Anti-oxidant response of Cucumis sativus L. to fungicide carbendazim

PESTICIDE Biochemistry & Physiology

Pesticide Biochemistry and Physiology 89 (2007) 54–59 www.elsevier.com/locate/ypest

Anti-oxidant response of Cucumis sativus L. to fungicide carbendazim Li Zhen Zhang a b

a,b

, Niu Wei c, Qiao Xiong Wu

b,*

, Ma Li Ping

b

Institute of Loess Plateau, Shanxi University, No. 36, Wucheng Road, Taiyuan, Shanxi Province 030006, PR China Shanxi Key Laboratory of Pesticide Science, Shanxi Academy of Agricultural Sciences, No. 64, North Nongke Road, Taiyuan, Shanxi Province 030031, PR China c Shanxi Academy of Agricultural Sciences, No. 2, Changfeng Road, Taiyuan, Shanxi Province 030006, PR China Received 6 November 2006; accepted 27 February 2007 Available online 7 March 2007

Abstract Our purpose in this research was to determine the response of anti-oxidative enzymes of cucumber (Cucumis sativus L.) when carbendazim applied as soil drench at 0, 5, 50, and 100 mg kg1. The changes in the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) enzymes were measured in roots, stems, and leaves of cucumber. The plants were sampling at cotyledon phase (14 days) and florescence phase (56 days). A strong correlation between anti-oxidant content and carbendazim concentration was observed. As concentration increased, the activities of SOD and CAT increased in roots and leaves. While in stems, SOD and CAT activities were increased in florescence phase and declined in cotyledon phase. The content of GPX increased in stems, and declined in leaves. Higher levels of SOD, CAT, and GPX were observed in cucumber cotyledons than the older leaves. The present study suggested that carbendazim treatments had different effects on cucumber anti-oxidant system in different tissues. It was concluded that cotyledons might play an important role for adaptation as the carbendazim concentration increased, and the ability of mature cucumber to maintain a balance between the formation and detoxification of activated oxygen species appeared likely to enhance. On the basis of our observations, we conclude that increased SOD, CAT and GPX activity provides plant with increased carbendazim stress tolerance. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Fungicide; Carbendazim; Anti-oxidant response; SOD; CAT; GPX; Cucumis sativus L.

1. Introduction Carbendazim is widely used at a large scale as agriculture and horticultural fungicide around the world [1]. It is strongly absorbed to soil organic material and remains in the soil for up to 3 years [2]. Most often fungicides have been applied as soil drenches, and with this approach it is possible to cause a phytotoxic reaction by a high dose fungicide. Carbendazim is uptaken by plants through the roots, seeds or leaves and, afterwards, is transferred to the whole plant [3]. And concentrations of carbendazim in plants increased with increasing dose rate [4]. Repeated field applications of this fungicide may produce a high *

Corresponding author. Fax: +86 351 7126215. E-mail address: [email protected] (Q.X. Wu).

0048-3575/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2007.02.007

residual concentration in the soil, leading to even more severe detrimental effects on plants. The accumulation effects of these chemical on plants evolution are limited; therefore this subject has attracted the interest of many researchers recently. The effects of carbendazim on different organism have been investigated. A series of test with administration of carbendazim to rats, result in adverse effects, including testicular injury, infertility, respiratory failure, and muscle cramps have been well documented [5–9]. And recently results indicate that carbendazim is a potential endocrine disruptor in humans [10]. Weyers et al. [11] findings indicate that high application rate of carbendazim should be assessed as high environmental risk in ecosystem. Sousa et al.’s [12] results suggest that higher carbendazim may cause a reduction in soil microbial. Koolhaas et al. [1]

