their aging and therefore in their
performance and in the stability of PTEs immobilization over the long term. However,
researches on the effect of the source of biochar type are very limited.
Cd and Cu are the major pollutants that contaminate farm lands near mines,
especially copper mines, and the mobility and bio-availability of these pollutants are of
great concern to China [24,25], that is the reason for the present study focused on paddy
soils close to copper mines and contaminated with Cd and Cu. We sought to find out
whether biochars derived from two sources (hardwood and corn straw) can
continuously reduce the availability of Cd and Cu in soil and their uptake by rice. The
oxidative aging of biochars under acid conditions was simulated in the laboratory to
gain insights into the aging process of two biochars. Specifically, the study (1)
compared the effect of two biochars on soil properties, on the mobility Cd and Cu in
soil, and on their uptake by rice plants over 3 years and (2) determined the variations
in the surface properties of the two biochars as a result of simulated aging.

2. Materials and methods

2.1. Preparation of biochar
Corn straw (Zea mays) and hardwood (oak, Quercus spp.) were pyrolyzed at
300 °C to produce corn-straw-derived biochar (CB) and hardwood-derived biochar
(HB). The preparation method for biochars is described by Qian et al. [14] and Yuan et
al. [22]. Briefly, the samples of corn straw and hardwood were air-dried initially and
then oven-dried overnight at 70 °C, ground fine enough to pass through a No. 10 mesh
sieve (pore size 2 mm). The raw biochars were then placed in airtight ceramic crucibles
with tight-fitting lids, pyrolyzed under oxygen-limited conditions in a muffle furnace
(MXQ1100-15, Chuangsai, China). Previous studies showed that the oxygen-
containing functional groups of biochar played central roles in binding heavy metal in
soils [17]. Higher pyrolysis temperature will decrease the amounts of oxygen-
containing functional groups in biochar surface, and then affect the adsorption capacity
of metal ions [14]. Moreover, some studies suggested that the pyrolysis temperature at
300 ºC for biochar could effectively immobilize the Cu, Cd and Pb in contaminated
soils [26,27]. Therefore, we set the pyrolysis temperature at 300 ºC. The pyrolysis
temperature was raised gradually, starting from room temperature, and increased by
5 °C per min to reach 300 °C (the final temperature), which was held constant for 6 h
for slow pyrolysis, then the biochar was allowed to cool and ground once again to pass
through a No. 60 mesh sieve (pore size 0.25 mm) to produce the final biochar samples.

2.2. Characterization of samples

Physical and chemical properties of the biochars, were determined as follows.
Carbon, hydrogen, and nitrogen contents of the samples were determined using a CHN
Element Analyzer (vario EL III, elementar Analysensysteme GmbH, Hanau, Germany).
The biochar samples were digested with HNO3 and H2O2, and the contents of major
inorganic elements were determined using graphite furnace atomic absorption
spectrometry (GFAAS) (Varian SpetrAA 220 Z, USA). The pH of biochar was
measured using a glass electrode dipped in a suspension of biochar in water (1:10). The
ash content of the biochars was calculated using the following equation: Ash content
(%) = (MAsh/MBiochar) ×100, where MAsh and MBiochar are the mass of the ash and biochar
respectively. The CEC of biochar was measured by a modified ammonium acetate
compulsory displacement method [28]. The Fourier Transform Infrared Spectroscopy
(FTIR) measurements were mounted on a Nexus spectrometer (Thermo Nicolet
Corporation, USA) in a KBr pellet at room temperature. Specific surface areas of the
samples were measured with Ommishop 100CX surface area analyzer (Beckman
Coulter, Brea, California, USA) using the N2 adsorption methods. The Zeta potential of
the obtained suspension was determined using a Zeta potential analyzer
(Zetasizer3000HSa, Malvern, UK). The elemental composition and the chemical
bonding state on the sample surface were determined using X-ray photoelectron
spectroscopy (XPS) (Thermo Scientific ESCALAB 250 spectrometer). The component
analysis of the corn straw and the hardwood was carried out using the procedure
reported by Li et al. [29].

2.3. Soil
 Surface samples of soil (0–20 cm) were collected from a rice-growing area around
a former copper mine site (Fenghuang Mountain, Tongling City, China; 30°56′42′′ N,
117°43′28′′ W). This soil was air-dried for 1 week and then passed through a No. 10
mesh sieve to remove larger pieces of debris and coarse impurities. Soil pH was
determined using a glass electrode dipped in a suspension of soil in water (1:2.5); soil
organic matter (SOM) was measured by the wet digestion method [30]; and cation
exchange capacity (CEC) was analyzed by NH4OAC leaching [31]. Soil texture was
measured using the method of Bowman and Hutka [32]. Total Cd and Cu contents of
the soil were determined by GFAAS following mixed-acid digestion (HNO3–HClO4–
HF) [33]. Certified sediment reference material (GBW07456) with known
concentrations of Cd (0.56 ± 0.04 mg kg–1) and Cu (54 ± 2 mg kg–1) was used as a
standard. Table 1 lists the basic physicochemical properties of the soil.

