Plant Production Science

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Effects of Ammonium Chloride Fertilizer and its

Application Stage on Cadmium Concentrations in
Wheat (Triticum aestivum L.) Grain

Naoyuki Ishikawa, Gen Ishioka, Mikiko Yanaka, Kanenori Takata & Masaharu
Murakami

To cite this article: Naoyuki Ishikawa, Gen Ishioka, Mikiko Yanaka, Kanenori Takata & Masaharu
Murakami (2015) Effects of Ammonium Chloride Fertilizer and its Application Stage on Cadmium
Concentrations in Wheat (Triticum aestivum L.) Grain, Plant Production Science, 18:2, 137-145,
DOI: 10.1626/pps.18.137

To link to this article: http://dx.doi.org/10.1626/pps.18.137

2015 Crop Science Society of Japan

Published online: 03 Dec 2015.

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 Plant Prod. Sci. 18(2): 137145 (2015)

Effects of Ammonium Chloride Fertilizer and its Application Stage

on Cadmium Concentrations in Wheat (Triticum aestivum L.) Grain
Naoyuki Ishikawa1, Gen Ishioka1, Mikiko Yanaka1, Kanenori Takata1 and Masaharu Murakami2

(1NARO Western Region Agricultural Research Center, 6-12-1 Nishifukatsu, Fukuyama 721-8514, Japan;
2
National Institute for Agro-Environmental Sciences, 3-1-3 Kannondai, Tsukuba 305-8604, Japan)

Abstract: Chloride (Cl) in saline soil increases the cadmium (Cd) concentration in crops. Here, we
conducted a field experiment to investigate changes in Cd concentrations in wheat grain after the
application of the Cl-containing fertilizer ammonium chloride (NH4Cl), with the aim of reducing
its potential health risk. Effects of the application stage of NH4Cl fertilizer and leaching treatment
(i.e., heavy rainfall) were also investigated in field and pot experiments. Both field and pot
experiments showed that the Cd concentration of wheat grain was higher with NH4Cl fertilizer
than with ammonium sulfate or urea fertilizers. Grain Cd concentration in wheat fertilized with
NH4Cl at the tilleringjointing and flowering stages in the field experiment was 0.223 mg kg1,
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which was about 1.5 times higher than that fertilized with urea. This finding is important because,
in Japan, compound fertilizers containing NH4Cl are commonly used in fields for wheat
cultivation. NH4Cl fertilizer application at the tilleringjointing and flowering stages had nearly
equal effects on the Cd concentration in wheat grain. Basal dressing with NH4Cl fertilizer
increased Cd concentrations in wheat grain to a greater extent than topdressing (at the tillering
jointing and flowering stage applications) in a pot experiment that was protected from rain.
Leaching treatment (assuming two lots of 100 mm rainfall) negated the effect of NH4Cl fertilizer
application on Cd concentration in wheat grain. We recommend the use of ammonium sulfate or
urea preferentially as the nitrogen fertilizer because heavy rainfall rarely occurs during this period
in Japan.

Key words: Ammonium chloride, Cadmium, Leaching, Wheat.

Cadmium (Cd) is an element that is highly toxic to
humans, and it is mainly acquired from food. In 2005, the
maximum permissible Cd concentration in wheat grain
was set at 0.2 mg kg1 globally (Codex Alimentarius
Commission, 2005a, 2005b). According to a survey on Cd
concentrations performed by the Ministry of Agriculture,
Forestry and Fisheries of Japan in 2000 2002, the Cd
concentration in wheat grain harvested in Japan was 0.072
mg kg1 on average, with a standard deviation of 0.057 mg
kg1, and a maximum concentration of 0.470 mg kg1
(WHO and FAO, 2006). The Cd concentration in wheat
grain is influenced by certain properties, including soil pH
(Singh et al., 1995; Oliver et al., 1998; Adams et al., 2004),
nitrogen fertilizer application (Mitchell et al., 2000;
Wngstrand et al., 2007; Gao et al., 2010; Perilli et al., 2010;
Li et al., 2011), soil Cd concentration (Ibaraki et al., 2005),
and wheat cultivars (Kubo et al., 2008). Of these
properties, soil salinity, particularly soil chloride (Cl)
concentration, is an important factor that influences the
accumulation of Cd in various crops, such as sunflower,
potato, Swiss chard, wheat, kenaf, and corn (Li et al., 1994;
McLaughlin et al., 1994; Smolders and McLaughlin, 1996;
McLaughlin et al., 1997; Smolders et al., 1998; Norvell et
al., 2000; Weggler-Beaton et al., 2000; Wu et al., 2002;
Weggler et al., 2004; Hattori et al., 2006; Ghallab and
Usman, 2007). These studies indicated that increasing Cl
concentration in the soil promotes the formation of CdCl+,
which increases Cd solubility in the soil and, hence, Cd
uptake by plants.
Given that soil Cl increases the uptake of Cd by plants, it
is expected that fertilizers containing ammonium chloride
(NH 4 Cl) influence Cd concentrations in wheat.
Matsuyama et al. (2003) conducted a pot experiment and
showed that NH4Cl fertilizer significantly increases Cd
concentrations in the shoots of Brassica rapa L. Perviridis
Group and Spinacia oleracea L. compared with urea or

