IJERD – International Journal of Environmental and Rural Development (2018) 9-1

Research article
erdInfluence of Percolation Patterns on Copper Uptake, and
Growth and Yield with Copper-polluted Stratified Paddy
FieldsJINHUN FANThe United Graduate School of Agricultural Sciences, Iwate University, Morioka, Iwate, JapanCHOICHI SASAKI*
Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, Aomori, Japan
Email: chsasaki@hirosaki-u.ac.jp

CHIHIRO KATO
Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, Aomori, JapanNOBUHIKO MATSUYAMA
Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, Aomori, Japan

TAKEYUKI ANNAKA
Faculty of Agriculture, Yamagata University, Tsuruoka, Yamagata, JapanAKIRA ENDO
Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, Aomori, JapanSONGTAO LI
The United Graduate School of Agricultural Sciences, Iwate University, Morioka, Iwate, Japan

KIICHI SASAKI
Aomori Prefectural Improvement Projects Federation, Aomori, Aomori, Japan
Received 30 November 2017

Accepted 10 May 2018(*Corresponding Author)

Abstract Copper(Cu), arsenic, and cadmium are designated as specific substances of theAgricultural Land Soil Pollution Prevention Act in Japan. It has been known that high Cu
concentrations in soil layers reduce rice crop production and therefore agricultural practices
such as soil dressing have been applied to minimize damage to crops by Cu pollution. In
this study, we investigated the effects of percolation patterns of the plowsole and the subsoil
on growth and yield, and Cu uptake of paddy rice. Four stratified paddy field models wereconstructed to conduct growth tests under the condition that the percolation patterns ofplowsole and subsoil were in an open or closed system. These models had a plow layer andan upper plowsole made with 12.5cm-thickness of non-polluted soil dressing (3.7 mg/kg)
and underlying 15cm-thickness of a polluted lower plowsole and a subsoil layer whose Cu
concentrations were either higher (approximately 250 mg/kg) or lower (approximately 70
mg/kg) than Japanese safety standards (125 mg/kg). During the tests, a constant waterponding system was adopted, and mid-summer drainage was not done. As a result, Cu
concentrations in the rice grains were 5% significantly higher in the open systempercolation models regardless of the original amount of Cu in the plowsole and subsoil. On
the other hand, we did not recognize the significant difference in growth and yield of rice
plants among the models. We concluded that the Cu concentrations in rice plants wereaffected by percolation patterns of the polluted plowsole and subsoil even though they werecovered with non-polluted soil dressing layers.
Keywords copper, rice, percolation patterns, soil dressingⒸ ISERD