L.Z. Zhang et al. / Pesticide Biochemistry and Physiology 89 (2007) 54–59

analysis reveale that a significant dose-related decreasing of soil microarthropod numbers with increasing carbendazim dosages. It is a threat to earthworms [14], and it will affected the decomposition of organic matter [15]. The harmful effect of carbendazim on Arbuscular mycorrhiza fungi is well documented [16–18]. But report concerning plants are very limited. Dumpe and Wokoma [19] observed that carbendazim at a lower concentration possesses germination stimulation of plantain hybrid seeds. Also, it is reported that carbendazim could improve the performance of seeds under PEG-6000 induced water stress conditions [20]. This inconsistency may be due to considerable variation in treatment time and manner, the setting of studies, purity of chemicals, and species tissues differences etc [21]. Plants, as well as other organisms, have evolved a complex anti-oxidative system in order to protect cellular membranes and organelles from the damaging effects of toxic concentrations of activated oxygen species [22]. It is widely accepted that reactive-oxygen species (ROS)1, such as hydrogen peroxide (H2O2), superoxide radicals ðO2  Þ, singlet oxygen(.O2), hydroxyl radicals (.OH), are responsible for various stress-induced damages to macromolecules and ultimately to cellular structures. It is now well known that chemical pollution are at the cellular, often involved in oxidative stress, which results from the production of ROS [23–26]. Some of these stress responses have been attributed to changes in the activities of ROS scavenging enzymes, such as superoxide dismutase (SOD; EC1.15.1.1), catalase (CAT; EC1.11.1.6) and Guaiacol peroxidase (GPX; EC1.11.1.7). SOD dismutation of superoxide radicals, whereas CAT eliminates hydrogen peroxide. GPX provide an efficient protection against oxidative damage and free radicals in the presence of GSH through reduction of both hydrogen peroxide and organic hydroperoxides [13]. Since their activities and transcripts are altered when plants are subjected to stress, changes in the levels of anti-oxidant enzymes have been used to assess the effect of different stressors including salinity [27], waterlogged [28], ionizing radiation [29], metals [30–32], herbicide mefenacet [26,33] and fungicide [37]. One aim in the present study was to investigate the effect of carbendazim stress in terms of the activities of SOD, CAT and GPX in cucumber. Thus, the objective was to examine changes in anti-oxidant enzyme activities (SOD, CAT, GPX) in the root, stem and leaf tissues of both cotyledon phase and florescence phase upon exposure to different dosages carbendazim. And an attempt was made to answer the following questions (i) Do different dose of carbendazim contamination significantly modify the activity of oxidative stress defense enzymes? (ii) Do different organ(root, stem, leaf)show the same enzy-

1 Abbreviations used: SOD, superoxide dismutase; CAT, catalase; GPX, glutathione peroxidase; ROS, reactive oxygen species.

55

matic response with respect to carbendazim contamination? and (iii) Do different phase (seedling and florescence) show the same enzymatic response with respect to carbendazim contamination? The present study was intended to increase the understanding of the adaptation and survival mechanisms of plants under natural environmental stress. 2. Materials and methods 2.1. Plant materials and treatments Cucumber (Cucumis sativus L. cv. No. 4 Jinyan) seeds were surface sterilized with 70% ethanol for 30 s and rinsed three times with distilled water, then drenched with 0.5% mercury bichloride for 10 min and rinsed three times with distilled water. After 3 days germination on moistened of 70–80%, the seeds were sown in 10-cm polyethylene bags containing pre-moistened, fungicide-drenched, unsterilized soils. The physicochemical characteristics of the soil were shown in Table 1. After soil preparation, the carbendazim was applied and well mixed. In the investigation a control (0 mg kg1) plus three treatment levels were chosen, including the recommended field rate (5 mg kg1), the usually practical greenhouse rate (50 mg kg1) and the accumulated greenhouse rate (100 mg kg1) according to our investigation and assay. Each treatment was conducted nine replications. The plants were kept inside a growth room maintain at 26 °C/20 °C day/night with 70–80% humidity and a 16 h photoperiod at the level of photosynthetically active radiation was 300 lmol m2 s1. In cotyledon phase and florescence phase, respectively, roots, stems and leaves were obtained for enzymes activity. Cotyledon phase, 14 days after sowing cucumber seeds in treated soils, which implied that there are only two cotyledons. Florescence phase, 56 days after sowing cucumber seeds in treated soils, which implied that cucumber have floured. 2.2. Measurement of protein Protein concentration of all samples were determined spectrophotometrically at A595 using the method of Bradford [34] with bovine serum albumin as standard. We could detect no protein concentration in root of cotyledon phase for control. This maybe because that it was too young and there was no protein accumulation.