2.4. Experimental design

The pot culture experiments were carried out in a greenhouse of the Anhui
Academy of Agricultural Sciences in Hefei City during the rice-growing season over
three years (2012 to 2014). The seeds of rice (Oryza sativa L., Xinliangyou 343) were
obtained from the Institute of Rice, Anhui Academy of Agricultural Sciences. In the
soil-incubating experiment, 3 kg of Cd- and Cu-contaminated soil was placed in each
plastic pot (25 cm in diameter and 20 cm in height) and amended with biochar (3% and
5% by weight). Soils without biochar served as controls. After amended with two
different biochars, each pot was covered with a plastic lid to minimize moisture loss
(although the lid had a few small holes to allow gaseous exchange) and incubated at
constant humidity (70% water holding capacity) for one month. In the rice-planting
experiment, one-month-old rice seedlings were transplanted into the pots. The pots
were arranged in a randomized complete block design with three replicates. The pot
soil was maintained under flooded conditions (with 2–3 cm of water above the soil
surface) during the whole growth period.

2.5. Collection and analysis of samples

 At maturity, the aboveground parts of rice were removed from the pots and divided
into shoots and grains. Both were oven-dried at 60 °C to a constant weight, and the
grains were polished with a rice polishing machine (LTJM-12, Huier, China). The oven-
dried samples of shoots and the polished grains were pulverized in a stainless steel
grinder (FW-80, Wanneng, China). The concentrations of Cd and Cu in the samples
were determined by GFAAS following digestion with HNO3–H2O2. A certified
reference sample of rice (GBW 10010) with known concentrations of Cd (0.087 ± 0.005
mg kg–1) and Cu (4.9 ± 0.3 mg kg–1) was used as a standard.
Soil samples were collected four times: on the day before the first planting
(planting 1) and after each of the three harvests (2012, harvest 1; 2013, harvest 2; 2014,
harvest 3). Soil pH, SOM, dissoluble organic carbon (DOC), and CaCl2- and TCLP-
extractable Cd and Cu were determined in different biochar treatments. The details of
measurements for DOC and CaCl2- and TCLP-extractable Cd and Cu are provided in
the supporting information.

2.6. Oxidation of biochar

The aging of two biochars in acidic soil was simulated using 10% HNO3 solution.
−
There are two important considerations for the selection of HNO3. One is NO3 is the

main anions in acid soil in China, and other is the oxidizing ability of HNO3 is moderate
among many oxidants, which make the simulation of the aging process more close to
real situation. Each biochar sample (20 g) was immersed in 400 mL of 10% HNO3. The
control samples were immersed in deionized water instead. The reaction flasks were
maintained at 25 °C for 7 days, and the samples were collected at 1, 3, 5, and 7 days,
respectively. To remove any residual acid on the biochar surface, the oxidized samples
were washed repeatedly with deionized water until a constant pH was obtained. The
samples were freeze-dried for 1 day and stored until required for analysis. The carbon
content was determined using a CHN Elemental Analyzer. The samples collected on
the 7th day were used for determining the elemental composition and the chemical
bonding state by XPS and oxygen-containing functional groups by FTIR and Boehm
titration, and the Zeta potential were ascertained using Zeta potential analyzer. The
oxidized CB was mixed and reacted with Cd and Cu solution for 48 h, and then analyzed
by FTIR. Another biochar sample was immersed in 400 mL of different concentration
HNO3 (1%, 3%, 5% and 10%), and the samples were collected at 7 days to measure the
oxygen content by XPS. In order to simulate the actual pH in soil, each biochar sample
was immersed in HNO3 solution (pH=4.5). The samples were collected on the 7th day,
the carbon content was determined using a CHN Elemental Analyzer.
The extent of carbon reduction was considered to measure the oxidation resistance
of the biochar. The rate at which carbon was lost from the samples was calculated using
the following equation:
W = (M0 × N0 － M × N) × 100 / (M0 × N0)
where M0 (mg) is the mass of control samples, N0 (%) is the carbon content of control
samples, M (mg) is the mass of oxidized samples, and N (%) is the carbon content of
oxidized samples.

2.7. Boehm titration

Oxygen-containing functional groups were determined using the Boehm titration
method: 1 g of biochar was added to 25 mL of 0.05 M alkali solutions (Na2CO3,
NaHCO3, and NaOH). After stirring for 24 h, the biochar suspension was filtered
through a Millipore filter (pore size 0.45 μm) and the supernatant was back-titrated with
0.05 M HCl [34, 35].

2.8. Data analysis

The data were analyzed using Excel 2007 and SPSS (ver. 16.0) for Windows. The
means of three replicates were subjected to one-way analysis of variance using Tukey’s
HSD (honest significant difference) test at a significance level of 0.05.

3. Results
3.1. Characterization of biochar
The basic characteristics of biochars derived from the two sources (CB and HB)
are given in Table 2. Both were alkaline, with a pH of 8.6 and 9.1, respectively. CB was
richer in nitrogen, hydrogen, and trace elements but poorer in carbon. The oxygen
contents of the two biochars were broadly similar (Table 2).