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INTRODUCTION
Copper (Cu), Cadmium (Cd) and Arsenic are recognized as the specific contaminant heavy metals
for agricultural lands and thus a variety of techniques for minimizing the heavy metals uptake ofcrops have been developed, for example, soil dressing, chemical measurement, phytoremediation,and breed improvement of rice plants. It has also been recommended to keep the soil in reduction
condition by flooding during the whole growing period in order to reduce Cd and Cu uptake(Yamane et al., 1997; Asami, 2005; Inahara et al., 2007; Akahane et al., 2013).
The studies of Cu pollution in agricultural lands have mainly been focused on damage to crops
such as growth inhibition, while Cd concentrations in brown rice itself have been anothersignificant issue of soil pollution. The problem of soil Cu pollution has been of relatively smallinterest compared to Cd pollution. It is probably because Cu pollution rarely directly affects humanhealth (Kobayashi, 1978, Takaishi et al., 2015) and the area affected by Cu pollution is not as large
as that of Cd contamination in Japan. It is reported that in apple orchards, Bordeaux mixtures,mixture of copper sulphate and calcium carbonate have been used for a long time and thus soil Cu
concentrations in some orchards are as high as several hundred mg/kg (dry soil) (Aoyama, 2009)
while the safety standard of soil Cu concentrations in Japan is 125 mg/kg. Since apple farming isreally hard work, elderly farmers, especially, tend to abandon their orchards. Some of the apple
orchards in lowland had once been converted from paddy fields and there is a possibility that they
will be restored to paddy fields, which require less labor. Therefore, it should be necessary andimportant to develop the technique of minimizing Cu uptake of paddy rice plants.
In Japan, soil dressing has mainly been applied for remediation of Cu polluted soil (Asami,2010). Recently, Paul et al. (2011a, b) and Sasaki et al. (2016a, b) clarified that variations of
percolation patterns of the plowsole and the subsoil using stratified paddy field models with soil
dressing layers resulted in significant differences in Cd concentrations in the brown rice. Paul et al.
(2011b) also showed that the percolation patterns affected the amount of Cu accumulation in rice
plants even though they used non-polluted soil (12.2 mg/kg). Since the solubility of Cu increases
under the oxidation condition and decreases in the reduction condition as in the case of Cd (Donget al., 2007), the percolation patterns in stratified paddy fields may affect the Cu uptake and growthand yields of rice plants.
It has been reported that Cu polluted soil is likely to induce a Cu accumulation in the roots of
paddy rice and a decrease in the number of panicles and the ratio of ripening (Chino et al., 1966;
Shibuya, 1979). Shibuya (1979) also mentioned that the yields of brown rice had decreased byapproximately 10% under the condition that Cu concentrations in the subsoil layer were higher than
200 mg/kg with a 15cm-thick soil dressing. These studies, however, did not consider utilizing thepercolation patterns.
From the above, the objective of this study is to clarify whether percolation patterns affect thegrowth, the yields, and the Cu uptake of rice plants under the conditions that Cu concentrations areeither under or above the Japanese safety standard (125 mg/kg). We prepared stratified paddy fieldmodels with approximately 70 mg/kg- and 250 mg/kg- Cu contaminated soil. The results were thatthe percolation patterns significantly changed Cu concentrations in the brown rice but neither thegrowth nor the yields.
METHODOLOGY
Table 1 shows the physical and chemical properties of the soils used in this study. Kanagi soil
(Loam), 3.7 mg/kg Cu concentration, was sampled from a plow layer of the paddy field in Kanagi
farm of Hirosaki University, Aomori prefecture. Bunkyou soil (Clay Loam) was made by adding asolution of CuCl2・2H2O to the soil which had been sampled from a plow layer of the paddy field
on Bunkyo campus of Hirosaki University, Aomori prefecture, and both were mixed well. Cu
concentrations in the soil of Bunkyo campus were originally 10.5 mg/kg. We produced two levelsof Cu contaminated soil, Cu concentrations in which were either lower (71 mg/kg) or higher (247mg/kg) than Japanese safety standard (125 mg/kg). These values are 15 and 50 times, respectively,Ⓒ ISERD

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as large as the average Cu concentration of non-contaminated paddy fields in Japan (4.47 mg/kg)(Asami, 2010). The organic matter content (OM) of the Kanagi soil and Bunkyou soil was 4.7%
and 6.6%, respectively. The gravel, which contained 0.8 mg/kg, was used for the lower layer of themodels since they were designed after the fashion of paddy fields near a river.
Table 1 Physical and chemical properties of soil samples and gravel
Density