Table 1 Physicochemical characteristics of the soil used for cucumber seed cultivation under greenhouse condition pH

Olsen-P (mg kg1)

Available K (mg kg1)

Total N (g kg1)

O.M. (g kg1)

8.41

1.2178

51.5

0.4445

4.142

56

L.Z. Zhang et al. / Pesticide Biochemistry and Physiology 89 (2007) 54–59

2.3. Enzyme extraction and activity determination Cucumber plants were taken from the polyethylene bags at random and separated into three parts, root, stem and leaf. They were prepared for assays of SOD, CAT, and GPX activity. The activity of SOD, CAT, GPX was determined spectrophotometrically according to the method of Nanjing Jiancheng Bioengineering Institute with a Microplate reader. The cut roots, stems and leaves of each treatment were carried in an icebox and ground in a mortar to form homogenized powder. Samples were prepared for SOD, CAT, GPX activity analyses by weighing 1.0 g roots, stems and leaves material, respectively, in 9.0 mL of an ice-cold 0.9% sodium chloride injection (Shandong Hualu Pharmaceutical Co., Ltd.). The homogenate (1:10 w/v) were centrifuged at 3000 rpm/min at 4 °C for 15 min. The supernatants were then collected in a fresh tube and stored on ice for determination of enzyme activity. The enzyme activity of SOD, CAT, and GPX were determined using the ‘‘Assay Kit’’ (Nanjing Jiancheng Bioengineering Institute) by the spectrophotometrical method. Each treatment was conducted in triplicate. SOD activity was assayed spectrophotometrically at 550 nm by use of a xanthine and xanthine oxidase system. The rate of the reduction with O2  is linearly related to the xanthine oxidase activity and is inhibited by SOD. Therefore, the 50% inhibition activity of SOD can be measured at 550 nm of absorbance. The specific activity of the SOD (inhibition rate) was calculated using the equation described in the protocol of the kit. One unit of SOD activity was defined as the amount of SOD required for 50% inhibition of the xanthine and xanthine oxidase system reaction in 1 mL enzyme extraction of per milligram of protein. The principle of the catalase activity was assayed spectrophotometrically at 405 nm by measuring the initial rate of the disappearance of H2O2. The specific activity of the CAT was calculated using the equation described in the protocol of the kit. One unit of CAT activity was defined as 1 lmoL H2O2 decomposed 1 mg of protein per second. GPX was assayed spectrophotometrically by use of glutathione as substrate by measurement of the decrease of enzymatic reaction of glutathione (except the effect of non-enzymatic reaction) at 412 nm in system of enzymatic reaction of 1 mg protein per minute. The specific activity of the GPX was calculated using the equation described in the protocol of the kit. One unit of GPX activity was defined as the decrease amount of 1 nmol L1 GSH (except the effect of non-enzymatic reaction) in system of enzymatic reaction of 1 mg protein per minute. 2.4. Statistical analysis Data of the experiment were analyzed statistically and the results were expressed as means ± SD of three independent measurements of SOD, CAT, and GPX activity. All analyses were carried out with SPSS [35] (Li et al., 2005).

Analysis of one-way ANOVA was made on the data obtained, and Student’s t-test was used for testing the significance differences. 3. Results The results of experiment showed that the treatment of cucumber plants with carbendazim caused changes in the activity of SOD, CAT and GPX in root, stem and leaf tissue in comparison to control cucumber plants. To found out the significance difference of anti-oxidant enzymes between different growing phase of cucumber plants, cotyledon phase (14 days) and florescence phase (56 days), on exposure to carbendazim, the data obtained have been subjected to Student’s t-test. According to the results, there were striking differences in SOD,CAT and GPX activity in three tissues between the two phases (p < 0.001). The carbendazim treatment caused a significant increase in SOD activity in comparison with control. In roots, stems and leaves for the florescence phase and leaves for the cotyledon phase, the highest enzymes activity was at 100 mg kg1 treatment and the lowest enzymes activity was at 0 mg kg1 control. Whereas in roots and stems for the cotyledon phase, the enzymes activity were higher at 50 mg kg1 than 100 mg kg1. The carbendazim treatment caused increase in the CAT activity for florescence phase in roots and leaves for florescence phase in comparison to control. While in stems of cotyledon phase CAT activity was decreased in comparison to control. The carbendazim treatment caused significant decrease in the CAT activity for cotyledon phase in stems in comparison to control.The carbendazim treatment caused significant increase in the GPX activity of stems in comparison to control, while in leaves GPX activity was decreased in comparison to control.