Received 17 February 2014. Accepted 4 August 2014. Corresponding author: N. Ishikawa (ishik@affrc.go.jp, fax +81-84-924-7893). This research
was supported in part by a fund Research project for ensuring food safety from farm to table from the Ministry of Agriculture, Forestry, and
Fisheries of Japan.
 138 Plant Production Science Vol.18, 2015
ammonium sulfate fertilizers. Ohtani et al. (2007) showed
that NH 4 Cl fertilizer significantly increased Cd
concentrations in rice and spinach shoots grown in Entisol-
filled pots compared with other nitrogen fertilizers, such as
ammonium sulfate, ammonium nitrate, and ammonium
dihydrogen phosphate. However, there was little difference
in the Cd concentration in barley shoots among the four
ammonium salts. Further, Cd concentrations in rice,
spinach, and barley grown in pots filled with Andisol were
similar in all four ammonium salts. Zhao et al. (2010) used
a pot experiment to show that the application of NH4Cl
fertilizer increased Cd concentration in wheat more than
the application of ammonium sulfate, ammonium nitrate,
or urea fertilizers. Moreover, Jiaka et al. (2010) obtained
the same results with rice. To the best of our knowledge,
field experiments evaluating the effects of NH4Cl fertilizer
application on Cd concentrations in wheat grain have not
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yet been reported, nor has the influence of the application
stage of NH4Cl fertilizer been investigated.
In Japan, compound fertilizers containing NH4Cl are
widely used on crops, including wheat. Therefore, it is
important to assess the effects of NH4Cl fertilizer on Cd
concentrations in wheat grain, with the aim of reducing
any potential health risks. In this study, we compared the
effect of NH4Cl fertilizer with ammonium sulfate and urea
fertilizers on the Cd concentration of wheat using field and
pot experiments. Furthermore, we investigated the effects
of the application stage of NH4Cl fertilizer, along with the
amount of leaching caused by rainfall, on the Cd
concentrations in wheat grain.
Materials and Methods
1.Field experiment
We conducted a field experiment in paddy fields (640
m2) at the NARO Western Region Agricultural Research
Center, Japan (N3430, E13323, 1 m above sea level).
The field soil was a fine-textured (light clay) Gray Lowland
soil. At the experimental site, paddy rice was cultivated
uniformly before wheat cultivation. Subsequently, the hard
red wheat (Triticum aestivum L.) cultivar Setokirara was
sown by drill seeding at 30-cm row spacing and at a sowing
rate of 6 g m2 (140 seeds m2) on 25 November 2011. The
wheat plants were harvested at crop maturity on 4 June
2012. Temperature and precipitation data during the
wheat cultivation period are shown in Table 1. The
experimental design was a randomized complete block
with four replicates.
All treatments were applied with the same basal
dressing: 150 g m2 magnesium lime (CaCO3 + MgCO3) on
9 November 2011, followed by 6.8 g m2 N as ammonium
sulfate and ammonium phosphate, 8.6 g m2 P2O5 as
ammonium phosphate, and 6.8 g m2 K2O as potassium
chloride at sowing on 25 November 2011. The basal
dressing (N, P 2 O 5 , and K 2 O) was applied as NPK
Table1.Average temperature and precipitation during the wheat
cultivation period at the experimental site.
Average temperature Precipitation
Month and year
(C) (mm)
December 2011 6.4 24
January 2012 4.0 7
February 2012 3.6 71
March 2012 8.0 98
April 2012 14.2 72
May 2012 18.9 67
compound fertilizer Aladdin 484. All treatments were
top-dressed with the same amount of N as NH4Cl or urea.
Specifically, 5.5 g m2 N was applied at the tillering and
jointing stages (termed tilleringjointing stage hereafter)
(5 February and 14 March 2012) and 6.5 g m2 N was
applied at the flowering stage (28 April 2012). Five types of
topdressing fertilizers were applied: (A) urea at all stages,
(B) urea + potassium chloride at all stages, (C) NK
compound fertilizer C54 (NKC54) at the tilleringjointing
stage and urea at the flowering stage, (D) urea at the
tilleringjointing stage and NKC54 at the flowering stage,
and (E) NKC54 at all stages. The fertilizer, NKC54,
contained 15% N as NH4Cl, 10% K2O as potassium
chloride, and 4% K2O as potassium silicate. All fertilizers
were surface broadcast. Application details are provided in
Table 2.
Soil samples were collected from the rooting zone (0
30 cm soil depth) during the grain filling period, nine days
after the flowering stage. The samples were air-dried at
40C, ground, and passed through a 2-mm sieve.
At crop maturity on 4 June 2012, the wheat plants were
harvested at ground level and air-dried. After the moisture
of the grains had decreased to < 13.5%, the plants were
threshed and the grains and straw (which comprised the
culm, leaf sheath, and rachis) were dried in an oven at
40C. The straw was ground with a Wiley mill.
2.Pot experiment
Soil was collected from the plow layer (0 15 cm depth)
of a paddy field at the NARO Western Region Agricultural
Research Center after rice harvest in November 2012. The
soil was a fine-textured (light clay) Gray Lowland soil. The
contents of water-extractable Cl and 0.1 mol L1 HCl-
extractable Cd were 94 and 0.13 mg kg1, respectively.
Approximately 4.2 kg of the wet soil (dry weight of
approximately 3.3 kg) was put in each pot, with a 200 cm2
soil surface area and 3.2 L soil volume. Water-extractable
Cl was 94 mg kg1 3.3 kg pot1 = 310 mg pot1, and 0.1
mol L1 HCl-extractable Cd was 0.13 mg kg1 3.3 kg pot1
= 0.43 mg pot1 before sowing. Before put in the pot, the
soil was fertilized with 0.38 g pot1 of P2O5 as magnesium
 Ishikawa et al.Effects of NH4Cl Fertilizer on Cadmium Concentrations in Wheat Grain 139

