Soil genotoxicity induced by successive ... - Wiley Online Library

Environmental Toxicology and Chemistry, Vol. 33, No. 5, pp. 1043–1047, 2014 # 2014 SETAC Printed in the USA

SOIL GENOTOXICITY INDUCED BY SUCCESSIVE APPLICATIONS OF CHLOROTHALONIL UNDER GREENHOUSE CONDITIONS XIANGXIANG JIN, NING CUI, WEI ZHOU, MAHDI SAFAEI KHORRAM, DONGHONG WANG, and YUNLONG YU* Institute of Pesticide and Environmental Toxicology, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China (Submitted 17 November 2013; Returned for Revision 15 December 2013; Accepted 21 January 2014) Abstract: Greenhouse production of vegetables has been developed rapidly in China. High temperature and humidity inside the greenhouse make this environment more suitable for fast reproduction of fungal diseases. Fungicides are among the chemicals used extensively in the greenhouse to prevent crops from invasive infections by phytopathogens; however, little is known about the accumulation of fungicides in soil and their effect on soil quality under greenhouse conditions. In the present study, the accumulation of the fungicide chlorothalonil (CT) and its toxic metabolite hydroxy-chlorothalonil (HCT) in soil as well as their related soil genotoxicity under greenhouse conditions was investigated. The results indicated that both CT and HCT accumulated in soil with repeated applications of CT, and the accumulation level was strongly correlated to application dosage and its frequency. In addition, soil genotoxicity, which was measured by Vicia faba, also increased with the accumulation of CT and HCT, and the main contributor to this phenomenon was CT rather than HCT. The data demonstrated that successive applications of fungicides may result in their accumulation in soil and thus a decline in soil quality. Environ Toxicol Chem 2014;33:1043–1047. # 2014 SETAC Keywords: Chlorothalonil

Hydroxychlorothalonil

Accumulation

Genetic toxicity

possibility of pesticide accumulation in soil and its potential for the induction of soil genotoxicity under greenhouse conditions. The results will help us to understand the potential contribution of pesticide applications in soil quality reduction under greenhouse conditions.

INTRODUCTION

Greenhouse production of vegetables has grown quickly in China, and vegetable cultivation, over-fertilization, and intensive applications of pesticides are among the most common operational methods used to reach maximum yields. Increasing concern has been expressed regarding soil quality under greenhouse conditions. Numerous studies have been conducted on changes in soil quality and on its physicochemical properties, fertility, and other qualities [1–6], and the results have indicated that over-fertilization has led to rapid accumulation of nutrients, acidification, and salinity of soils, thus degrading soil quality [1,2]. However, little attention has been paid to soil toxicity, such as genotoxicity induced by the intensive applications of pesticides in greenhouses. The greenhouse environment is favorable for the growth of crops as well as of pests and diseases [7–9]. To protect crops against phytopathogens or pests in the greenhouse, pesticides (especially fungicides) are frequently applied every 7 d to 10 d. Pesticide dissipation on crops and inside the soil might be retarded by a reduction of airflow and rainfall, enclosed construction (limited volatilization and leaching), and the filtration of sunlight by a cover layer for photodegradation in greenhouse. This has been confirmed by numerous investigations performed in greenhouses with various types of crops and soils [10–13]. Therefore, pesticide accumulation in soil under greenhouse conditions probably occurs rapidly and thus inducing soil toxicity. In the present study, chlorothalonil (CT) was successively applied onto a pakchoi-soil system, as is usual in greenhouses. The accumulation of the fungicide and its main metabolite, hydroxy-chlorothalonil (HCT), in soil, and their genotoxicity, evaluated by the micronucleus of Vicia faba, were monitored. The aim of the present study was to assess the

MATERIALS AND METHODS

Chemicals

Chlorothalonil (99.5%) was purchased from Dr. Ehrenstorfer, GmbH. Augsburg, Germany. Hydroxy-chlorothalonil (99.5%) was provided by X. Ou (National Engineering Research Center for Agrichemicals, Hunan, P. R. China). Methanol was high-pressure liquid chromatography gradient grade, and all other chemicals and solvents used in the present study were of analytical grade. The V. faba seeds were provided by the Department of Biology, Central China Normal University, Wuhan, P. R. China. Field experiments