3.2. Effect of biochar on soil properties

The soil pH in the control treatment remained unchanged throughout the
experiment (Table S1) whereas the biochar-amended soil showed significantly higher
pH values (P < 0.05): after three years, the pH had increased from 4.35 to 5.35 in HB
and to 5.15 in CB (Table S1), and the difference between the two were insignificant.
The contents of SOM and DOC were significantly higher in the soil treated with
CB or HB (P < 0.05), and the extent of increase was roughly proportional to the amount
of biochar added (Table S1). There was little change in SOM during the three-year
period, indicating that both types of biochar had remained stable in the soil in the long
term; DOC contents, on the other hand, changed over time, increasing steadily up to
harvest 1, and then decreasing over harvest 2 and harvest 3 (Table S1).

3.3. Extractable Cd and Cu

The effect of CB and HB on the immobilization of metals was examined by
monitoring the levels of CaCl2- and TCLP-extractable Cd and Cu (Fig. 1 and Table S2):
both had decreased significantly and stably in the CB-treated soils over the three years,
and the extent of reduction was proportional to the additive amount of biochar. By the
end of the three years, CB at 5% level had lowered the levels of CaCl2-extractable Cd
to 46.4% and those of Cu to 33.2% of those in the controls (Fig. 1A and 1C). The
corresponding figures for TCLP-extractable Cd and Cu were 61.1% and 49.0%,
respectively (Fig. 1B and 1D). In the case of HB at 5% addition rates, CaCl2-extractable
Cd and Cu in harvest 1 were 42.1% and 36.2% of those in the controls respectively, but
had increased in following two years (Fig. 1A and 1C). The CaCl2-extractable Cd
increased from 0.17 mg kg–1 to 0.24 mg kg–1 and Cu from 10.6 mg kg–1 to 18.6 mg kg–
1
between 2012 and 2014. The trend of TCLP-extractable Cd and Cu was identical to
that of CaCl2-extractable Cd and Cu in HB. These results demonstrated that CB was
superior to HB in terms of steady and long-term immobilization of Cd and Cu in
contaminated soils.

3.4. Uptake of Cd and Cu

There was a stable reduction of Cd and Cu uptake in rice shoot and polished grain
during three years in CB-treated soil (Fig. 2), which is consistent with the results of
extractable metal contents in the soil samples (Fig. 1). The application of CB at 5%
lowered the Cd and Cu contents to 38.8% and 49.4% of those in the controls in the
polished grain, respectively, in last collection. The corresponding figures for shoots
were 36.8% and 48.8%, respectively (Fig. 2). Such stable decrease in Cd and Cu uptake
in rice revealed efficient and continuous immobilization of Cd and Cu in the soil
amended with CB. In the soil amended with 5% HB, the Cd and Cu contents of the
polished grains were decreased to 36.4% and 43.7% of those in the controls in 2012,
respectively, and increased in next two years. The corresponding values were 55.3%
and 53.8% in 2013, and 69.4% and 78.7% in 2014, respectively. The higher Cd and Cu
contents of the polished grain in the last two harvests when compared to that in the first
harvest matched the pattern seen in the shoot (Fig. 2). The increase in Cd and Cu uptake
in rice could be attributed to the elevated mobility of metals in the HB-treated soil.

3.5. Aging of biochar

In the experiment that simulated the aging of biochar, the carbon content of CB
and HB decreased with time initially and plateaued after the fifth day using 10% HNO3
solution, thus high concentration of acid solution could reflect the surface properties of
the biochar under the status of oxidation equilibrium (Fig. 3). The decrease of the
carbon content in CB was markedly higher than that in HB throughout (Fig. 3). It was
identical to the results using HNO3 solution (pH=4.5), and the carbon loss rate (3.2%)
of CB was more than HB (1.4%). The result showed the higher antioxidant ability of
HB in acid condition.
The kinetic changes of oxygen content by CB and HB in 10% HNO3 solution was
showed in Fig. S1a. The variation of oxygen content could be divided into two steps:
in the first step a rapid increase occurred, the second step being slower before reaching
the equilibrium. The slope of line in CB was higher than that of HB, which indicated
that the oxidation process of CB was more rapid than HB (Fig. S1a). The plot in Fig.
S1a, showing two line with different slopes, suggested that CB was more ready to
oxidize in acid condition. These results indicated that the CB included more oxygen-
containing groups than HB after HNO3 oxidation.