Soil
Texture

MgO

CaO

K2O

Cu

T-C

T-N

C/N

OM

L
CL
-

229
219
-

(mg/kg)
531
306
1848
373
-

3.7
10.5
0.8

(%)
2.74
3.84
0.04

(%)
0.18
0.26
0.01

15.40
14.50
4.00

(%)
4.7
6.6
0.1

3

Kanagi Soil
Bunkyou Soil
Gravel(g/cm )
2.58
2.61
2.68

Experimental Design
According to the previous report (Sasaki et al., 2016a), two types of stratified paddy field models
were used for the experiment: the open-system percolation model and the closed-systempercolation model. The percolation patterns were determined by Sasaki et al. (1992). Each stratified
paddy field model was constructed in an iron box (30 cm×50 cm×70 cm) filled with three layers ofsoil. The plow layer was from 0cm to 10cm deep with non-polluted Kanagi soil (dry density in
puddling condition was 1.04 g/cm3). The plowsole was from 10cm to 20cm deep with non-polluted
and polluted soil (dry density at the depth from 10cm to 12.5cm [non-polluted Kanagi soil] andfrom 12.5cm to 20cm [Cu mixed Bunkyou soil] were 1.23 g/cm3 and 0.75 g/cm3, respectively). The
subsoil was from 20cm to 55cm deep with polluted Bunkyou soil and non-polluted gravel (drydensity at the depth from 20cm to 27.5cm [polluted Bunkyou soil] and from 27.5cm to 55cm [thegravel] was 0.75 g/cm3 and 1.40 g/cm3, respectively). Those layers were formed by compaction.
The authors defined O-70 and C-70 as the setting value of 71 mg/kg of the stratified paddy fieldmodes. Similarly, O-250 and C-250 were defined as the setting value of 247 mg/kg of Cu
concentrations (‘O’ and ‘C’ stand for the open-system and the closed-system percolation,
respectively). The ground water levels of the open-system and the closed-system percolation
models were controlled at 57.5cm and 12.5-20cm depth, respectively. In the closed-systempercolation models, the holes in the side walls of iron box were blocked in order to prevent thepenetration of the atmosphere. On the other hand, in the open-system percolation models, the holes
in the side walls of the iron box were open in the lower part of the plowsole and the upper part ofthe subsoil in order to aerate those layers.
After the two types of models were prepared, fifteen paddy seedlings (the plant length and theleaf stage were from 12.5 to 17.5 cm and from 4.4 to 5.0 leaves, respectively) named ‘Oryza sativa
L., Tsugaru Roman’ were transplanted. The paddy seedlings were transplanted by 10cm intervals.
As for fertilizer, 2g of N, 2g of P2O5 and 2g of K2O were administered per model and mixed withthe whole plow layer before transplanting. During the cultivation period, the water ponding
condition was constantly adopted but the mid-summer drainage was not done. Transplanting of the
paddy seedlings and harvesting were conducted at the end of May and at the end of September,respectively. The experiment with the stratified paddy field models was conducted in a greenhouseon the university campus.
Measuring Method
The examination of rice plants such as plant length, leaf stage, the number of stems and panicles,
the weight of straw, the number of grains of brown rice and the weight of brown rice was done by
the standard method of Iwate Agricultural Experimental Station (1981). The quantitative analysis
of Cu concentrations in leaves, root, brown rice and soils extracted by HCl solution was carried out
with atomic absorption spectroscopy (MAFF, 1979). Other measurements were also conducted inⒸ ISERD

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standard methods used in Japan. The Oxidation-Reduction Potential (ORP) meter (Central Kagaku
Co., Ltd., model UC-203) was used for measuring oxidation-reduction potential (Eh). The ORP
sensors were installed at depths of 5, 15, 20, 27.5, 37.5 and 47.5 cm of each model.
RESULTS AND DISCUSSION
Oxidation-reduction Potential (Eh)The temporal changes of Eh are shown in Figs. 1~4. The plow layer of O-70 and O-250 became
reduction layers (under -100 mV) while the plowsole and the subsoil became oxidation layers (over400 mV). On the other hand, Eh values measured at the depths of C-70 and C-250 were gradually
decreased after transplanting, and in due time all the layers became reduction layers as Eh values
showed under 0 mV. This means that, in this study, the polluted soil layers in O-70 and -250 were
under oxidation condition while those layers in C-70 and C-250 were under reduction condition. It
has been pointed out that the Cu uptake in rice is affected by the oxidation-reduction environment(Matsunaka, 2014) and, therefore, in this study, Cu solubility was probably high in the models of
O-70 and O-250 (Takaishi et al., 2015). We decided on the oxidation and reduction condition on
the basis of Yamane (1982), who had defined the oxidation layer as Eh value as 300 mV or more
and reduction layer as < 300 mV.