4. Discussion In this study, carbendazim was preferred because information on its effects on plants is very limited, especially higher dosage exposure. The data collected in this study were all from two time-points (14 and 56 days) of the experiment. We found that the treatment changed the production of anti-oxidant enzyme activities in various cucumber tissues. However, cucumber plants did not show visible symptoms of toxicity after cucumber exposure up to 100 mg kg1 carbendazim. This may be explained that plants possess effective enzymatic and non-enzymatic detoxifying systems continuously involved in the cellular protection against ROS coming from both in the environment and cell metabolism [23]. The induction protein accumulation in roots of cotyledon phase under carbendazim-treated plants maybe attribute the properties cytokinins of carbendazim [24]. The highest experimental dose in these studies was 20 times higher than the recommended field rate. A change

L.Z. Zhang et al. / Pesticide Biochemistry and Physiology 89 (2007) 54–59

57

in SOD, CAT, and GPX activity suggest that carbendazim causes oxidative stress in cucumber plants, possibly by generating reactive-oxygen in the bodies. To counter the carbendazim stress, anti-oxidant enzymes become activated and may activate the defense system by stimulating the expression of the respective enzymes. SOD is an essential component of a plant 0 s anti-oxidative defense system. It plays an important role in dismutation of free hydroxyl radicals by the formation of hydrogen peroxide. Information on SOD activity is shown in Table 2. The SOD activities in the leaves were significantly higher in the cotyledon phase than in the florescence phase (p < 0.001). These results suggest that two cotyledons have a greater capacity to scavenge free hydroxyl radicals by more rapidly developing anti-oxidative defense system than mature leaves. This phenomenon is mainly based on the fact that ageing leaf contain lower anti-oxidant levels than younger leaf [37]. However, in florescence phase the higher level of generation of SOD in root and stem tissues was observed. These results suggest that mature cucumber plants enhanced the free hydroxyl radicals by increasing the content of superoxide dismutase. A similar case that SOD activities have strong correlation with growing phase was also reported in microbe [33]. The SOD activity increased significantly (p < 0.01) for 5–100 mg kg1 carbendazim, but decreased in stems of cotyledon phase. This suggested that the adjustments of enzymic activities and anti-oxidant levels in stems might be different from other tissues. In roots, stems and leaves for the florescence phase and leaves for the cotyledon phase, SOD activity increased with increasing carbendazim concentration, suggesting that SOD was stimulated by scavenging O2  to protect cucumber plants from carbendazim toxicity. And the SOD activity was sufficient to cope with an increased concentration of such radicals at low carbendazim concentration. Higher concentration of carbendazim resulted in inhibition of SOD activity. This result showed that there were no a sufficient increased in SOD activity to scavenge excess O2  that accumulated in