Table2.Effects of applied fertilizer on soil, wheat straw, and wheat grain properties in a field experiment.
Applied fertilizer Cl in fertilizer (g m2) Soil Straw Grain
Treat- Tillering Basal Tillering Cl Cl Cd Cd
Flower-
ment jointing Flowering dress- jointing Total pH EC conc. conc. conc. Yield Protein conc.
stage ing stage
stage ing stage (dS m1) (mg kg1) (g kg1) (mg kg1) (g m2) (%) (mg kg1)
A Urea Urea 5.0 0.0 0.0 5.0 6.72 0.091 10.4 c 7.9 c 0.204 576 10.5 0.150 b
B Urea + KCl Urea + KCl 5.0 3.9 4.6 13.5 6.73 0.107 22.5 bc 10.3 b 0.164 597 10.3 0.155 b
C NKC54 Urea 5.0 16.7 0.0 21.7 6.68 0.092 24.7 bc 12.0 ab 0.227 639 10.7 0.180 ab
D Urea NKC54 5.0 0.0 19.7 24.7 6.77 0.128 37.8 ab 10.4 b 0.176 597 11.1 0.178 ab
E NKC54 NKC54 5.0 16.7 19.7 41.4 6.55 0.105 48.5 a 13.8 a 0.213 631 11.2 0.223 a
ANOVA ns ns *** *** ns ns ns **
KCl: potassium chloride fertilizer.
NKC54 is NK compound fertilizer C54 containing 15% N as NH4Cl, 10% K2O as KCl, and 4% K2O as potassium silicate.
As the basal dressing, 6.7 g m2 N as ammonium sulfate and ammonium phosphate, 8.6 g m2 P2O5 as ammonium phosphate, and 6.7 g m2
K2O as KCl were applied to all treatments.
As the topdressing, 2.5 g m2 N was applied at the tillering stage (5 February), 3.0 g m2 N was applied at the jointing stage (14 March), and 6.5
g m2 N was applied at the flowering stage (28 April) of all treatments. The amount of K in the KCl applied for treatment B was the same as the
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amount of K in the NKC54 applied for treatment E.