Experiments were conducted in a 6.3 m 30 m plasticcovered greenhouse located in Jiaxing Academy of Agricultural Sciences, Jiaxing, Zhejiang, China. The soil was classified as heavy loam, and its properties were as follows: sand, 7.6%; silt, 69.3%; clay, 23.1%; organic matter, 5.02%; cationic exchange capacity, 21.3 cmol/kg; and pH, 6.52. The experiment was implemented from 27 November 2011, with pakchoi var. Su Zhouqing. A random block scheme was used, plots of 1.2 m 3 m each were designed for test treatments, and all experiments were performed in triplicate. Chlorothalonil (75% wettable powder [WP], Syngenta Biotechnology) was separately sprayed at single and double dosages (3000 g/ha and 6000 g/ha) every 12 d. Soil samples (1 kg) were collected 12 d after each application. The collected samples were air-dried and passed through a 2-mm sieve to remove plant roots, stones, and other particles, and thereafter used for the extraction of residual CT and the micronucleus test.

* Address correspondence to [email protected]. Published online 29 January 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/etc.2538 1043

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Environ Toxicol Chem 33, 2014

Analysis of residual chlorothalonil in soil

To extract residual CT in soil, a sample (10 g dry wt) was placed in a 150-mL Erlenmeyer’s flask and mixed with 40 mL n-hexane–dichloromethane (1:1, v/v). After ultrasonication for 20 min, the supernatant was decanted, filtered, and collected in a flat-bottom flask through anhydrous sodium sulfate. The residue was extracted again with the procedure described previously. The collected filtrate was concentrated on a rotary evaporator at 45 8C, dried under nitrogen flow, and finally dissolved in 10 mL n-hexane. Determination of CT was performed by an Agilent 6890 N gas chromatograph (Agilent Technologies) equipped with an electron capture detector, and an HP-5 capillary column (30 m 0.32 mm 0.25 mm, Agilent Technologies) was used for the separation of CT. Injector and detector temperatures were 250 8C and 300 8C, respectively, and nitrogen was used as the carrier gas at a constant flow of 3.3 mL/min. The oven was used with a temperature program of 80 8C for 1 min increased to 260 8C at 25 8C/min and held at 260 8C for 3.8 min. A splitless volume of 1 mL was injected. Analysis of HCT in soil

To extract HCT from soil, a sample (20 g dry wt) was added into a 150-mL Erlenmeyer’s flask and mixed with 50 mL acetone. After ultrasonication for 30 min, the supernatant was decanted onto a Buchner funnel and collected in a flat-bottom flask. The residue was rinsed 3 times with 60 mL acetone. The filtrates were combined and concentrated on a rotary evaporator at 50 8C, dried under nitrogen flow, and finally dissolved in 10 mL methanol. Determination of HCT was carried out with high-pressure liquid chromatography (Agilent Technologies 1200) equipped with a diode array detector. The column used was a Hewlett Packard stainless steel analytical column (Eclipse XDB-C18, 15 cm 4.6 mm 5 mm). The extraction (20 mL) was injected into the high-pressure liquid chromatography system, separated with a mobile phase of methanol and 0.1% phosphoric acid (55:45, v/v) at a constant flow of 1.2 mL/min, and recorded at 242 nm. Micronucleus test

A soil sample (10.0 g dry wt) was mixed with 100 mL deionized water in a 150-mL Erlenmeyer’s flask, and then horizontally shaken in the dark at 150 rpm for 2 h. After resting for 30 min, the supernatant was carefully collected for the micronucleus test. A soil sample taken from a field near the experimental field was used as a control. The micronucleus test was conducted according to Cotelle et al. [14]. Dry V. faba seeds were soaked in deionized water for 24 h and then allowed to germinate on moistened cotton for 3 d in the dark (25 8C). Seeds with primary roots of approximately 2 cm to 3 cm were chosen and suspended in the prepared soil solution for 5 h followed by recovery for a 24-h period on moistened cotton. Root tips were rinsed with distilled water and cut off at approximately 1 cm to 2 cm, fixed in Carnoy’s solution (glacial acetic acid: ethanol, 1:3) at 25 8C for 24 h, and rinsed again with distilled water. The treated tips were hydrolyzed with 5 N HCl at 25 8C for 25 min, Feulgen stained for 2 h at the same temperature in the dark, and squashed on a slide in 45% acetic acid. The interphase cells were scored for micronucleus frequencies at 1000 magnification. Six slides were prepared for each treatment, and 1000 cells (at least) were counted per seedling. The micronucleus frequency was expressed in terms of

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the number of cells with micronucleus per 1000 scored cells, resulting from at least 18 000 cells per treatment. Data analysis