cells of cucumber plants at high carbendazim concentrations. Thus, carbendazim generated oxidative stress damage in cells, and SOD could not protect cucumber plants from carbendazim toxicity at high carbendazim concentration. CAT, located in peroxisomes, mitochondrial and cytosol, can scavenge H2O2 with co-substrates [32]. It is another important enzyme against oxidative stress, being able to scavenging H2O2, which is the major product produced by SOD[36].Information on CAT activity is given in Table 3. The CAT activities leaves were significantly higher in the cotyledon phase than in the florescence phase (p < 0.001). This is consistent with the tendency of SOD. This indicated that H2O2 generated by SOD was removed by the induced activity of CAT. In root and leaf tissues in the florescence phase, CAT activity increased in compareison with control, suggesting that, possibly, CAT mediated the removal of H2O2 and toxic peroxide in cucumber roots and leaves in the florescence phase. However, CAT activity at 100 mg kg1 treatment in the roots and leaves were lower than the other treatments. This result showed that increased CAT activity was not enough to scavenge excess H2O2 that accumulated in cells of cucumber plants at 100 mg kg1 carbendazim accumulated concentrations. In contrast, in stem tissues in the florescence phase, the CAT activity decreased in response to increased carbendazim concentration. This result showed that CAT in mature stems could not cope with an increased concentration of H2O2 under carbendazim treatments. Carbendazim toxicity to CAT was severe in mature stems even at low carbendazim concentration. Information on GPX activity is shown in Table 4. Constitutive levels of GPX of all tissues were higher in cotyledon phase than florescence phase, which indicated that lower activities of CAT in stems probably compensate for by GPX. The results observed for GPX activity indicate an enhancement of GPX activity in stem tissues upon exposure to carbendazim when compare with control, suggesting that this enzyme serves as an intrinsic defense tool to

Table 2 SOD activities in control and carbendazim-treated groups (means ± SD)

Table 3 CAT activities in control and carbendazim-treated groups (means ± SD)

Groups (n:3)

Tissue

Day 14 (cotyledon phase)

Groups (n:3)

Tissue

Day 14 (cotyledon phase)

Control

Root Stem Leaf

— 119.41 ± 3.59 62.53 ± 2.55

537.39 ± 1.17 431.80 ± 1.53 0.52 ± 0.03

Control

Root Stem Leaf

— 715.74 ± 7.78 176.60 ± 1.72

68.19 ± 0.85 821.99 ± 9.03 74.23 ± 2.01

5 mg kg1

Root Stem Leaf

109.60 ± 4.36 76.94 ± 2.30 73.24 ± 2.50

684.78 ± 2.41 521.71 ± 1.75 1.12 ± 0.27

5 mg kg1

Root Stem Leaf

4697.35 ± 29.44 354.16 ± 3.37 199.65 ± 10.20

93.05 ± 1.92 122.60 ± 5.43 79.71 ± 1.93

50 mg kg1

Root Stem Leaf

133.49 ± 6.67 403.79 ± 6.70 73.24 ± 2.50

843.47 ± 5.24 2235.74 ± 18.45 6.26 ± 0.46

50 mg kg1

Root Stem Leaf

815.30 ± 10.36 403.79 ± 6.70 178.71 ± 6.71

378.22 ± 3.32 676.60 ± 5.69 86.90 ± 1.41

100 mg kg1

Root Stem Leaf

96.93 ± 10.90 132.65 ± 1.89 394.52 ± 9.30

610.72 ± 2.46 1676.71 ± 21.11 3.7 ± 0.38

100 mg kg1

Root Stem Leaf

96.93 ± 10.62 350.66 ± 3.09 148.10 ± 2.88

337.35 ± 1.83 1113.80 ± 10.16 82.25 ± 1.84

Day 56 (florescence phase)

Day 56 (florescence phase)

58

L.Z. Zhang et al. / Pesticide Biochemistry and Physiology 89 (2007) 54–59

Table 4 GPX activities in control and carbendazim-treated groups (means ± SD)

possible H2O2 build-up could be attended by increased in activities CAT and GPX. Decreased CAT activity in stems is probably compensated for by GPX. Induced activities of SOD, CAT and GPX in root, GPX in stems and SOD, CAT in leaves, might be attributed to the strategies adopted by the cucumber plants to overcome the toxicity of carbendazim. Further investigation, using molecular biology techniques, is needed to determine the mechanisms involved in enzyme induction under pesticide-contaminated conditions.