Soil samples were collected at the grain filling period, nine days after the flowering stage, from 0 30 cm soil depth. Straw and grain samples
were collected after harvest.
Values followed by the same letters within a column are not significantly different based on Tukeys multiple comparison at the 5% level.
ns: not significant at P = 0.05; ** and ***: significant at P = 0.01 and 0.001, respectively.

superphosphate and 0.50 g pot1 of K2O as potassium
silicate. Six seeds of hard red wheat (T. aestivum L.) cultivar
Setokirara were seeded in each pot on 3 December 2012
and the seedlings were thinned to two plants per pot on 25
December 2012. The pots were placed outside under a
glass roof to protect them from the rain, and were irrigated
to maintain soil moisture tension at pF 1.8 2.4, except
when the leaching treatment was applied. No water ran
out or moved to the bottom of the pot, except during the
leaching treatment.
Four different NH4Cl treatments were applied: (1)
without NH4Cl at all stages, (2) with NH4Cl at germinating
stage (25 December), (3) with NH4Cl at the tillering
jointing stage (9 February and 11 March), and (4) with
NH4Cl at flowering stage (10 and 21 April). When NH4Cl
was not applied, ammonium sulfate was applied. NH4Cl
and ammonium sulfate were of fertilizer grade. At each
stage, 0.22 g nitrogen per pot was applied. Each treatment,
including the leaching treatments, was replicated 6 10
fold (with 6 10 pots per each treatment.)
The leaching treatments were combined with the
fertilizer treatments, except for the flowering-stage NH4Cl
treatment. The leaching treatment might drain off both Cl
and nitrogen from the soil, with nitrogen deficiency
causing retarded wheat growth. To avoid growth
retardation, the leaching treatments were conducted just
before the subsequent application of fertilizer. At the
experimental site, the maximum precipitation in a given
rainfall event during wheat cultivation period in 2005
2012 was approximately 100 mm, corresponding to 2 L
water per pot. Thus, the same quantity of water was added
to each pot at the pre-tillering (29 January) and pre-
flowering (28 March) stages. Water ran down through the
soil, and excess water (approximately 1.7 L) ran out of the
pots within 17 h. The leachate was collected and analyzed.
Pots without leaching treatment were irrigated with the
same amount of water as that retained by treated pots
(0.3 L).
On 25 May 2013, after the crop had reached maturity,
the wheat plants were harvested at ground level and
processed as part of the field experiment. Soil samples
were collected after the wheat was harvested and were
passed through a 2-mm sieve. The moisture of these soil
samples was approximately 16% by weight. Analyses were
conducted on each pot.
3.Analytical procedure
Soil pH was measured using a glass electrode in a settled
1:2.5 (field experiment) or 1:5 (pot experiment) soil/
water (w/w) suspensions (dry matter weight basis). Soil
electrical conductivity (EC) was measured using a compact
conductivity meter (B-771, HORIBA, Ltd., Kyoto, Japan) in
a 1:5 soil/water (w/w) extract. Soil Cl was measured using
an ion chromatography system (TOSOH IC-2010
equipped with a TSKgel SuperIC-AZ column, Tosoh
Corporation, Tokyo, Japan) in a 1:5 soil/water (w/w)
extract. Soil Cd extracted with 0.1 mol L 1 HCl was
determined using inductively coupled plasma mass
spectrometry (ICP-MS) (PerkinElmer ELAN DRC II,
PerkinElmer Japan Co., Ltd., Yokohama, Japan). The Cl
 140 Plant Production Science Vol.18, 2015

Fig.1.Relationship between grain Cd concentrations and (a) soil Cl concentration at the grain
filling period, (b) straw Cl concentration at maturity in a field experiment.
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* and **: significant at P = 0.05 and 0.01, respectively.