All data are presented by the means of triplicate samples for 3 independently performed experiments as means standard deviation. Statistical significance was determined using analysis of variance and t test. A p value of 0.05 or less was considered significant. The correlation of the residue and micronucleus was proved using Origin 8.0 and its accessory logistic equation. RESULTS

Validation of CT and HCT extraction

The average recoveries of CT and HCT from soil are presented in Table 1. The recoveries of CT from soil ranged from 73.0% to 99.2%, with a relative standard deviation less than 3.7%. The recovery of HCT was from 93.3% to 105.8%, with the relative standard deviation less than 3.8%. The detection limits for CT and HCT in soil were 0.01 mg/kg and 0.05 mg/kg, respectively. These data demonstrated that the extraction methods are satisfactory for the extraction of CT and HCT in soil. CT accumulation in soil

Residual CT in soil measured 12 d after each application at single and double dosages are shown in Figure 1. The initial deposit concentrations of CT in soil measured 2 h after its first application at single and double dosages were 2.76 0.23 mg/ kg and 4.29 0.17 mg/kg, respectively. In a single dosage, CT concentration in soil collected 12 d after the first spray was estimated as 0.48 mg/kg. A slight reduction in the concentration appeared in the soil after the second spray. However, it increased gradually to 0.89 mg/kg and 2.08 mg/kg after the third and fourth sprays, respectively. Then it varied approximately 1.65 mg/kg after the fifth to 1.92 mg/kg after the sixth spray. The accumulation factor, expressed as CT concentration in collected soil when the sixth spray concentration was divided by the concentration of the first spray, was 4.0. In the case of double dosage, the variation in CT concentration with the application number was similar to what was observed in a single dosage with an accumulation factor of 6.2. The accumulation seemed to be related to the application rate. HCT accumulation in soil

The variation pattern of HCT concentration in soil with the applications of CT at single and double dosages is given in Figure 2. Approximately 0.085 mg/kg HCT was found in soil collected 12 d after the first introduction of CT at the recommended dosage. No significant difference in HCT concentration was observed after the second and third applications of CT. Subsequently, the concentration increased Table 1. Recoveries of chlorothalonil and hydroxylchlorothalonil amended in soil Pesticide Chlorothalonil Hydroxyl-chlorothalonil

Amendment level (mg/kg)

Mean recovery standard deviation (%)

10.0 1.0 0.1 5.0 1.0 0.05

99.2 3.67 80.4 2.25 73.0 1.90 105.8 4.02 93.3 2.33 96.8 0.58

7 6 5 4 3 2 1 0

1 dose 2 dose

1st 2nd 3rd 4th 5th 6th Application times

to 0.14 mg/kg after the fourth application and remained at similar levels, ranging from 0.18 mg/kg at the fifth to 0.15 mg/kg at the sixth spray. The accumulation factor of HCT was estimated as 1.8. For the application of chlorothalonil at double dosage, the accumulation occurred after the second spray. More visible accumulation appeared in this case, with an accumulation factor of 3.5. Soil genotoxicity

The genotoxicity of soil solutions, extracted from the collected samples after CT applications, was estimated by the V. faba micronucleus test and is presented in Figure 3. At recommended dosage, micronucleus frequency was increased gradually with the introduction of CT to 4.04 at the first and 5.92 at the fifth application. More marked genotoxicity was observed when double the recommended dosage was applied. The micronucleus frequency increased rapidly with CT application frequency. Compared with the control, the micronucleus frequency was enhanced by 1.54, 2.08, 2.56, and 2.85 times after the first, second, third, and fourth introductions of CT, respectively. Afterward, with the last two introductions of CT, the micronucleus frequency remained at approximately 7.81 to 8.66.

Hydroxychlorothalonil in soil (mg/kg)

0.4 1 dose 2 dose

0.2 0.1 0.0

1st

2nd 3rd 4th

11 10 9 8 7 6 5 4 3 2 1

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1 dose 2 dose

Control 1st

2nd 3rd 4th

5th 6th

Application times

Figure 1. Chlorothalonil in soil samples collected 12 d after each spray. Error bars represent the means standard error with the same case.

0.3


Micronucleus frequency (‰)

Chlorothalonilin soil (mg/kg)

Genotoxicity of repeated chlorothalonil applications in greenhouse

5th

6th

Application times Figure 2. Hydroxy-chlorothalonil in soil samples collected 12 d after each spray. Error bars represent the means standard error with the same case.

Figure 3. Micronucleus frequency of Vicia faba treated with soil solution extracted from the samples collected 12 d after each spray. Error bars represent the means standard error with the same case.