The zeta potentials of unoxidized and oxidized samples of CB and HB at varying

values of pH are shown in Fig. 4. The results indicated that the surface charges of
biochar were negative at all pH values, and the zeta potential of biochar become more
negative with pH increase. Protonation and deprotonation of functional groups can
create a net charge on the surfaces of the colloid particles, which can form an electrical
double-layer in the solution phase near the surfaces. Zeta potential values were
measured as a function of solution pH for each of the biochar samples. There were large
−
amounts of oxygen-containing functional groups (e.g. –COO , –COH and –OH) on the
−
biochars. Among them, –COO and –OH contributed considerably to surface charge of
− − +
the biochars. The association of –COO and –O with H may have reduced the
negative charge of the biochars with the decline of pH, and the zeta potentials of the
biochar samples became less negative. On the contrary, zeta potential can become more
negative with the pH increase. The negative charges in the unoxidized samples of both
CB and HB were nearly similar; however, the charge on CB was more negative than
that on HB at any given pH after the oxidation treatment. It indicated that CB had a
greater number of negatively charged functional groups on its surface than HB when
both had been subjected to the same oxidation treatment.
The XPS analysis of C1s, O1s, and N1s contents on the surface of the unoxidized
and oxidized biochars are presented in Fig. S2. The C1s, O1s and N1s represent the
first orbital electron of C, O and N respectively, and further identify the presence of
chemical element (C, O and N) in two samples. In XPS spectrum (Fig. S2), three peaks
are observed at 291.6, 536.3 and 404.8 eV corresponding to the binding energies of C1s,
O1s and N1s, respectively. After the HNO3 treatment, the oxygen contents on the
surface of CB were higher than those on the surface of HB (Fig. S2C and S1D),
suggesting that part of the carbon in CB was more easily oxidized by HNO3, thus lead
to the higher oxygen content of CB (32.4%) than that of HB (26.1%). In addition, XPS
analysis of C1s binding energies on the surface of the oxidized biochars showed that
the peaks of C-O, C=O, and COOH were strengthened after oxidation, and that CB
showed higher proportions of C-O, C=O, and COOH than HB (Fig. 5 and Table S2). In
particular, the contents of carboxylic groups in CB (6.8%) were almost 1.7 times those
in HB (3.9%) after the HNO3 treatment (Table S2).
The results of Boehm titration of unoxidized and oxidized samples for BC and HB
are shown in Table 3. After HNO3 oxidation, the contents of carboxylic, lactone, and
phenolic hydroxylic groups in BC increased by 77.1%, 32.0%, and 42.6%, respectively;
the corresponding values in HB were 14.4%, 21.1%, and 17.8%. The oxidized samples
of CB showed a greater proportion of oxygen-containing groups than oxidized HB.
More carboxyl groups were found on the surface of CB (1.36 meq g–1) than that of HB
(0.81 meq g–1), an observation consistent with the results of XPS. These results indicate
that the source of biochar has a remarkable impact on the number of oxygen-containing
function groups during the aging of biochar.

4. Discussion
The application of biochar can influence soil properties (i.e. pH, SOC and DOC)
significantly. The increase in soil pH was consistent with the alkaline nature of the
biochars (Table S1). Base cations in the biomass were transformed into oxides,
hydroxides, and carbonates that were mixed with biochar during pyrolysis [22,36]. The
dissolution of these alkaline substances in biochars increased the soil pH [37,38]. As
the pH increased, the hydrolysis of metal cations increased, and precipitates of metal
(oxy)hydroxides formed. Two biochars have an alkaline pH (Table 2), and thus
incorporation of biochar increased the pH of acidic soil as described above (Table S1).
The pH of the soil used in this study increased by 1 units when the addition of 5% CB
and HB, respectively, which promoted the formation of precipitates of these metals.
The biochars were rich in stable carbon; therefore the addition of CB and HB increased
the content of SOC, which can act as a stable source of carbon [39]. The increase in
DOC can be ascribed to the dissolution of biochar organic carbon [40]. The alteration
in soil properties due to the addition of amendments can also affect the stable retention
of PTEs in soil.
The two biochars differed significantly in their long-term effect on Cd and Cu
immobilization over the three years (Table S2). HB lowered the availability of Cd and
Cu in soil in the first year, but their activity increased in the next two years. The
increased concentration of Cd and Cu in CaCl2 and TCLP is associated with elevated
concentrations of DOC in the soil. Earlier studies have reported that Cd and Cu form
complexes with available ligands within the natural organic matter, which increases the
mobility of Cd and Cu in soil interstitial water. This suggests co-mobilization [41].
Beesley et al. [42] reported that the application of hardwood-derived biochar increased
the concentrations of DOC, which has an adverse effect on the immobilization of Cu.
Therefore, the elevated labile pool of Cd and Cu in the last two years is probably due
to the increase in DOC following the addition of HB.
The CaCl2- and TCLP-extractable fractions of Cd and Cu in the soil were stably
immobilized by CB over the three years. The variation in the degree of stability of the
immobilized Cd and Cu probably stems from the differences in the way the two
biochars undergo aging. Chemical aging (oxidation) in the highly acidic soil is the most
critical factor affecting biochar properties over time, and thus the aging of the CB and
HB was simulated by acid oxidation. The greater decrease in the carbon content of CB
during the simulation (two different concentration of acid solution) was due to its
composition. The main constituents of plant are cellulose, hemicellulose, and lignin.
Lignin represents the polyphenols present in the plant, which are efficient antioxidants
of natural origin [43], and hardwood contained much more lignin (31.3%) than corn
straw (7.6%). Therefore, CB has stronger resistant ability to oxidation than HB under
acid conditions. In turn, CB and HB showed various quantities of oxygen-containing
functional groups on their surface due to different degree of oxidation, which
subsequently affected the immobilization stability of Cd and Cu in soil.
Oxidation causes significant variation in the surface properties of CB and HB. The
XPS analysis indicated that oxidation led to a greater number of oxygen-containing
functional groups (C-O, C=O, and COOH) on the surface of CB than HB (Fig. 5 and
Table S2). The zeta potential of oxidized CB was more strongly negative than that of
oxidized HB at a given pH, confirming that CB had a greater number of negatively
charged functional groups on its surface; and a higher O/C ratio (35.9%) of CB than
HB (17.7%) after the oxidation treatment is a main cause for the enhanced
immobilization of Cd and Cd for three years (Fig. S2). Uchimiya et al. noted [17] that
oxygen-containing functional groups on the biochar surface controlled their capacity to
adsorb PTEs, and that the stabilization ability of PTEs was positive correlated to the
O/C ratio. It has also been found that acid-oxidized biochars exhibited significantly
greater ability to retain Pb, Cu, and Zn in contaminated soil than unoxidized biochars
[16]. The increase in the number of new oxygenated adsorption sites in CB due to acid
oxidation could increase the adsorption of Cd and Cu, which depends on the enhanced
electrostatic interaction and surface complexation reaction. Moreover, the
incorporation of biochar led to the increases in soil CEC and made soil negative surface
charge more negative. The increase of soil CEC depended on the CEC of the biochar
samples [20]. In present study, the CEC of CB and HB increased from 143 cmol kg–1
and 129 cmol kg–1 to 275 cmol kg–1 and 177 cmol kg–1 respectively after oxidation
treatment, and thus incorporation of CB led to the greater increase in soil CEC than HB,
suggesting that the variation of CEC in biochar was an important factor influencing the
immobilization of Cd and Cu.

