Joint stress of chlorimuron-ethyl and cadmium on wheat Triticum aestivum at biochemical levels.

Abstract

Biochemical responses to joint stress of chlorimuron-ethyl and cadmium (Cd) in wheat Triticum aestivum were examined. The joint action of chlorimuron-ethyl and Cd weakened the inhibition of Cd or chlorimuron-ethyl on the formation of chlorophyll. It was deduced that wheat plants had the capability to protect themselves by increasing the activity of the antioxidant enzyme peroxidase (POD) with the exposure time. The joint effect of chlorimuron-ethyl and Cd on the superoxide dismutase (SOD) activity in leaves was additive, while the joint effect on the SOD activity in roots was determined by the interaction of chlorimuron-ethyl and Cd in wheat. It was also concluded that the change of malondialdehyde (MDA) content in wheat might not be a good biomarker in the oxidative damage by chlorimuron-ethyl, while a decrease in the soluble protein content and POD activity in roots could be considered as a biomarker in the damage of wheat by chlorimuron-ethyl and Cd.

Full text

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Joint stress of chlorimuron-ethyl and cadmium on wheat
Triticum aestivum at biochemical levels
Mei-E Wanga,b, Qi-Xing Zhoua,c,*
aKey Laboratory of Terrestrial Ecological Process, Institute of Applied Ecology, Chinese Academy of Sciences,
Shenyang 110016, People’s Republic of China
bGraduate School of the Chinese Academy of Sciences, Beijing 100039, People’s Republic of China
cCollege of Environmental Science and Engineering, Nankai University, Tianjin 300071, People’s Republic of China
Received 18 June 2005; received in revised form 25 December 2005; accepted 11 January 2006
Soluble protein content and peroxidase activity in seedlings were the biomarkers indicating joint stress of chemicals.
Abstract
Biochemical responses to joint stress of chlorimuron-ethyl and cadmium (Cd) in wheat Triticum aestivum were examined. The joint action of
chlorimuron-ethyl and Cd weakened the inhibition of Cd or chlorimuron-ethyl on the formation of chlorophyll. It was deduced that wheat plants
had the capability to protect themselves by increasing the activity of the antioxidant enzyme peroxidase (POD) with the exposure time. The joint
effect of chlorimuron-ethyl and Cd on the superoxide dismutase (SOD) activity in leaves was additive, while the joint effect on the SOD activity
in roots was determined by the interaction of chlorimuron-ethyl and Cd in wheat. It was also concluded that the change of malondialdehyde
(MDA) content in wheat might not be a good biomarker in the oxidative damage by chlorimuron-ethyl, while a decrease in the soluble protein
content and POD activity in roots could be considered as a biomarker in the damage of wheat by chlorimuron-ethyl and Cd.
? 2006 Elsevier Ltd. All rights reserved.
Keywords: Biochemical response; Chorimuron-ethyl; Cd; Joint stress; Wheat Triticum aestivum
1. Introduction
Chlorimuron-ethyl is an important herbicide used in soy-
bean production in northeast China to combat weeds (Zou
et al., 2001). Its prolonged use may lead to pollution of soil
as well as surface and ground water by the herbicide itself
and its metabolisms, which have an adverse effect on
wateresoileplant systems (Ying and Williams, 2000; Zhou
and Huang, 2001; Yen et al., 2003). It was shown in our pre-
vious studies (Wang and Zhou, in press) that the antioxidative
defensive system in plants were damaged and the defensive
function of antioxidative enzymes were lost due to stress of
chlorimuron-ethyl in soils. Meanwhile, cadmium (Cd) is
widely used in modern industry. Since Cd is an unessential el-
ement with high phytotoxicity, its pollution of water-soile
plant systems is becoming the focus of ecological studies
(Zhou, 2003; Zhou et al., 2001, 2002). It has been reported
that toxic action of Cd cause oxidative stress, which result
in enzymatic and non-enzymatic anti-oxidative responses of
plants and stimulation of lipid peroxidation (Horva `th et al.,
1996; Hegedu ¨s et al., 2001; Markkola et al., 2002). Results
of our previous work (unpublished data) also demonstrated
that the accumulation of Cd in wheat Triticum aestivum treated
with both chlorimuron-ethyl and Cd was more complicated
compared with Cd only treatment. On the whole, the concen-
tration of Cd in shoots, roots, and glumes of wheat with joint
stress of Cd and chlorimuron-ethyl was lower than that with
stress of Cd only. The Cd accumulation in roots was higher
than that in other tissues of wheat, accounting for 70e90%
* Corresponding author. Key Laboratory of Terrestrial Ecological Process,
Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang
110016, People’s Republic of China Tel.: þ86 24 8397 0373; fax: þ86 24
8397 0436.