Fig.1 The temporal changes of Eh in the stratified paddy field model (O-70)

Fig.2 The temporal changes of Eh in the stratified paddy field model (C-70)Ⓒ ISERD

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Fig.3 The temporal changes of Eh in the stratified paddy field model (O-250)

Fig.4 The temporal changes of Eh in the stratified paddy field model (C-250)
Copper Concentrations in Rice Plants
The results of Cu concentrations in rice plants are listed in Table 2.
Rice grains: Cu concentrations in brown rice ranged from 2.8 to 4.3 mg/kg which were similar tothe values reported by Asami (2010). Their studies, however, did not make a distinction between
open and close percolation patterns. Paul et al. (2011b) reported that the significant difference wasobserved in Cu concentrations in brown rice due to differences in percolation patterns. In addition,in their study, despite the low soil Cu concentrations, Cu concentrations in brown rice ranged from
2.5 to 4.2 mg/kg, similar to the results of our experiment. Thus the Cu concentrations in the lower
layer did not make much difference in Cu concentrations in brown rice. From these results, it was
revealed that the differences in Cu concentrations in brown rice due to differences in percolation
patterns were confirmed as those in Cd concentrations found in the experiments conducted by
Sasaki et al. (2016a, b).
Stems and leaves: It confirmed that statistically significant differences in the Cu concentrations instems and leaves were between O-250 (1.6 mg/kg) and C-250 (0.8 mg/kg). However, there was no
significant difference between O-70 and C-70. This discrepancy might be caused by the soil Cu
concentrations, but this requires further elucidation.
Roots: In Cu concentrations in roots, significant differences in the high concentration models of O250 (20.3 mg/kg) and C-250 (13.7 mg/kg) were recognized. However, significant differences in the
low Cu concentration models due to the percolation patterns were not confirmed. Here, further
discussion is needed to clarify its mechanism as in the case of stems and leaves.
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Cu concentrations in the rice plants were in the order of roots > brown rice > stems and leaves.
This order was similar to those of Shibuya (1979) and Paul et al. (2011b) who used rice plants, andto those of Li et al. (2017) who used soybeans. This result is probably due to the transportcharacteristics of Cu and Cd in the rice plants.
Table 2 Cu concentrations in rice grains, stems and leaves androots in plow layer (mg/kg)
Model
O-70
C-70
O-250
C-250