Groups (n:3)

Tissue

Day 14 (cotyledon phase)

Day 56 (florescence phase)

Control

Root Stem Leaf

— 122.95 ± 2.58 263.95 ± 2.94

137.93 ± 0.34 108.41 ± 3.78 15.86 ± 0.74

5 mg kg1

Root Stem Leaf

12019.38 ± 20.86 445.67 ± 0.62 243.87 ± 2.88

113.96 ± 1.67 206.25 ± 5.48 9.96 ± 0.54

50 mg kg1

Root Stem Leaf

1271.20 ± 95.99 1098.91 ± 21.42 229.54 ± 7.48

216.10 ± 1.99 743.83 ± 3.76 10.47 ± 0.70

Acknowledgment

Root Stem Leaf

770.86 ± 19.58 1274.34 ± 22.84 231.68 ± 4.56

869.04 ± 1.38 444.44 ± 3.54 13.82 ± 1.25

We are grateful Zhang Qiang for his helpful efforts to provide experimental soil.

100 mg kg1

resist carbendazim oxidative damage in stem tissues. However in leaf tissues, carbendazim treatments resulted in a decrease in GPX capacity when compare with control. Carbendazim toxicity to GPX in leaf tissues was severe in cucumber plants even at carbendazim recommended field concentration. Dixit et al. [30] also reported that GPX could be activated relatively more in roots than in leaves of Ca2+-treated pea plants. Despite the decreased GPX activity with an increased in carbendazim, abound capacity of the anti-oxidative system was apparent from increased levels of SOD and CAT. It appears that SOD and CAT activity involvement in the carbendazim detoxification mechanism in leaf tissues during cucumber growing phase. Zhang et al. [26] determined that rice seedling in the presence of herbicide mefenacet showed increase in the activities of SOD and CAT. Wu and Tiedemann [37] results indicated that the fungicides azoxystrobin and epoxiconazole significantly enhance SOD and CAT activity. This work show, for the first time, that carbendazim treatment at higher dosage has significant effect on antioxidant enzymes activities that are also involved in oxidative stress conditions in cucumber plants. This minimizes the direct effects of carbendazim on plants. The major defense strategy against ROS resulting from the presence of the higher carbendazim in cucumber plants appears to be the removal of O2  . In our study, patterns of response to carbendazim stress were obtained as judged by activities of SOD, CAT, and GPX enzymes. The altered balance of the anti-oxidant enzymes caused by an increase in most anti-oxidant enzymes in all the carbendazim-treated except SOD in stems of cotyledon phase and CAT in stem, GPX in leaves. The increased activities of SOD, CAT and GPX may be a response to increased production O2  and H2O2 under carbendazim stress. This indicates that H2O2 generated by SOD was removed by the induced activity of CAT and GPX. In contrast to the observed increased in SOD activity of roots and leaves, a decrease recorded of stems in cotyledon phase may influence the generation of H2O2. Correspondingly, a decrease of CAT but increase of GPX in stems was observed. Thus, in cucumber plants the