To find the relationship between soil genotoxicity and CT, HCT, or their combination, a logistic equation was adopted for the statistical analysis. In the case of recommended dosage, soil genotoxicity varied with CT, HCT, and CT þ HCT with determination coefficients (r2) of 0.508, –0.47, and 0.508, respectively. More significant relations (or correlations) were found at double the recommended dosage, with r2 ¼ 0.978, 0.942, and 0.976, respectively. The data demonstrated that genotoxicity of CT-contaminated soil is mainly caused by CT itself rather than its metabolite HCT. DISCUSSION

Dissipation and accumulation of CT in soil

The fungicide CT has been considered a nonpersistent compound in the soil environment, with half-lives of 1 d to 6 mo under different conditions [15–17]. The application of CT in the field will not lead to its accumulation in soil. However, the accumulation of CT resulting from its repeated applications under greenhouse conditions was observed in the present study, and our findings suggested that the observed accumulation might have originated from the inhibited degradation resulting from its successive applications and slow dissipation rate in the greenhouse environment. This was in agreement with previous studies [17–19] in which Singh et al. [18] and Motonaga et al. [20] indicated that the degradation rate of CT in the soil was reduced by its repeated applications. Wu et al. [17] also stated that the accumulation of CT in soil with the decline in dissipation rate resulted from its second and third introductions. Moreover, other limiting factors such as reduced photodegradation, leaching, surface runoff, and evaporation of the fungicide in the greenhouse decreased the dissipation of CT in soil [13]. Hydroxy-chlorothalonil is the main metabolite of CT degradation in soil [21–23]. This compound is expected to be more persistent in soil and more toxic than its parent compound [15,21]. Suppression of CT degradation by HCT was found in previous studies [15,21,24]. A sharp increase in HCT concentration occurred in the soil sample 12 d after the third spray at the recommended dosage. With the appearance of HCT accumulation in soil after the third spray, a significant increase in CT concentration was subsequently observed in soil after the fourth spray. Dissipation of CT and HCT in soil is

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mainly attributable to biodegradation by microorganisms [22]. When microorganisms acclimate to the compounds and acquire the ability to metabolize them, dissipation of CT and HCT will be enhanced. However, it was not observed throughout the experiment in the present study, and no significant reduction in the concentrations of CT and HCT was found in soil samples after the latter applications. These data imply that repeated applications of CT in greenhouses probably result in accumulation of CT and its metabolites in soil and thus have a direct or indirect lasting effect on soil quality. Soil genotoxicity induced by successive applications of CT

Chlorothalonil has been deemed a carcinogenic compound to humans by the International Agency for Research on Cancer, and it has been placed in category 3 (i.e., sufficient available data to evaluate carcinogenicity) [25,26]. The data obtained in the present study showed mutagenic activity of the soil solutions extracted from the samples after the application of CT. The micronucleus frequency estimated with V. faba was positively related to CT (Figure 1), HCT (Figure 2), and their combination (Figure 4). In agreement with this observation, Dearfield et al. [27] indicated that both sister-chromatid exchanges and chromosomal aberrations in Chinese hamster ovary cells were induced by CT treatment. Lebailly et al. [28] found dosedependent DNA damage in human peripheral blood lymphocytes caused by CT. The micronucleus of the V. faba root tip is an appropriate indicator to monitor genotoxic potential of environmental pollutants and has been used widely as an indicator for soil genotoxicity [29–31]. The data provided herein indicated that soil genotoxicity was induced by accumulation of CT, and HCT resulted from its repeated applications. The induction of micronucleus of V. faba seems more sensitive than classical endpoints (e.g., germination rate or time, root elongation), and it provides a predictive biomarker of long-term deleterious effects in plants [32]. Based on our results, one could conclude that genotoxicity increased by repeated applications of pesticides might be an important factor in declining soil quality. Agricultural and environmental implications

Conditions in greenhouses are favorable for the development of arthropod pests and plant diseases. Excessive applications of pesticides are often required to ensure crop yield under greenhouse conditions. Findings of this study revealed clearly that successive applications of CT under greenhouse conditions

Figure 4. Sum of chlorothalonil (CT) and hydroxy-chlorothalonil (HCT) in soil samples collected 12 d after each spray. Error bars represent the standard error of the sum of the 2 compounds with the same case.