The interaction between the aging of biochar and PTEs affects not only the
adsorption capacity but also the stability of immobilization of Cd and Cu in soil. The
immobilization of metals by biochar may occur through both specific and non-specific
adsorption [26]. Specific adsorption of PTEs is more stable, because the adsorption of
metals depends on the formation of coordinate bonds, which hold PTEs more tightly
than physical adsorption [44]. In the present study, oxidized CB showed a higher
proportional increase (81.5%) in carboxyl groups than that shown by oxidized HB
(29.3%, Table S2) , a result confirmed by Boehm titration (Table 3). The complexation
of oxygen-containing functional groups (especially carboxyl) played a crucial role in
the stable binding of metal ions with biochar [45]. The infrared spectra of oxidized CB
before and after the interaction with Cd and Cu are shown in Fig. 6. The wavenumber
−
of –COO was 1698 cm−1 in the absence of Cd and Cu, and changed to 1678 cm−1 after
−
Cd and Cu association [14]. Furthermore, the intensity of band at 1698 cm−1 (–COO )
decreased after the Cd and Cu loading, which resulted from the decrease of number of
the carboxylic groups. These results indicate that the carboxylic groups on the surface
of oxidized CB serve as adsorption sites for Cd and Cu. The aging process increases
the quantity of carboxylic groups which can affect the adsorption capacity of Cd and
Cu by the surface complexation. Earlier research has shown that activated plant biochar
(from pecan shells) complexed and stabilized Cu and Pb in the presence of competing
acetate ligands which were able to combine Cu and Pb to increase the soluble
concentrations, and the stable retention of PTEs in soil could be ascribed to the increase
in the functional carboxyl groups after activation. However, plant biochars that had not
been activated (from cottonseed hulls) increased the concentrations of dissolved Cu and
Pb in soils by forming soluble complexes with biochar-borne DOC [46]. Therefore,
enhanced surface complexations resulted in more stable immobilization of Cd and Cu
by CB in soil, which may reduce the possibility of the two metals being released into
the environment in future. The activation of CB in acidic soils could further enhance
the stability of immobilization of Cd and Cu even the increase in DOC.
Over the three years, the Cd and Cu in rice tissue decreased significantly and
steadily in CB-treated soils, whereas HB proved less efficient in decreasing the uptake
of Cd and Cu by the rice plants. The aging increased the number of oxygen functional
groups on the surface of CB and kept them active year by year, allowing CB to continue
retaining PTEs in the soil, which explains the decrease of Cd and Cu uptake in rice over
the three years. These results indicate that biochar type has a marked effect on the long-
term stabilization of immobilized Cd and Cu in acid soils.

5. Conclusion
There were not significant variations of soil properties (pH, SOM and DOC) after
the application of two biochar samples (CB and HB), and CB proved superior to HB in
reducing the bio-availability of Cd and Cu in contaminated soil over three years. While
the Cd and Cd content in rice plant tissues was consistently and significantly reduced
by CB, depending on biochar application rate, these values increased in HB treatment
as the time period. Analyses of oxidized CB and HB showed a greater number of
oxygen-containing groups (especially carboxyl) on the surface of CB than that on HB
under the same acidic conditions. The oxygen-containing groups continued to lower the
availability of Cd and Cu in soil year after year. Lignin was mainly responsible for the
difference in the degree to which the two biochars were oxidized. The study
demonstrated the link between biochar type and long-term immobilization of Cd and
Cu in acidic paddy soils and showed the importance of choosing the right type of
biochar, which promotes the activation of biochar surface in acidic soils. It is
recommended that the readily oxidized biochar could be considered to apply in acid
soils, which could make a sustained contribution to the reduction of PTEs risks in
contaminated farmland.