E-mail addresses: zhouqixing2003@yahoo.com, zhouqx@nankai.edu.cn
(Q.-X. Zhou).
0269-7491/$ - see front matter ? 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envpol.2006.01.024
Environmental Pollution 144 (2006) 572e580
www.elsevier.com/locate/envpol

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of total Cd accumulation in wheat. It was suggested that chlor-
imuron-ethyl might inhibit Cd accumulation in wheat. Al-
though there are many studies on toxic mechanisms of either
chlorimuron-ethyl or Cd, little is known about joint effects
of the two on physiological and biochemical mechanisms in
plants.
Higher plants act as one of the key producers in ecosystems
with important roles in sustaining the integrality of ecosys-
tems. However, increasing application of chemicals in contem-
porary agriculture, especially the use of various herbicides,
inevitably damages some normally physiological and bio-
chemical metabolisms in plants. As a result, the growth of
plants is inhibited and the survival of plants threatened. These
toxic effects of agrochemicals on higher plants not only bring
out many of uncertain and adverse changes through biochem-
ical mechanisms such as toxic transportation and magnifica-
tion in the food chain, but also decrease the diversity of
species with the death of some plant species. Some herbicides
have more toxic effects on plants than on other living organ-
isms. Miller et al. (1985) compared the toxicity of heavy
metals with herbicides to phytoplankton, zooplankton, bacte-
ria, plant seeds, and earthworms. Results showed that the tox-
icity of heavy metals to phytoplankton was the strongest,
while the toxicity of herbicides to plant seeds was the stron-
gest (Miller et al., 1985). It was suggested the root elongation
of lettuce was more sensitive to the toxicity of wastewater than
an alga Selenastrum capricornutum, the standard specie for the
alga toxic test (Thomas et al., 1986). Other toxic tests for
chemicals also demonstrated that higher plants were more sen-
sitive than phytoplankton in the toxic assessment of herbicides
(Hoffman et al., 2003). Thus, toxic stress of agrochemicals on
plants is a key challenge in ecosystem health. The study on
toxic effects of agrochemicals on plants at biochemical levels
will provide basic information with safe usage and ecological
risk assessment of agrochemicals.
Some species of plants are usually used as biomonitors to
determine the ecological toxicity of heavy metals and agro-
chemicals (Wang and Zhou, 2004, in press; Zhou et al.,
2001, 2004). Additionally, many biomarkers of soileplant sys-
tems have been developed (Wang and Zhou, 2004; Zhou et al.,
2004). Soileplant biomarkers, including a decrease of protein
content and antioxidant enzyme activity along with an in-
crease of malondialdehyde (MDA), which is a product of the
damage of membrane lipids in response to agrochemicals
and heavy metals, have shown promising applications (Jin
et al., 2002; Pang et al., 2001; Wu and Von Tiedemann,
2002). It was shown in our previous study (Wang and Zhou,
in press) that the effect of chlorimuron-ethyl on physiological
mechanisms in wheat include variation on the contents of
MDA, chlorophyll, and soluble protein as well as on the activ-
ity of superoxide dismutase (SOD) and peroxidase (POD) in
roots and leaves of wheat that was markedly significant except
for the effect on the content of MDA in roots (Table 1). In this
work, wheat (Triticum aestivum) was selected as a mode plant
to further investigate joint stress of chlorimuron-ethyl and Cd
on the activity of SOD and POD along with the concentration
of chlorophyll, MDA, and soluble protein in comparison to our
previous study on the interactive effect of chlorimuron-ethyl
and Cd on seed germination and shoot and root elongation
of wheat (Wang and Zhou, 2005). The purpose of this work
is to seek sensitive biomarkers for diagnosing potential ad-
verse effects on ecosystems at biochemical levels under joint
stress of agrochemicals and heavy metals.