References [1] J.E. Koolhaas, C.A.M. Van Jestel, J. Ro¨mbke, A.M.V.M. Soares, S.E. Jones, Ring-testing and field validation of a terrestrial model ecosystem(TEM)—an instrument for testing potentially harmful substances: effects of carbendazim on soil microarthropod communities, Ecotoxicology 13 (2004) 75–88. [2] World Health Orhanisation, 1993. Carbendazim. Environmental Health Criteria 149. Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organisation, and the World Health Organisation, Geneva, Switzerland, pp.1–125. [3] M.J.G. de la Huebra, P.H.O. Nieto, Y. Ballestero, L. Herna´ndez, Determination of carbendazim in soil samples by anodic stripping voltammetry using a carbon fiber ultramicroelectrode, Fresenius J. Anal. Chem. 367 (2002) 474–478. [4] S.E. Jones, D.J. Williams, P.J. Holliman, N. Taylor, J. Baumann, B. Fo¨rster, C.A.M. Van Gestel, J.M.L. Rodrigues, Ring-testing and field validation of a terrestrial model ecosystem (TEM)-an instrument for testing potentially harmful substances: fate of the model chemical carbendazim, Ecotoxicology 13 (2004) 29–42. [5] J. Lim, M.G. Miller, Role of testis exposure levels in the insensitivity of prepubertal rats to carbendazim-induced testicular toxicity, Fundam. Appl. Toxicol. 37 (1997) 158–167. [6] M. Nakai, R.A. Hess, B.J. Moore, R.F. Guttroff, L.F. Strader, R.E. Linder, Acute and long-term effects of a single dose of the fungicide carbendazim (methyl 2-benzimidazole carbamate) on the male reproductive system in the rat, J. Androl. 13 (1992) 507–518. [7] B. Uludag, S. Tarlaci, N. Yuceyar, N. Arac, A transient dysfunction of the neuromuscular junction due to carbendazim intoxication, J. Neurol. Neurosurg. Psychiatry 70 (2001) 563–567. [8] L.M. Correa, M.G. Miller, Microtubule depolymerization in rat seminiferous epithelium is associated with diminished tyrosination of -tubulin, Biol. Reprod. 64 (2001) 1644–1652. [9] L.M. Correa, M. Nalai, C.S. Strandgaard, R.A. Hess, M.G. Miller, Microtubules of the mouse testis exhibit differential sensitivity to the microtubule disruptors carbendazim and colchicines, Toxicol. Sci. 69 (2002) 175–182. [10] H. Morinaga, T. Yanase, M. Nomura, T. Okabe, K. Goto, N. Harada, H. Nawata, A benzimdazole fungicide, benomyl, and its metabolite, carbendazim, induce aromatase activity in a human ovarian granulose-like tumor cell line (KGN), Endocrinology 145 (4) (2004) 1860–1869. [11] A. Weyers, B.S. Klu¨ttgen, T. Knacker, S. Martin, C.A.M. Van Gester, Use of terrestrial model ecosystem data in environmental risk assessment for industrial chemicals, biocides and plant protection products in the EU, Ecotoxicology 13 (2004) 163–176. [12] J.P. Sousa, J.M.L. Rodrigues, S. Loureiro, A.M.V.M. Soares, S.E. Jones, B. Fo¨rster, V.A.M. Van Jestel, Ring-testing and field validation

L.Z. Zhang et al. / Pesticide Biochemistry and Physiology 89 (2007) 54–59

[13]

[14]

[15]

[16]

[17]

[18]

[19] [20] [21]

[22] [23]

of a terrestrial model ecosystem (TEM)-an instrument for testing potentially harmful substances: effect of carbendazim on soil microbial parameters, Ecotoxicology 13 (2004) 43–60. Y. Tuluce, I. Celik, Influence of subacute and subchronic treatment of abcisic acid and gibberellic acid on serum marker enzymes and erythrocyte and tissue antioxidant defenses and lipid peroxidation in rats, Pestic. Biochem. Physiol. 86 (2006) 85–92. J. Ro¨mbke, C.A.M. VanJestel, S.E. Jones, J.E. Koolhaas, J.M.L. Rodrigues, T. Moser, Ring-testing and field validation of a terrestrial model ecosystem (TEM)-an instrument for testing potentially harmful substances: effect of carbendazim on earthworms, Ecotoxicology 13 (2004) 105–118. C.A.M. Jestel, J.E. Koolhaas, G. Nentwig, J.M.L. Rodrigues, J.P. Sousa, S.E. Jones, T. Knacker, Ring-testing and field validation of a terrestrial model ecosystem (TEM)-an instrument for testing potentially harmful substances: effect of carbendazim on organic matter breakdown and soil fauna feeding activity, Ecotoxicology 13 (2004) 129–141. J.H. Kough, V. Gianinazzi-Pearson, S. Gianinazzi, Depressed metabolic activity of vesicular-arbuscular mycorrhizal fungi after fungicide applications, New Phytol. 106 (1987) 707–715. I. Thingstrup, S. Rosendahl, Quantification of fungal activity in arbuscular mycorrhizal symbiosis by polyacrylamide gel electrophoresis and densitomety of malate dehydrogenase, Soil Biol. Biochem. 26 (1994) 1483–1489. M. Kling, I. Jakobsen, Direct application of carbendazim and propiconazole at field rates to the external mycelium of three arbuscular mycorrhizal fungi species:effect on 32P transport and succinate dehydrogenase activity, Mycorrhiza 7 (1997) 33–37. S. Nayyar, H. Nayyar, Carbendazim alleviates effects of water stress on chickpea seedlings, Biologia. Plantarum 49 (2) (2005) 289–291. B.D. Dumpe, E.C.W. Wokoma, Factors influencing germination of hybrid plantain seeds in soil, Plant Foods Hum Nutr. 58 (2003) 1–11. I. Celik, Y. Tuluce, Effects of indoleacetic acid and kinetin on lipid peroxidation and antioxidant defense in various tissues of rats, Pestic. Biochem. Physiol. 84 (2006) 49–54. H.X. Ren, Z.L. Wang, X. Chen, Y.L. Zhu, Antioxidative responses to different altitudes in Plantago major, Environ. Exp. Bot. 42 (1999) 51–59. B.D. Banerjee, V. Seth, A. Bahattacharya, Biochemical effects of some pesticides on lipid peroxidation and free-radical scavengers, Toxicol. Lett. 107 (1999) 33–47.