Acknowledgments
This research was supported by grants from National Major Research Program of
China (2013CB934302), National Natural Science Foundation of China (No. 41301347
and No. 21177132), Anhui Provincial Natural Science Foundation (1408085MKL61),
Natural Science Foundation of Anhui Academy of Agricultural Sciences (14A1009),
Scientific and Technical Key Research Program of Auhui, and CAS/SAFEA
International Partnership Program for Creative Research Teams of Chinese Academy
of Sciences, China.

References
[1] X. Hang, H. Wang, J. Zhou, C. Ma, C. Du, X. Chen, Risk assessment of potentially toxic element
pollution in soils and rice (Oryza sativa) in a typical area of the Yangtze River Delta, Environ.
Pollut. 157 (2009) 2542–2549.
[2] X.X. Ye, H.Y. Li, Y.B. Ma, L. Wu, B. Sun, The bioaccumulation of Cd in rice grains in paddy
soils as affected and predicted by soil properties, J. Soil Sed. 4 (2014) 1407–1416.
[3] J.G. Liu, M. Qian, G.L. Cai, J.C. Yang, Q.S. Zhu, Uptake and translocation of Cd in different
rice cultivars and the relation with Cd accumulation in rice grain, J. Hazard. Mater. 143 (2007)
 443–447.
[4] X.X. Ye, S.H. Kang, H.M. Wang, H.Y. Li, Y.X. Zhang, G.Z. Wang, H.J. Zhao, Modified natural
diatomite and its enhanced immobilization of lead, copper and cadmium in simulated
contaminated soils, J. Hazard. Mater. 289 (2015) 210–218.
[5] J.H. Park, G.K. Choppala, N.S. Bolan, J.W. Chung, T. Chuasavathi, Biochar reduces the
bioavailability and phytotoxicity of heavy metals, Plant Soil 348 (2011) 439–451.
[6] A. Brennan, E.M. Jiménez, M. Puschenreiter, J.A. Alburquerque, C. Switzer, Effects of biochar
amendment on root traits and contaminant availability of maize plants in a copper and arsenic
impacted soil, Plant Soil 379 (2014) 351–360.
[7] D.Y. Xu, Y. Zhao, K. Sun, B. Gao, Z.Y. Wang, J. Jin, Z.Y. Zhang, S.F. Wang, Y. Yan, X.T. Liu,
F.C. Wu, Cadmium adsorption on plant-and manure-derived biochar and biochar-amended
sandy soils: impact of bulk and surface properties, Chemosphere 111(2014) 320–326.
[8] R.J. Bian, S. Joseph, L.Q. Cui, G.X. Pan, L.Q. Li, X.Y. Liu, A.F. Zhang, H. Rutlidge, S.W.
Wong, C. Chia, C. Marjo, B. Gong, P. Munroe, S. Donne, A three-year experiment confirms
continuous immobilization of cadmium and lead in contaminated paddy field with biochar
amendment, J. Hazard. Mater. 272 (2014) 121–128.
[9] N. Karami, R. Clemente, E. Moreno-Jiménez, N.W. Lepp, L. Beesley, Efficiency of green waste
compost and biochar soil amendments for reducing lead and copper mobility and uptake to
ryegrass, J. Hazard. Mater. 191 (2011) 41–48.
[10] B.T. Nguyen, J. Lehmann, J. Kinyangi, R. Smernik, S.J. Riha, M.H. Engelhard, Long-term
black carbon dynamics in cultivated soil, Biogeochemistry 89 (2008) 295−308.
[11] S.E. Hale, K. Hanley, J. Lehmann, A. Zimmerman, G. Cornelissen, Effects of chemical,
biological, and physical aging as well as soil addition on the sorption of pyrene to activated
carbon and biochar, Environ. Sci. Technol. 45 (2011) 10445−10453.
[12] C.H. Cheng, J. Lehmann, J.E. Thies, S.D. Burton, M.H. Engelhard, Oxidation of black carbon
by biotic and abiotic processes, Org. Geochem. 37 (2006) 1477–1488.
[13] J.D. Mao, R.L. Johnson, J. Lehmann, D.C. Olk, E.G. Neves, M.L. Thompson, K. Schmidt-
Rohr, Abundant and stable char residues in soils: implications for soil fertility and carbon
sequestration, Environ. Sci. Technol. 46 (2012) 9571−9576.
[14] L.B. Qian, B.L. Chen, Interactions of aluminum with biochars and oxidized Biochars:
implications for the biochar aging process, J. Agric. Food Chem. 62 (2014) 373−380.
[15] K.T. Klasson, L.H. Wartelle, J.E. Rodgers, I.M. Lima, Copper (II) adsorption by activated
carbons from pecan shells: Effect of oxygen level during activation, Ind. Crop. Prod. 30 (2009)
72–77.
[16] M. Uchimiya, D.I. Bannon, L.H. Wartelle, Retention of heavy metals by carboxyl functional
groups of biochars in small arms range soil, J. Agric. Food Chem. 60 (2012) 1798−1809.
[17] M. Uchimiya, S. Chang, K.T. Klasson, Screening biochars for heavy metal retention in soil:
Role of oxygen functional groups, J. Hazard. Mater. 190 (2011) 432−441.
[18] J.W. Chen, D. Zhang, M. Wu, P. Wang, Comparison of oxidation resistance of two biochars,
Environ. Chem. 33 (2014) 943−948.
[19] A. Mukherjee, A.R. Zimmerman, W. Harris, Surface chemistry variations among a series of
laboratory-produced biochars, Geoderma 163 (2011) 247–255.
[20] J.H. Yuan, R.K. Xu, W. Qian, R.H. Wang, Comparison of the ameliorating effects on an acidic
ultisol between four crop straws and their biochars, J. Soil Sed. 11 (2011) 741–750.
[21] M.J. Gundale, T.H. DeLuca, Temperature and source material influence ecological attributes
of ponderosa pine and Douglas-fir charcoal. Forest Ecol. Manag. 231 (2006) 86–93.
[22] J.H. Yuan, R.K. Xu, H. Zhang, The forms of alkalis in the biochar produced from crop residues
at different temperatures, Bioresour. Technol. 102 (2011) 3488–3497.
[23] B. Singh, B. P. Singh, A. L. Cowie, Characterisation and evaluation of biochars for their