Soil Cl was measured in three out of four replications. Grain Cd and straw Cl were each
measured in four replications.

and Cd concentrations of leachate were determined using
the ion chromatography system and ICP-MS (PerkinElmer
ELAN 6000, PerkinElmer Japan Co., Ltd.), respectively.
Straw Cl was extracted from the ground straw by
autoclaving a 1:100 straw/water (w/w) suspension at 120C
for 20 min, followed by measurement using the ion
chromatography system. The Cd concentration in the
grains and straw were measured using ICP-MS (Agilent
7500ce, Agilent Technologies Japan, Ltd., Tokyo, Japan)
after incinerating and dissolving them in HCl and nitric
acid. The protein concentration in the grains was
measured with a near infrared spectrometer (Infratec 1241
Grain Analyser, FOSS Japan Ltd., Tokyo, Japan) using a
protein conversion factor of 5.70 on a 13.5% moisture
basis.
Analyses of variance and correlation were performed
using the SAS Add-In 5.1 for Microsoft Office connected
with SAS 9.3 (SAS Institute Japan Ltd., Tokyo, Japan).
fertilizer application (Table 2). Straw Cd concentration,
grain yield, and grain protein concentration did not show
any significant differences among the treatments (Table 2).
Grain Cd concentration was not affected by the application
of potassium chloride fertilizer (Table 2, treatment A vs. B).
Grain Cd concentration in treatment E (NH4Cl containing
fertilizer NKC54, applied at the tilleringjointing and
flowering stages) was 0.223 mg kg1, which was about 1.5
times higher than that in treatment A (urea, applied at the
tilleringjointing and flowering stage) (Table 2). There
was no significant difference in grain Cd concentration
between treatment C (NKC54, applied at the tillering
jointing stage) and treatment D (NKC54, applied at the
flowering-stage) (Table 2).
Grain Cd concentration was significantly correlated with
soil Cl concentration at the grain filling period, nine days
after the flowering stage (r = 0.664**), and straw Cl
concentration at maturity (r = 0.509*) (Fig. 1).

Results 2.Pot experiment

1.Field experiment
The concentrations of soil Cd extracted with 0.1 mol L1
HCl were 0.16 and 0.06 mg kg1 at 0 15 cm and 15 30
cm soil depth, respectively, using soil mixture of equal
parts of 20 treatment plots at the grain filling period, nine
days after flowering (the data are not shown in tables and
figures).
Soil pH and EC at the grain filling period were not
significantly influenced by fertilizer treatments (Table 2).
Soil Cl concentration at the grain filling period increased
as the amount of Cl in the applied fertilizer increased
(Table 2). Straw Cl increased after NH4Cl-containing
Soil pH at maturity was not significantly influenced by
fertilizer treatments (Table 3). Soil EC at maturity
increased slightly after NH4Cl fertilizer application (Table
3). Soil Cl concentration at maturity markedly increased
after NH4Cl fertilizer application, but was similar at the
three application stages (Table 3). Straw Cl concentration
increased after NH 4Cl fertilizer application at the
germinating and tilleringjointing stages, but not by NH4Cl
application at the flowering stage (Table 3). Straw Cd
concentration showed no significant difference among
treatments (Table 3). Grain yield increased slightly after
NH4Cl application at the germinating stage (Table 3).
 Ishikawa et al.Effects of NH4Cl Fertilizer on Cadmium Concentrations in Wheat Grain 141

Table3.Effects of NH4Cl application stage on soil, wheat straw, and wheat grain properties at maturity in a pot experiment without
leaching treatment.