2. Materials and methods
2.1. Basic tested materials
All reagents used in the study were of analytical grade. The tested herbi-
cide chlorimuron-ethyl was bought from the duPont de Nemours and Co.,
USA and its type is 25% of a dispersible granule. The molecular formula of
chlorimuron-ethylisC15H15ClN4O6S.
CdCl2$ 2.5H2O. The variety of the tested wheat (T. aestivum) is Liaoning
Spring No. 14 and was obtained as seeds from Shenyang Agricultural Univer-
sity, China.
Thetested formofCd was
2.2. Plant culture
Seeds of wheat (T. aestivum) were surface-sterilized in the 3.0% sodium
hypochlorite for 5 min, washed several times with sterilized de-ionized water
and germinated in darkness at 25?C for 72 h. Further growth occurred using
solution hydroponics in a growth chamber (LRH-250-A, made in Guangdong)
operated with 12-h light/12-h dark cycles at a constant temperature of 25?C.
Air-dried soil (500 g) was placed in a pot to about 20 cm in the depth and
mixed solution of chlorimuron-ethyl and Cd was poured on the surface of
the soil and filtered for24 h. The seedlings were incubated for 7 days then
transferred to the pots and placed in the growth chamber operated at the
same conditions as mentioned above. The solution concentrations of the joint
stress was 0, 150 and 300 mg/kg for chlorimuron-ethyl and 0, 10 and 100 mg/kg
for Cd. The final humidity of the test soil was about 20%. After incubation for
time intervals of 0, 1, 2, 3, and 4 days, samples were taken and analyzed.
2.3. Determination of chlorophyll and lipid peroxidation
The content of chlorophyll in wheat (T. aestivum) was determined in 80%
acetone extract of 0.1-g leaf tissues as described by Hegedu ¨s et al. (2001) and
expressed as mg g?1fresh weight (FW).
MDA content was also determined as described by Hegedu ¨s et al. (2001)
and expressed as nmol g?1FW.
2.4. Preparation of tissue extract
About 0.1 g of leaf or root tissues was ground with 1.5 ml 50 mM Na-
phosphate buffer (pH 7.8) that was precooled in an ice bath, 0.1 mM EDTA
and 1.0% (w/v) PVPP. The filtered tissue extract was centrifuged at
13,000 rpm for 30 min at 4?C. The supernatant was used for further analyses.
2.5. Determination of total soluble protein
The protein concentration in the supernatant part was determined by the
dye-binding method according to Bradford (1976) and expressed as mg g?1
FW.
2.6. Assay of enzyme activity
All enzyme activity data are related to plant fresh weight (FW) and the ac-
tivity of enzymes is expressed as kU g?1FW. The activity of POD was deter-
mined using guaiacol substrates as described by Wu and Von Tiedemann
(2002). The activity of SOD was also determined as described by Wu and
Von Tiedemann (2002). One enzyme unit is defined as 50% inhibition of
the colorimetric reaction.
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2.7. Statistical analysis
All measurements were replicated four times in independent experiments
and the determination of enzyme activity was carried out with three parallel
samples in all samples. All data were subjected to analysis of variance
(ANOVA) with factors of pollutant concentrations and five time intervals to
determine the difference followed by the multiple comparison procedure
(LSR test) at 1% and 5% of significance level. Standard deviation (SD) was
also calculated.
3. Results
3.1. Dynamic changes of chlorophyll content
in wheat leaves
The chlorophyll content in wheat leaves was significantly
affected by joint stress of chlorimuron-ethyl and Cd (Fig. 1).
On the whole, the influence on the chlorophyll content in
wheat leaves by the joint stress was stronger than that by either
chlorimuron-ethyl or Cd. When Cd concentration was 10 mg/kg,
the chlorophyll content in leaves with joint stress of chlori-
muron-ethyl and Cd was obviously lower than that in the Cd
only treatment or in the control. However, there were no sig-
nificant (P > 0.05) changes in the chlorophyll content among
all the treatments after the 1-day exposure. After a 4-day ex-
posure, the chlorophyll content in leaves with joint stress of
chlorimuron-ethyl and Cd was higher than that treated with
Cd only. When Cd concentration was up to 100 mg/kg, there
were no regular changes in the chlorophyll content in leaves
with increasing chlorimuron-ethyl concentration. The chloro-
phyll content in leaves treated with 100 mg/kg Cd and 5 mg/kg
chlorimuron-ethyl was significantly higher than that with other
treatments including the control after a 1-day exposure. After-
wards, the chlorophyll content in leaves treated with both
chlorimuron-ethyl and Cd was lower than in the Cd only treat-
ment or in the control. After a 4-day exposure, the chlorophyll
content in leaves treated only with 100 mg/kg Cd and 5e
150 mg/kg chlorimuron-ethyl was significantly (P < 0.05)
lower than in the Cd only treatment or in the control.