Rice grains
n=10
4.3±0.3a
3.4±0.4b
4.1±0.4a
2.8±0.2c

Stem and leaves
n=5
1.5±0.4a
1.2±0.4ab
1.6±0.2a
0.8±0.1b

Root of plow layer
n=816.0±2.3a
17.8±2.7a
20.3±1.4b
13.7±0.5a

Growth and Yield of Rice Plants
Experimental results for the growth and yield of rice plants are shown in Tables 3 and 4,
respectively.
Growth of rice plants: The average plant height (n = 10) of each model was almost equal, at 90
cm level (Table 3). Leaf age of each model was about 14, showing a small difference between them.
Total straw weight was 12.3～15.0 g/hill. No significant difference was observed in the plantheight, leaf age and total straw weight regardless of the percolation patterns. Previous research(Shibuya, 1979) reported that Cu concentrations in the Cu polluted soil layer had an influence on
the growth of rice plants. In this study, however, influence of the Cu concentrations on the growthof rice plants was not noticeable, which may well to have resulted from the application of soil
dressing.
Yield of rice plants: An average number of panicles per unit hill in each model was 8.6～9.6 /hill(Table 4). Likewise, averages of the weight of one panicle and the number of grains of brown rice
per unit hill were 1.9～2.2 g / panicle and 616～680 grains /hill, respectively. In addition, averagesof the percentage of ripening and the 1,000 grain weight of brown rice were 86.5～93.7% and 19.1
～19.9 g, respectively. No significant differences were found in any of the items of the models withdifferent percolation types. Paul et al. (2011a) reported that yield components of the closed-systempercolation model were significantly higher than those of the open-system percolation model
though their experiment was conducted by using a different soil type for Cd polluted soil layers.
Sasaki et al. (2016a) conducted an experiment using stratified paddy field models with a Cd
polluted soil layer and reported that there was no significant difference between the models withdifferent percolation patterns. Soil dressing was presumed to be one of the reasons why nosignificant difference was observed in that study. In a previous study (Shibuya, 1979), it wasreported that Cu concentrations had an influence on the number of panicles and the percentage ofripening. In this study, however, the influence of the Cu concentrations on the growth of rice plants
was not remarkable, which may be attributed to the soil dressing.
Table 3 Parameters of rice plant growth
Model
O-70
C-70
O-250
C-250

Plant length(cm)
99.6±5.1a
99.6±3.8a
95.6±4.4a
99.7±5.5a

Leaf age(leaf)
14.3±0.5a
14.3±0.5a
14.0±0.0a
14.3±0.5a

Weight of dry straw(g/hill)13.3±2.4a
15.0±2.2a
13.3±3.0a
12.3±1.8a

Note: Tukey-Kramer test was performed at 5% level; letter indicates significant difference.
The numerical value of ± shows standard deviationⒸ ISERD

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Table 4 Parameters of rice plant yield
Model

Weight of one
panicleNo. of Panicles

Percentage ofripening

Number of brownrice per unit hill

(Panicles/hill)（g）（%）（grains/hill）
O-70
1.9±0.3a
9.4±1.1a
92.3±2.2a644.3±116.0a
C-70
2.0±0.2a
9.6±1.2a
86.5±4.1b616.9±118.8a
a
a
a
O-250
2.2±0.3
8.6±1.3
93.1±1.5
680.5±163.9a
a
a
a
C-250
2.0±0.2
9.6±1.893.7±1.5
627.9±65.0a
Note: Tukey-Kramer test was performed at 5% level; letter indicates significant difference.
The numerical value of ± shows standard deviation

1000 grain weightof brown rice（g）
19.1±0.9a
19.7±0.5a
19.9±0.8a
19.4±0.8a

CONCLUSION
Using four types of Cu polluted stratified paddy field models, we conducted an experiment toclarify the effects of percolation patterns in the sub-layer (both plowsole and subsoil) on the Cu
concentrations in the rice plants and their growth and yield. The models had a 15-cm thick Cu
polluted soil layer and a 12.5-cm thick non-polluted soil dressing. For the Cu polluted soil layer,
two different Cu concentrations of 71 mg/kg and 247 mg/kg were prepared.
The results of our experiment showed that in the open system percolation models the sublayers became oxidation layers and those in the closed system percolation models the sub-layers
became reduction layers. Cu concentrations in the brown rice of the open system percolation
models were significantly larger by 5% than those of the closed system percolation models. In themodels with concentrations of Cu (247 mg/kg), the Cu concentrations in stems and leaves androots showed significantly different values between the percolation patterns. However, there wasno statistically significant difference in the growth and yield of rice plants between the percolation
patterns.
Under the above conditions, difference in percolation patterns of the stratified paddy fieldmodels did not affect the growth and yield of rice plants, while it had an influence on the Cu
concentration in the rice plants.
ACKNOWLEDGEMENTS
The authors appreciate the cooperation of Toikawa Yoshito, Okada Hiroaki, and Fujita Masayoshi
who cooperated in carrying out this research. In addition, this research was conducted under the
support of Grant-in-Aid for Scientific Research (No. 26660188). We would like to thank everyone
concerned.
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