Soil Straw Grain

NH4Cl Cl Cl Cd Cd
application pH EC conc. conc. conc. Yield Protein conc.
(dS m1) (mg kg1) (g kg1) (mg kg1) (g pot1) (%) (mg kg1)
Without NH4Cl 6.45 0.37 b 50 b 9.3 c 0.103 a 24.4 b 11.2 0.080 c
Germinating stage 6.40 0.42 a 186 a 11.6 a 0.131 a 26.0 a 11.1 0.112 a
Tilleringjointing stage 6.43 0.41 ab 187 a 10.8 ab 0.131 a 25.9 ab 10.9 0.099 b
Flowering stage 6.40 0.41 ab 191 a 10.2 bc 0.108 a 24.4 ab 11.2 0.095 b
ANOVA ns * *** *** * * ns ***
Grain yield and protein concentrations are shown on 12.5% and 13.5% moisture basis, respectively. Soil Cl and straw Cl concentrations
are shown on a dry-matter basis. Grain Cd and straw Cd concentrations are shown as recorded (grain moisture was about 9%).
Values followed by the same letters within a column are not significantly different based on Tukeys multiple comparison at the 5% level.
ns: not significant at P = 0.05; * and ***: significant at P = 0.05 and 0.001, respectively.
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Fig.2.Relationship between grain Cd concentrations and (a) soil Cl concentration at maturity, (b) straw
Cl concentration at maturity in a pot experiment.
***: significant at P = 0.001.
Each parameter was measured in each pot, with 6 10 pots per treatment.

Grain protein concentration was not significantly different
among treatments (Table 3). Grain Cd concentration
applied with NH4Cl was 0.112 0.095 mg kg1, which was
1.4 1.2 times higher than that without NH4Cl (Table 3).
Grain Cd concentration was higher when NH4Cl was
applied at the germinating stage than at the tillering
jointing and flowering stages (Table 3). Grain Cd
concentration was not significantly different between the
tilleringjointing application and flowering-stage
application (Table 3).
Grain Cd concentration was significantly correlated with
soil Cl concentration at maturity (r = 0.818***) and straw
Cl concentration (r = 0.744***) (Fig. 2).
Leaching treatment had no or minimal influence on
soil pH (Table 4). Leaching treatment caused a marked
decrease in soil EC, soil Cl, and straw Cl concentrations,
along with a slight decrease in grain yield (Table 4). Straw
Cd and grain protein concentrations were reduced by
leaching treatment when NH4Cl was applied (Table 4).
Leaching treatment reduced the grain Cd concentration
when NH4Cl was applied, almost negating the effects of
NH4Cl at increasing grain Cd (Table 4).
Leaching treatment removed relatively large amounts of
Cl. In the pots applied with NH4Cl at the germinating
 142 Plant Production Science Vol.18, 2015

Table4.Effects of the leaching treatment on soil, wheat straw, and wheat grain properties at maturity in a pot experiment.
Soil Straw Grain
Leaching
NH4Cl Cl Cl Cd Cd
treat-
application pH EC conc. conc. conc. Yield Protein conc.
ment
(dS m1) (mg kg1) (g kg1) (mg kg1) (g pot1) (%) (mg kg1)
No 6.45 0.37 50 9.3 0.103 24.4 11.2 0.080
Without NH4Cl ns *** *** *** ns * ns ns
Yes 6.45 0.31 27 7.4 0.095 22.8 10.9 0.075
No 6.40 0.42 186 11.6 0.131 26.0 11.1 0.112
Germinating stage ns *** *** *** * ** * **
Yes 6.45 0.29 55 8.7 0.097 23.0 10.5 0.087
No 6.43 0.41 187 10.8 0.131 25.9 10.9 0.099
Tilleringjointing stage * *** *** ** * * * **
Yes 6.39 0.32 101 9.1 0.099 24.1 10.5 0.088
Grain yield and protein concentrations are shown on a 12.5% and 13.5% moisture basis, respectively. Soil Cl and straw Cl concentrations are
shown on a dry-matter basis. Grain Cd and straw Cd concentrations are shown as recorded (grain moisture was about 9%).
*, **, and ***: significant at P = 0.05, 0.01, and 0.001, respectively.

Table5.Amount of Cl and Cd leached out of the soil in the pots by the leaching treatments.
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Application at Application at
germinating stage tilleringjointing stage Cl Cd
Leaching Volume of in in
Cl Cl
treatment stage Applied Applied leachate leachate leachate
in fertilizer in fertilizer (L pot1)
fertilizer fertilizer (mg pot1) ( g pot1)
(mg pot1) (mg pot1)
Ammonium sulfate 0 1.715 134 0.134
Pre-tillering stage
NH4Cl 557 1.700 400 0.192
Ammonium sulfate 0 Ammonium sulfate 0 1.593 5 0.000
Pre-flowering stage NH4Cl 557 Ammonium sulfate 0 1.556 37 0.005
Ammonium sulfate 0 NH4Cl 557 1.601 101 0.014
All pots were treated twice: once at the pre-tillering stage and once at the pre-flowering stage.
Water-extractable Cl and 0.1 mol L1 HCl-extractable Cd in the soil before sowing were 310 and 0.43 mg pot1, respectively.