59

[24] K.G.M. Skene, Cytokinin-like properties of the systemic fungicide benomyl, J. Hort. Sci. 47 (1972) 179. [25] J. Nicholas, J.S. Wood, Catalase and superoxide dimutase activity in ammonia-oxidising bacteria, FEMS Microbiol. Ecol. 38 (2001) 53–58. [26] C.D. Zhang, S.K. Han, A.Q. Zhang, Effect of herbicide mefenacet on response of active oxygen scavenging system in rice plant, Agroenvironment. Protect. 20 (2001) 411–413 (in chinese). _ Tu¨rkan, Comparative lipid peroxidation, antioxidant [27] T. Demiral, I. defense systems and protein in roots of two rice cultivars differing in salt tolerance, Environment. Exp. Bot. 53 (2005) 247–257. [28] K.H.R. Lin, C.H. Weng, H.F. Lo, J.T. Chen, Study of the root antioxidative system of tomatoes and eggplants under waterlogged conditions, Plant Sci. 167 (2004) 355–365. [29] R. Zaka, C.M. Wandecasteele, M.T. Misset, Effect of low chronic doses of ionizing radiation on antioxidant enzymes and G6PDH activities in Stipa capillata (Poaceae), J. Exp. Bot. 376 (53) (2002) 1979–1987. [30] V. Dixit, V. Pandey, R. Shyam, Differential antioxidative responses to cadmium in roots and leaves of pea (Pisum Sativum L. cv. Azad), J. Exp. Bot. 358 (52) (2001) 1101–1109. [31] S.M. Gallego, M.P. Benavides, M.L. Tomaro, Effect of cadumium ions antioxidant defense system in sunflower cotyledons, Biol. plant 42 (1) (1999) 49–55. [32] A. Hegedu¨s, S. Erdei, G. Horva´h, Comparative studies of H2O2 detoxifying enzymes in green and greening barley seedlings under cadumium stress, Plant Sci. 160 (2001) 1085–1093. [33] Y.F. Ye, H. Min, Z.M. Lu, Effect of herbicide mefenacet pollution on antioxidant enzyme and ATPase of Sphingobacterium multivorum Y1, Acta. Scientiae Circumstantiae 26 (1) (2006) 151–156 (in Chinese). [34] M.M. Brodford, A rapid and sensitive method for the quantification of microgram quantities of protein using the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [35] Z.H. Li, P. Luo, W.J. Wu, SPSS for windows (second edited), Publishing of electrical industry, Beijing, 2005, pp.130-156. [36] K. Asada, Ascorbate peroxidase-a hydrogen peroxide scavenging enzyme in plants, Plant Physiol. 85 (1992) 235–241. [37] Y.X. Wu, A. van Tiedemann, Impact of fungicides on active oxygen species and antioxidant enzymes in spring barley (Hordeum vulgare L.) exposed to ozone, Environ. Pollut. 116 (2002) 37–47.