Chemosphere 111 (2014) 209–217

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Degradation of kresoxim-methyl in soil: Impact of varying moisture, organic matter, soil sterilization, soil type, light and atmospheric CO2 level Ashish Khandelwal a, Suman Gupta a,⇑, Vijay T. Gajbhiye a, Eldho Varghese b a b

Division of Agricultural Chemicals, Indian Agricultural Research Institute, New Delhi 110012, India Indian Agricultural Statistics Research Institute, New Delhi 110012, India

h i g h l i g h t s  Kresoxim-methyl rapidly forms acid metabolite in soil.  Total residues (kresoxim methyl + metabolite) dissipated with T1/2 of 3.9–56.8 d.  Degradation faster under anaerobic condition compared to aerobic condition.  Increased degradation with light exposure, sludge amendment and elevated CO2 level.  Degradation faster in Inceptisol compared to Ultisol.

a r t i c l e

i n f o

Article history: Received 29 October 2013 Received in revised form 30 January 2014 Accepted 7 March 2014

Handling Editor: I. Cousins Keywords: Kresoxim-methyl Soil dissipation Moisture Organic matter Light Sterilization and CO2 level

a b s t r a c t In the present investigation, persistence of kresoxim-methyl (a broad spectrum strobilurin fungicide) was studied in two different soil types of India namely Inceptisol and Ultisol. Results revealed that kresoximmethyl readily form acid metabolite in soil. Therefore, residues of kresoxim-methyl were quantified on the basis of parent molecule alone and sum total of kresoxim-methyl and its acid metabolite. Among the two soil types, kresoxim-methyl and total residues dissipated at a faster rate in Inceptisol (T1/2 0.9 and 33.8 d) than in Ultisol (T1/2 1.5 and 43.6 d). Faster dissipation of kresoxim-methyl and total residues was observed in submerged soil conditions (T1/2 0.5 and 5.2 d) followed by field capacity (T1/2 0.9 and 33.8 d) and air dry (T1/2 2.3 and 51.0 d) conditions. Residues also dissipated faster in 5% sludge amended soil (T1/2 0.7 and 21.1 d) and on Xenon-light exposure (T1/2 0.5 and 8.0 d). Total residues of kresoximmethyl dissipated at a faster rate under elevated CO2 condition (550 lL L 1) than ambient condition (385 lL L 1). The study suggests that kresoxim-methyl alone has low persistence in soil. Because of the slow dissipation of acid metabolite, the total residues (kresoxim-methyl + acid metabolite) persist for a longer period in soil. Statistical analysis using SAS 9.3 software and Duncan’s Multiple Range Test (DMRT) revealed the significant effect of moisture regime, organic matter, microbial population, soil type, light exposure and atmospheric CO2 level on the dissipation of kresoxim-methyl from soil (at 95% confidence level p < 0.0001). Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Soil act as a sink for pesticides used in agriculture as a considerable proportion of the foliar applied pesticide falls on soil. In soil, it is acted upon by various biotic and abiotic factors and undergoes degradation forming number of metabolites. From soil, pesticides ⇑ Corresponding author. Tel.: +91 11 25841390; fax: +91 11 25843272. E-mail addresses: [email protected] (A. Khandelwal), drsumangupta2002 @yahoo.com (S. Gupta), [email protected] (V.T. Gajbhiye), [email protected] com (E. Varghese). http://dx.doi.org/10.1016/j.chemosphere.2014.03.044 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.

and their metabolites move to other environmental compartment like water and air as a result of leaching, run off and volatilization. Physico-chemical properties of the pesticides, soil type and other environmental conditions like temperature, light, moisture, etc. are some of the factors which strongly influence the fate of these xenobiotics in the environment. Kresoxim-methyl {methyl (E)-2-methoxyimino-2-[2-(o-tolyloxymethyl) phenyl] acetate} (Fig. 1a) is a broad spectrum foliar fungicide of the strobilurin group. It is a systemic compound for use against a wide range of plant pathogenic fungi on several crops

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CH3

CH3

O

O

H3CO

HO

OCH3

OCH3 N

N O

O

(a)

(b)

Fig. 1. Chemical structures of (a) kresoxim-methyl and (b) acid metabolite of kresoxim-methyl.