3.2. Dynamic changes of MDA content in wheat
leaves and roots
MDA formation is considered the general indicator of lipid
peroxidation. After a 1-day exposure, there were irregular
changes of MDA content in the leaves with the stress of
both single Cd and chlorimuron-ethyl combined with Cd.
Moreover, there was no obvious difference in the MDA con-
tent in leaves among all the treatments. After 2e3 days of ex-
posure, the MDA content in leaves with joint stress of
chlorimuron-ethyl and Cd was lower than that in the Cd
only treatment or in the control. After a 4-day exposure except
for 10 mg/kg Cd and 300 mg/kg chlorimuron-ethyl, the MDA
content in leaves treated with chlorimuron-ethyl and Cd was
higher than that in the Cd only treatment or in the control.
When Cd concentration was up to 100 mg/kg, the MDA con-
tent in leaves increased with increasing chlorimuron-ethyl
concentration (Fig. 2A,C).
Table 1
A variance analysis of joint effects of chlorimuron-ethyl (CE) and Cd on the contents of MDA, chlorophyll and soluble protein as well as on the activity of SOD
and POD in wheat roots and leaves
Pollutant MDA POD activitySOD activity Soluble proteinChlorophyll
LeafRoot Leaf RootLeafRootLeafRootLeaf
Cd
CEa
Cd ? CE
aData on the single effect of CE is published elsewhere (Wang and Zhou, in press).
P < 0.01
P < 0.01
P < 0.01
P < 0.01
P > 0.05
P < 0.01
P < 0.01
P < 0.01
P < 0.01
P < 0.01
P < 0.01
P < 0.01
P < 0.01
P < 0.01
P > 0.05
P < 0.01
P < 0.01
P < 0.01
P < 0.01
P < 0.01
P < 0.01
P < 0.01
P < 0.01
P < 0.01
P < 0.01
P < 0.01
P < 0.01
Fig. 1. Joint effects of chlorimuron-ethyl and Cd on the content of chlorophyll
in wheat seedlings (data presented as mean ? SD). Letters under x axis refer to
the difference at significance level P < 0.05 (LSR test).
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Compared with single Cd stress and the control, the MDA
content in roots with joint stress of chlorimuron-ethyl and Cd
decreased significantly (P < 0.01). After a 4-day exposure, the
content of MDA in roots with joint stress of chlorimuron-ethyl
and Cd was lower than that with stress of Cd only as well as
the control (Fig. 2B,D).
3.3. Dynamic changes in POD activity
in wheat leaves and roots
Among various enzymes involved in the elimination of ac-
tive oxygen species (AOS), guaiacol peroxidase can be consid-
ered one of the key enzymes, since both of its extra- and
intracellular forms are participating in the breakdown of
H2O2. Changes in the POD activity in leaves with joint stress
of chlorimuron-ethyl and Cd are depicted in Fig. 3A and C.
Compared with the control, single stress of 10 mg/kg Cd sig-
nificantly (P < 0.05) decreased the POD activity in leaves af-
ter 1- and 2-day exposure, and significantly (P < 0.05)
increased it after 3e4 days of exposure. Joint stress of
10 mg/kg Cd and 5 mg/kg chlorimuron-ethyl lowered the
POD activity in leaves in the first 3 days of exposure, and
then increased it significantly (P < 0.01) compared with single
Cd stress and the control after a 4-day exposure. Joint stress of
10 mg/kg Cd and 150e300 mg/kg chlorimuron-ethyl also
decreased the POD activity in leaves on the first day of treat-
ment, and afterwards increased it gradually. The POD activity
in leaves exposed to single stress of 100 mg/kg Cd was lower
than that in the control at the first 3 days of exposure, but signif-
icantly (P < 0.01) higher than that in the control after a 4-day
exposure. The POD activity in leaves treated with 100 mg/kg
Cd and 5 mg/kg chlorimuron-ethyl was significantly lower
than that in the control after 1- and 2-day exposure while
the difference became insignificant after 3e4 days of expo-
sure. There were no significant differences in the POD activity
in leaves between joint stress of 100 mg/kg Cd and 150e
300 mg/kg chlorimuron-ethyl and the control during first
2 days of exposure. Afterwards, the POD activity in leaves
withthe joint stress increased.