stage, 400 mg Cl had leached out by the pre-tillering stage
leaching treatment, whereas 557 mg Cl had added by
NH4Cl application at the germinating stage (Table 5).
Likewise, in the pots applied with NH4Cl at the tillering
jointing stage, 101 mg Cl had leached out by the pre-
flowering stage leaching treatment (Table 5). In contrast, a
maximum of just 0.2 g Cd per pot leached out, which was
equivalent to <1/2000th of 0.1 mol L1 HCl-extractable Cd
(0.43 mg pot1) (Table 5).
Discussion
Both field and pot experiments showed that NH4Cl
fertilizer increased wheat Cd concentration compared to
ammonium sulfate and urea fertilizers. At the experimental
site, the concentrations of soil Cd extracted with 0.1 mol
L1 HCl were 0.16 and 0.06 mg kg1 at 0 15 cm and 15
30 cm soil depth, respectively. These values are common
across Japan, with soil Cd concentrations of 109 out of 227
samples collected from wheat fields across Japan being 0.1
0.2 mg kg1 (Ministry of Agriculture, Forestry, and
Fisheries of Japan, 2004). Even in these fields, the grain Cd
concentration of wheat fertilized with NH4Cl at the
tilleringjointing and flowering stages was 0.223 mg kg1,
which exceeded the maximum permissible concentration
of 0.2 mg kg1 set by Codex Alimentarius Commission
(2005a, 2005b). This finding is important because
compound fertilizers containing NH4Cl are commonly
applied for wheat cultivation in Japan. For wheat
cultivation, fertilizers are usually applied three to five times
from the sowing to flowering stages of each crop. NH4Cl
fertilizer applications at the tilleringjointing and flowering
stages have nearly equal effects on wheat grain Cd
concentration. Basal dressing (applied at the germinating
stage in our pot experiment) increased grain Cd
concentration more than topdressing (applied at the
tilleringjointing or flowering-stage) in pots that were
protected from the rain. This result may be explained by
the persistence of Cl applied as basal dressing, allowing it
to influence Cd mobility throughout the entire crop
growth period.
Rainfall might cause the leaching of Cl in the fields;
although, we did not examine the effects of leaching in
fields. Addiscott et al. (1978) reported that 79.5% of the
applied Cl on 9 October moved below 50 cm depth by the
 Ishikawa et al.Effects of NH4Cl Fertilizer on Cadmium Concentrations in Wheat Grain
end of May in Rothamsted (UK), with 564 mm precipitation
during this period. Saso et al. (2012) reported that over
70% of applied Cl was lost to deep drainage (below 80 cm
depth) from November to April in Ontario (Canada), with
478 mm precipitation during this period. In our study,
46% (400 mg pot1) of initially existed Cl (initial Cl 310 mg
pot1 + applied Cl 557 mg pot1 = 867 mg pot1) was
leached by the pre-tillering stage leaching treatment
(assuming 100 mm rainfall) in the NH4Cl-applied pots. In
contrast, just 0.2 g pot1 Cd at the maximum leached out
of the soil, which was less than 1/2000th of 0.1 mol L1
HCl-extractable Cd (0.43 mg pot1). Thus, the leaching
treatment negated the effects of NH 4 Cl fertilizer
application on wheat Cd concentration. Although 100 mm
rain rarely occurs at any one time during the wheat
cultivation period in Japan, these results indicate that
abundant rainfall would decrease the effects of basal
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dressing with NH4Cl fertilizer, preventing an increase in
wheat Cd concentration. Additional studies are required to
assess the degree of leaching caused by rainfall. In any
case, we recommend the use of ammonium sulfate or urea
in place of NH4Cl as the nitrogen fertilizer.
Yet, Cl is essential for plants. From a practical
perspective, a certain level of Cl in soil must be maintained
for wheat production. Soil Cl concentrations sufficient for
optimal wheat yield have been reported to be 43.5 kg ha1
(at 0 60 cm soil depth at sowing) by Fixen et al. (1986),
33 kg ha1 (at 0 60 cm soil depth at around sowing) by
Engel et al. (1998), and 13.2 mg kg1 (at 0 20 cm soil
depth at sowing) by Daz-Zorita et al. (2004). In our field
experiment, the concentration of Cl in soil (at 0 30 cm