(EFSA, 2013; Navalon et al., 2002). It is found highly effective against botrytis blight, late and early blight, turcicum leaf blight, powdery and downy mildew, cercospora leaf spot, neck blast, fusarium wilt and rust in gladiolus, sunflower, sugar beet, potato, peach, paddy, wheat, maize, etc. (Chakraborty and Roy, 2012; Karadimos and Karaoglanidis, 2006; Kumbhar et al., 2012; Nenad et al., 2010; Pszczolkowska et al., 2013; Singh et al., 2011; Sudisha et al., 2010; Sunder et al., 2010; Yang et al., 2011). This compound is absorbed through the roots or through leaf surfaces and binds to quinol oxidation (Qo) site (or ubiquinol site) of cytochrome b in mitochondria. Kresoxim methyl was first discovered in 1983 by BASF AG. It was the first synthetic analogue of strobilurin A and was synthesized by substituting the methoxyacrylate group in natural strobilurin A with methoxyiminoacetate group (Balba, 2007). It was introduced in India by Rallis India Ltd as Ergon 44.3 SCÒ and is presently registered for control of blast and sheath blight in paddy and downy and powdery mildew in grape at recommended doses of 250–350 g a.i./ha (CIBRC, 2013). Kresoxim-methyl is considered a reduced risk fungicide with low mammalian toxicity and a benign profile for avians as compared to that of conventional fungicide, except some aquatic (Wang and Pan, 2005). Environmental fate data on kresoxim-methyl is generated by various regulatory agencies. As per European Food Safety Authority (EFSA) report kresoxim-methyl exhibited low persistence in laboratory incubations studies. In soil, the DT50-values for kresoxim-methyl were reported to be less than 1 day in various field trials, whereas under laboratory conditions, at 20 °C and 40% water holding capacity, DT50-values were in the range of 0.5–3.1 d. Literature search also revealed that in soil kresoxim-methyl readily dissipate into acid metabolite {(E)-2-methoxyimino-2-[2-(o-tolyloxymethyl) phenyl] acetic acid} (Fig. 1b). Reported DT50-values for acid metabolite in different soils under laboratory and field conditions varied from 22.8–85.7 d and 2.9–37.4 d respectively. The residue definition of kresoxim-methyl also includes the residues of acid metabolite (BF 490-1) for risk assessment purpose of processed commodities of plant origin and food of animal origin (ruminant matrices, milk). Acid metabolite was also taken into account for monitoring of commodities of plant and animal origin, soil and water. The metabolite was found to be more toxic than the parent compound in the acute oral rat study (EFSA, 2010). With the GUS scoring of >2.8, acid metabolite is classified as ‘probable leacher’ (APVMA, 2000). In the present study, dissipation behavior of kresoxim-methyl in Inceptisol and Ultisol soils of India was investigated. Effect of soil moisture level, organic matter, microbial population, light exposure and carbon dioxide concentration on dissipation of kresoxim-methyl from Inceptisol was also studied. Keeping in mind the toxicological importance of acid metabolite, in the present study, quantification has been done on the basis of residues of kresoxim methyl alone and the total residues (sum of parent + acid metabolite).

2. Materials and methods 2.1. Soil Soils used in the study were collected from two different agro-climatic zones of India namely Indian Agricultural Research Institute, Delhi (Inceptisol) and Thrissur district of Kerala (Ultisol). Surface soil from plough layer (0–15 cm depth) was collected from the cultivated fields having no previous history of kresoximmethyl application. The soils were air-dried by spreading on aluminium sheet and allowing the moisture to evaporate under natural room conditions. It was then ground and sieved through 2 mm mesh screen. To increase the organic carbon content of the Delhi soil, it was amended with sludge at 5% level. Sludge which was used in the study was collected from the water treatment plant, Keshopur, Delhi. It was ground and sieved through 2 mm mesh screen before mixing with the soil. To get 1 kg of 5% amended soil, 950 g of Delhi soil was mixed thoroughly with 50 g of grinded sludge. The physico-chemical properties of all the three test soils namely Inceptsol, Ultisol, sludge amended Inceptisol and sludge were determined using standard procedures described by Singh et al. (2005) and are listed in Table 1. 2.2. Reagents Analytical grade kresoxim-methyl (purity >98.4%) and its commercial formulation Ergon 44.3 SCÒ were supplied by the Rallis India Ltd., Mumbai. Laboratory grade dichloromethane and hexane were purified by distilling at their boiling points of 40 and 68 °C, respectively. Acetone was refluxed with KMnO4 for 2 h prior to distillation at 56 °C. HPLC grade water was taken from Millipore water purification unit (Milli-Q, Model Academic). It was filtered through 0.22 lm filter and sonicated before use. Sodium sulphate and sodium chloride were washed with acetone, air-dried and then