application as a soil amendment, Aus. J. Soil Res. 2010, 48, 516–525
[24] H.B. Cui, J. Zhou, Y.B. Si, J.D. Mao, Q.G. Zhao, G.D. Fang, J.N. Liang, Immobilization of Cu
and Cd in a contaminated soil: one- and four-year field effects, J. Soil Sed. 14 (2014) 1397–
1406.
[25] L. Cang, D.M. Zhou, Q.Y. Wang, D.Y. Wu, Effects of electrokinetic treatment of a heavy metal
contaminated soil on soil enzyme activities, J. Hazard. Mater. 172 (2009) 1602–1607.
[26] T.Y. Jiang, J. Jiang, R.K. Xu, Z. Li, Adsorption of Pb(II) on variable charge soils amended with
rice-straw derived biochar, Chemosphere 89 (2012) 249–256.
[27] J. Jiang, R.K. Xua, T.Y. Jianga, Z. Li, Immobilization of Cu(II), Pb(II) and Cd(II) by the
addition of rice straw derived biochar to a simulated polluted Ultisol, J. Hazard. Mater. 229–
230 (2012) 145–150.
[28] J.W. Gaskin, R.A. Speir, K. Harris, K.C. Das, R.D. Lee, L.A. Morris, D.S. Fisher, Effect of
peanut hull and pine chip biochar on soil nutrients, corn nutrient status, and yield. Agron. J.
102 (2010) 623–633.
[29] S.G. Li, S.P. Xu, S.Q. Liu, C.Y., Q.H. Lu, Fast pyrolysis of biomass in free-fall reactor for
hydrogen-rich gas, Fuel Process. Technol. 85 (2004) 1201–1211.
[30] D.W. Nelson, L.E. Sommers, Total carbon, organic carbon, and organic matter. In Methods of
Soil Analysis. Part 3-Chemical Methods. Soil Science Society of America, Inc., American
Society of Agronomy, Inc., Madison, Wisconsin, USA, 2001, pp. 961–1010.
[31] R.K. Lu, Soil and Agro-chemical Analysis Methods. Agricultural Science and Technology
Press, Beijing, China, 2000, pp. 205–266.
[32] G.M. Bowman, J. Hutka, Soil physical measurement and interpretation for land evaluation.
Australian Soil and Land Survey Handbooks. CSIRO Publishing, Collingwood, Australia, 2002,
pp. 224–239.
[33] M.C. Amacher, Nickel, cadmium, and lead. In: Methods of Soil Analysis: Part 3. Chemical
Methods. In: Sparks DL, editor. SSSA Book Series 5. Soil Science Society of America, Inc.,
American Society of Agronomy, Inc., Madison, Wisconsin, USA, 2001, pp. 739–768.
[34] Z.G. Liu, F.S. Zhang, Removal of lead from water using biochars prepared from hydrothermal
liquefaction of biomass, J. Hazard. Mater. 167 (2009) 933–939.
[35] T.J. Bandosz, J. Jagiello, C. Contescu, J.A. Schwarz, Characterization of the surfaces of
activated carbons in terms of their acidity constant distributions, Carbon 31 (1993) 1193–1202.
[36] J.M. Novak, W.J. Busscher, D.L. Laird, M. Ahmedna, D.W. Watts, M.A.S. Niandou, Impact
of biochar amendment on fertility of a southeastern coastal plain soil, Soil Sci. 174 (2009) 105–
112.
[37] L.V. Zwieten, S. Kimber, S. Morris, K. Chan, A. Downie, J. Rust, S. Joseph, A. Cowie, Effects
of biochar from slow pyrolysis of papermill waste on agronomic performance and soil fertility,
Plant Soil 327 (2010) 235–246.
[38] D. Houben, L. Evrard, P. Sonnet, Mobility, bioavailability and pH-dependent leaching of
cadmium, zinc and lead in a contaminated soil amended with biochar, Chemosphere 92 (2013)
 1450–1457.
[39] X.D. Cao, L. Ma, Y. Liang, B. Gao, W. Harris, Simultaneous immobilization of lead and
atrazine in contaminated soils using dairy-manure biochar, Environ. Sci. Technol. 45 (2011)
4884–4889.
[40] L. Beesley, E. Moreno-Jiménez, J.L. Gomez-Eyles, Effects of biochar and greenwaste compost
amendments on mobility, bioavailability and toxicity of inorganic and organic contaminants in
a multi-element polluted soil, Environ. Pollut. 158 (2010) 2282–2287.
[41] Y. Mason, A.A. Ammann, A. Ulrich, L. Sigg, Behavior of heavy metals, nutrients, and major
components during roof runoff infiltration, Environ. Sci. Technol. 33 (1999) 1588–1597.
[42] L. Beesley, N. Dickinson, Carbon and trace element fluxes in the pore water of an urban soil
following green waste compost, woody and biochar amendments, inoculated with the
earthworm Lumbricus terrestris. Soil Biol. Biochem. 43 (2011)188–196.
[43] D. Tatiana, T. Galina, J. Vilhelmina, V. Uldis, Characterization of the radical scavenging
activity of lignins−natural antioxidants, Bioresour. Technol. 95 (2004) 309–317.
[44] M. Uchimiya, K.T. Klasson, L.H. Wartelle, I.M. Lima, Influence of soil properties on heavy
metal sequestration by biochar amendment: 1. copper sorption isotherms and the release of
cations, Chemosphere 82 (2011) 1431–1437.
[45] Y.W. Xue, B. Gao, Y. Yao, M. Inyang, M. Zhang, A.R. Zimmerman, K.S. Ro, Hydrogen
peroxide modification enhances the ability of biochar (hydrochar) produced from
hydrothermal carbonization of peanut hull to remove aqueous heavy metals: Batch and column
tests, Chem. Eng. J. 200–202 (2012) 673–680
[46] M. Uchimiya, D.I. Bannon, Solubility of lead and copper in biochar-amended small arms range
soils: influence of soil organic carbon and pH, J. Agric. Food Chem. 61 (2013) 7679−7688.
Fig. 1. The concentrations of Cd and Cu in the CaCl2 (A and C) and TCLP (B and D)
extraction from untreated and different biochar treated soils in successive collections
over the three years.