soil depth at the grain filling period), which had been top-
dressed with urea only, was 10.4 mg kg1, corresponding to
31 kg ha1. Because straw Cl concentration was 7.9 g kg1
and plant dry weight (above ground plants excluding
grain) was 8000 kg ha1 (the data are not shown in tables
and figures), the amount of Cl in the wheat plants was
estimated to be 7.9 g kg1 8000 kg ha1 = 63 kg ha1.
Therefore, the soil Cl concentration in the field experiment
at sowing was expected to be sufficient. In our pot
experiment, the Cl concentration in the soil before sowing
was 94 mg kg1, corresponding to 155 kg ha1, which was
expected to be sufficient. Therefore, this study was
completed under the conditions where soil Cl was sufficient
for wheat production.
Like NH4Cl, potassium chloride is also a commonly used
fertilizer containing Cl for wheat cultivation. However, the
reported results of experiments investigating the effects of
potassium chloride fertilizer on Cd concentration in crops
are inconsistent. For instance, Sparrow et al. (1994)
reported that potatoes grown using potassium sulfate
fertilizer had 20 30% lower tuber and petiole Cd
concentrations than those grown using potassium chloride
fertilizer at four out of six sites in northern Tasmania,
143
Australia. In contrast, McLaughlin et al. (1995) reported
that changing the potassium fertilizer form from potassium
chloride to potassium sulfate had no effect on potato tuber
Cd concentrations in an irrigated field in southern Australia.
Furthermore, Zhao et al. (2003) reported that Cd uptake
by spring wheat plants grown in pots was similar for
potassium chloride and sulfate fertilizer applications. Gao
et al. (2011) reported that the application of potassium
chloride fertilizer did not consistently influence the grain
Cd concentration of bread and durum wheat grown in
fields in Manitoba, Canada. In our experiment, the effects
of potassium chloride fertilizer were not significant. This
result was obtained because the Cl content in the applied
potassium chloride fertilizer was only one fourth of that in
the applied NH4Cl fertilizer. The ratio of Cl to K2O in
potassium chloride fertilizer is 0.75, whereas that of Cl to N
in NH4Cl fertilizer is 2.53. Moreover, the amount of K2O
fertilizer needed for wheat production in Japan is generally
less than that of N fertilizer (Oyanagi, 2011). Thus,
potassium fertilizer is not as important as nitrogen fertilizer
with respect to the concentration of Cd in wheat grain.
Lpez-Chuken et al. (2012) showed that adding sodium
chloride to the soil enhances the solubility of Cd and
increases the concentration of CdCl+ and CdCl2 in soil
pore water. Many researchers have suggested that this
phenomenon explains the increasing Cd concentration in
crops grown in fields rich in Cl. In contrast, Ozkutlu et al.
(2007) showed that leaf-applied sodium chloride promotes
Cd accumulation in durum wheat grain. In the present
study, the application of NH4Cl fertilizer increased Cl
concentrations in both the soil and wheat straw, with grain
Cd concentration being correlated with both soil and straw
Cl concentrations. Thus, it is possible that Cl enhances the
transfer of Cd from the root/shoot to the grain, in
addition to increasing the solubility of soil Cd. Cd
translocation from roots to shoots is considered to be
important for the accumulation of Cd in wheat grain
(Chan and Hale, 2004; Greger and Lfstedt, 2004; Harris
and Taylor, 2004). Further studies are required to elucidate
the mechanisms underlying the effects of Cl on grain Cd
accumulation.
In this study, we demonstrated that the application of
NH4Cl fertilizer increases the Cd concentration in wheat
grain, unless massive leaching occurs due to heavy rainfall.
It is important to regulate the quantity of Cl in the soil to
reduce the health risk to humans and to maintain existing
wheat grain yields. In addition to fertilization, the effect of
implementing of various field-management techniques,
such as irrigation and drainage, should be investigated as a
means of controlling soil Cl concentrations and, hence,
the Cd concentrations in wheat grain.
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