Table 1 Physico-chemical properties of test soils. Location

Delhi

Kerala

Sludge amended Delhi soil

Sludge

Order Texture

Inceptisol Sandy loam 8.15 0.37 0.23

Ultisol Sandy clay loam 4.9 1.23 0.09

Inceptisol –



7.9 2.46 0.34

6.25 47.2 16.0

46.2 19.6 34.2

– – –

– – –

pH Organic Carbon (%) EC (dS m 1)

Particle size distribution (%) Sand (%) 54.4 Silt (%) 23.3 Clay (%) 22.3 – Not determined.

A. Khandelwal et al. / Chemosphere 111 (2014) 209–217

heated at 300 °C for 4 h. These were cooled and stored in desiccators. Adsorbents like Silica gel (60–120 mesh) and Florisil™ were activated by heating at 110 °C for 4 h in oven. Other solvents and reagents like HPLC grade acetonitrile, methanol, buffer tablets (analytical grade), etc. were purchased from Merck India Ltd. Kresoximmethyl acid metabolite (Fig. 1b) was prepared in the laboratory by hydrolysis of kresoxim-methyl with ethanolic KOH solution. For hydrolysis kresoxim-methyl (2 g) was taken in round bottom flask and 10% ethanolic sodium hydroxide aqueous solution (30 mL) was added to it. The mixture was refluxed for 30 min using air condenser. The aqueous solution was made acidic (pH 5) by adding dilute HCl drop wise and then extracted with dichloromethane (3  30 mL). The organic phase was passed through anhydrous Na2SO4 and concentrated using rotary evaporator. Crude acid metabolite (pale yellow solid) was re-crystallized using acetone:hexane mixture. After recrystallization 0.8 g of acid metabolite was obtained. The purity of the metabolite was checked using thin layer chromatography (TLC) which showed single spots at RF 0.85 and 0.53 for kresoxim-methyl and acid metabolite respectively in 15% methanol:chloroform developing solvent. Observed melting points were 101.3–102.7 °C for kresoxim-methyl and 136.0– 136.7 °C for acid metabolite. On the basis of HPLC profile, the purity of the prepared metabolite was assigned as 95.6%. The structure of acid metabolite was elucidated using various spectroscopic techniques. Following characteristic peaks were observed for acid metabolite: IR 3454 cm 1 (OAH stretch), 1699 cm 1 ([email protected] stretch); 1 H NMR (DMSO, 400 MHz): d 2.13 (s, 3H, ArACH3), 3.88 (s, 3H, NOCH3), 4.89 (s, 2H, OCH2), 6.80 (d, 1H, J = 8.4 Hz), 6.87 (m, 1H, Harom), 7.11 (m, 2H, Harom), 7.21 (dd, 1H, J1 = 7.6 Hz, J2 = 1.2 Hz, Harom), 7.38–7.45 (m, 2H, Harom), 7.54 (d, 1H, J = 7.6 Hz, Harom); 13C NMR (DMSO, 400 MHz): d 16.19 (ArACH3), 63.5 (NOCH3), 68.3 (OCH2), 111.5, 120.9, 126.4, 127.3, 128.2, 129.1, 129.6, 130.8, 135.3 (all Carom), 156.5, 150.2 ([email protected]), 164.2 ([email protected]) and LC/MS/MS (electron spray, positive mode): 299.70 (molecular ion peak). 2.3. Preparation of stock solution Kresoxim-methyl/acid metabolite (50 mg) was weighed accurately and transferred into a 50 mL capacity volumetric flask. The solid was first dissolved in 5 mL acetonitrile and then the volume was made up to the mark with additional acetonitrile. This gave a stock solution of 1000 lg mL 1. One milliliter each from stock solutions (1000 lg mL 1) of kresoxim-methyl and acid metabolite were taken in 10 mL volumetric flask. The flask was filled up to the mark with acetonitrile to get 100 lg mL 1 standard mixture with respect to kresoxim-methyl and acid metabolite. Working standard solutions of lower concentrations were prepared from the stock solution by serial dilutions. 2.4. HPLC analysis method Kresoxim-methyl and its acid metabolites were simultaneously quantified by using Shimadzu Ultra High Performance Liquid Chromatograph (UHPLC, Nexera™) equipped with PhenomenaxÒ RP-18 column (250 X 4.60 mm, 5 lm) and Photo Diode Array (PDA) detector set at 210 nm. HPLC grade solvents (water and acetonitrile) were filtered through 0.22 lm filter and degassed before use. Mixture of acetonitrile and water (80:20 v/v) was used as a mobile phase with a flow rate of 1 mL min-1. The injection volume was 10 lL. Under the standardized conditions, kresoxim-methyl and its metabolite were eluted at 4.52 and 1.83 min respectively. The calibration curve was linear over a range of 0.001 to 10 lg mL 1 with R2 value of 0.99 for both the compounds. Instrument limit of detection (LOD) of kresoxim-methyl was found to be 0.1 ng (0.01 lg mL 1 with 10 lL injection volume) and of its metabolite was 0.05 ng (0.005 lg mL 1 with 10 lL injection volume). Recovery