Fig. 2. The contents of Cd (A and B) and Cu (C and D) in rice grains and shoots across
3 years pot experiment (mean ± S.D., n = 3). Different letters indicate significant
differences in three harvests under the same treatment (P < 0.05).
Fig. 3. The oxidation kinetics of HB and CB in 10% HNO3 solution. Different lowercase
letters indicated significant differences among different times (P < 0.05)

Fig. 4. The zeta potential of HB and CB without and with oxidation treatment in 10%
HNO3 solution.
Fig. 5. XPS analysis of C1s binding energy and corresponding carbon bonds on HB and
CB surfaces without (A and B) and with (C and D) oxidation treatment in 10% HNO3
solution.
Fig. 6. FTIR spectrum of oxidized CB before (A) and after (B) reaction with Cu and Cd

in aqueous solution.

Table 1 Basic physico-chemical properties of the soil.

SOMa CECb Total concentration (mg kg–1)

pH Clay (%)
(g kg–1) (cmol kg–1) Cd Cu

4.43 29.8 11.3 26.2 2.2 452

a
SOC: soil organic carbon.

b
CEC: cation exchange capacity.
 Table 2 Basic properties of two different biochars used in this study.

Surface area CEC Ash content Elemental analysis (%)

Sample pH
(m2 g–1) (cmol kg–1) (%) C H O a
N P K Ca Mg F
HB 9.1 5.6 129 1.1 73.3 2.6 22.9 0.3 0.35 0.35 0.12 0.03 0.0
CB 8.6 6.6 143 5.8 67.7 4.1 23.3 1.6 0.4 2.27 0.33 0.12 0.0

a
Determined by weight difference supposing that the total weight of the samples consist of the
tested elements only.

b
< 0.01%.
Table 3 Boehm titration results for HB and CB without and with oxidation treatment
in 10% HNO3 solution.

Carboxylic group Lactone group Phenolic hydroxylic

Samples
(meq g–1) (meq g–1) group (meq g–1)
HB 0.71 0.19 0.66
Oxidized HB 0.81 0.23 0.78
CB 0.77 0.25 0.61
Oxidized CB 1.36 0.33 0.87