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could lead to the accumulation of this fungicide and its metabolite (HCT) in soil and then induce soil genotoxicity. This implies that with the intensive applications of different types of pesticides one after another in a greenhouse, parent compounds and their metabolites may accumulate in greenhouse soils as combined contamination (cocktails) and thus become a threat not only to soil quality but also to groundwater and surface water because the covers are usually removed from greenhouses in summer, coinciding with the rainy season. Acknowledgment—The present study was supported by the National High Technology R&D Program of China (2013AA102804 & 2012AA06A204), Zhejiang Provinicial Natural Science Foundation (LZ13D010001), the National Natural Science Foundation of China (21177111), and National Key Technology R & D Program of China (NC2010BF0006). REFERENCES 1. Shi WM, Yao J, Yan F. 2009. Vegetable cultivation under greenhouse conditions leads to rapid accumulation of nutrients, acidification and salinity of soils, and groundwater contamination in South-Eastern China. Nutr Cycl Agroecosys 83:73–84. 2. Moritsuka N, Nishikawa T, Yamamoto S, Matsui N, Inoue H, Li KZ, Inamura T. 2013. Changes in soil physicochemical properties following land use change from paddy fields to greenhouse and upland fields in the southeastern basin of Dianchi Lake, Yunnan Province, China. Pedosphere 23:169–176. 3. Malhi SS, Lemke R, Wang ZH, Chhabra BS. 2006. Tillage, nitrogen and crop residue effects on crop yield, nutrient uptake, soil quality, and greenhouse gas emissions. Soil Till Res 90:171–183. 4. Ju XT, Kou CL, Christie P, Dou ZX, Zhang FS. 2007. Changes in the soil environment from excessive application of fertilizers and manures to two contrasting intensive cropping systems on the North China Plain. Environ Pollut 145:497–506. 5. Lou Y, Xu M, Wang W, Sun X, Liang C. 2011. Soil organic carbon fractions and management index after 20 yr of manure and fertilizer application for greenhouse vegetables. Soil Use Manage 27: 163–169. 6. Ge T, Nie S, Wu J, Shen J, Xiao H, Tong C, Huang D, Hong Y, Iwasaki K. 2011. Chemical properties, microbial biomass, and activity differ between soils of organic and conventional horticultural systems under greenhouse and open field management: A case study. J Soils Sediments 11:25–36. 7. Van Lenteren JC. 2000. A greenhouse without pesticides: Fact or fantasy. Crop Protection 19:375–384. 8. Rial-Otero R, Arias-Estevez M, López-Periago E, Cancho-Grande B, Simal-Gándara J. 2005. Variation in concentrations of the fungicides tebuconazole and dichlofluanid following successive applications to greenhouse-grown lettuces. J Agric Food Chem 53:4471–4475. 9. Katsoulas N, Boulard T, Tsiropoulos N, Bartzanas T, Kittas C. 2012. Experimental and modeling analysis of pesticide fate from greenhouses: The case of pyrimethanil on a tomato crop. Biosys Eng 113:195– 206. 10. Da Silva JP, Da Silva AM. 1998. Comparative study of the dissipation of triadimefon in greenhouse and field conditions. Toxicol Environ Chem 66:229–236. 11. Hatzilazarou SP, Charizopoulos ET, Papadopoulou-Mourkidou E, Economou AS. 2004. Dissipation of three organochlorine and four pyrethroid pesticides sprayed in a greenhouse environment during hydroponic cultivation of gerbera. Pest Manag Sci 60:1197–1204. 12. Omirou M, Vryzas Z, Papadopoulou-Mourkidou E, Economou A. 2009. Dissipation rates of iprodione and thiacloprid during tomato production in greenhouse. Food Chem 116:499–504. 13. Wu CW, Sun JQ, Zhang AP, Liu WP. 2013. Dissipation and enantioselective degradation of plant growth retardants paclobutrazol and uniconazole in open field, greenhouse, and laboratory soils. Environ Sci Technol 47:843–849. 14. Cotelle SJ, Masfaraud FJ, Ferard F. 1999. Assessment of the genotoxicity of contaminated soil with the Allium/Vicia-micronucleus and the Tradescantia-micronucleus assays. Mutat Res 426:167–171. 15. Potter TL, Wauchope RD, Clubreath AK. 2001. Accumulation and decay of chlorothalonil and selected metabolites in surface soil following foliar application to peanuts. Environ Sci Technol 35:2634–2639. 16. Chaves A, Shea D, Cope WG. 2007. Environmental fate of chlorothalonil in a Costa Rican banana plantation. Chemosphere 69:1166–1174.

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