(P < 0.01) higher than that in the Cd only treatment or in
the control after a 4-day exposure.
Joint stress of chlorimuron-ethyl and Cd as well as single
stress of Cd decreased the POD activity in roots with exposure
time significantly (P < 0.01). The POD activity in roots with
joint stress of chlorimuron-ethyl and Cd decreased with
increasing chlorimuron-ethyl concentration during the 1-day
exposure. The POD activity in roots treated with joint stress
of 10 mg/kg Cd and 5e150 mg/kg chlorimuron-ethyl was
higher than that treated with single stress of 10 mg/kg Cd after
4 days of treatment. Nevertheless, the POD activity in roots
It wassignificantly
Fig. 2. Joint effects of chloronmium-ethyl and Cd on the MDA content in leaves (A and C) and roots (B and D) of wheat seedlings (data presented as mean ? SD).
Letters under x axis refer to the difference at significance level P < 0.05 (LSR test).
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treated with 10 mg/kg Cd and 300 mg/kg chlorimuron-ethyl
was the lowest during the exposure time. The POD activity
in roots with single and joint stress of 100 mg/kg Cd decreased
greatly. There were almost no significant differences among
the treatments after 2e4 days of exposure.
3.4. Dynamic changes of SOD activity
in wheat leaves and roots
As shown in Fig. 4, the SOD activity in leaves treated with
chlorimuron-ethyl and 10 mg/kg Cd was significantly (P <
0.05) lower than in the Cd only treatment or in the control
after a 1-day exposure. Afterwards, the SOD activity in leaves
treated with chlorimuron-ethyl and 10 mg/kg Cd increased and
significantly (P < 0.05) higher than in the Cd only treatment
or in the control. Only after a 2-day exposure, the SOD activity
in leaves increased with increasing chlorimuron-ethyl concen-
tration. When Cd concentration was up to 100 mg/kg, the SOD
activity in leaves treated with chlorimuron-ethyl and Cd was
significantly (P < 0.05) higher than in the Cd only treatment
or in the control after a 2-day exposure. After 4 days of treat-
ment, with an increase of chlorimuron-ethyl concentration, the
SOD activity in leaves increased and joint stress of chlori-
muron-ethyl and Cd elevated the SOD activity in leaves
more than the single stress of Cd only.
The SOD activity in roots with joint stress of chlorimuron-ethyl
and Cd decreased with increasing chlorimuron-ethyl concen-
tration after a 1-day exposure. After a 4-day exposure,
although higher than in the control, the SOD activity in roots
with joint stress of chlorimuron-ethyl and Cd was lower than
that with single stress of Cd.
3.5. Dynamic changes of soluble protein
in wheat leaves and roots
Changes in the content of soluble protein in leaves treated
with chlorimuron-ethyl and Cd are described in Fig. 5A and
C. When Cd concentration was 10 mg/kg, the soluble protein
content in leaves treated with joint stress of chlorimuron-ethyl
and Cd was lower than in the Cd only treatment or in the con-
trol. After 3 and 4 days of treatment, the soluble protein content
with joint stress of 10 mg/kg Cd and 150e300 mg/kg chlori-
muron-ethyl was lower than that with single stress of 10 mg/kg
Cd. When the concentration of Cd was up to 100 mg/kg, there
were no significant (P > 0.05) differences in soluble protein in
leaves among all the treatments after a 1-day exposure. After
a 3- and 4-day exposure, soluble protein in leaves treated
with joint stress of chlorimuron-ethyl and 100 mg/kg Cd was
higher than that with single stress of Cd.
Fig. 3. Joint effects of chloronmium-ethyl and Cd on the POD activity in leaves (A and C) and roots (B and D) of wheat seedlings (data presented as mean ? SD).
Letters under x axis refer to the difference at significance level P < 0.05 (LSR test).
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M.-E Wang, Q.-X. Zhou / Environmental Pollution 144 (2006) 572e580