211

studies were conducted from soil matrix at 1.0 and 0.01 lg g 1 fortification levels. The extraction and clean up method was standardized for best recovery of both the compounds from soil matrix. It was observed that extraction of samples with 20% aqueous acetone using dipping and shaking method, followed by acidification of aqueous phase and then partitioning with dichloromethane gave best recoveries. At 1 lg g 1 fortification level, average recovery of 91.5 ± 2.6% and 84.4 ± 0.9% was obtained for kresoxim-methyl and acid metabolite, whereas at 0.01 lg g 1 level, mean recovery of 86.4 ± 0.9% and 82.7 ± 1.4% was obtained for kresoxim-methyl and acid metabolite respectively. Limit of quantification (LOQ) was 0.01 lg g 1 for both the compounds in soil matrix (Sample size 20 g, final dilution 2 mL). Control sample of soil did not give any interfering peaks under the standardized parameters (Fig. 2).

2.5. Experimental setup In all the treatments, soil fortification has been done on the basis of the dry weight of soil. The effect of moisture regimes on dissipation of kresoxim-methyl was studied under air-dry, field capacity and submerged conditions in Delhi soil (Inceptisol) at 1 lg g 1 fortification level. The treated soil (20 g) along with untreated control samples were transferred to beakers. For treatments under field capacity moisture regime, the calculated amount of water was added to bring the soil to field capacity moisture level, while in case of submerged conditions enough water was added to raise the level of water to about 3 cm above the soil surface. No water was added in air-dry treatments. The effect of organic matter on dissipation of kresoxim-methyl was studied in 5% sludge amended Inceptisol under field capacity conditions at 1 lg g 1 fortification level. The treated soil (20 g) along with untreated control samples were transferred to beakers and then brought to field capacity moisture level. Effect of microorganisms on persistence of kresoxim-methyl was studied in sterilized Delhi soil. Delhi soil was sterilized using autoclave. The soil was taken in conical flask. The mouth of the flask was plugged with cotton ball. The soil was subjected to high pressure saturated steam at 121 °C, 1.035  105 Pa pressure for about 1 h. The process of soil sterilization was repeated for three consecutive days. The sterilized soil was fortified at 1 lg g 1 fortification level by treating with kresoxim-methyl standard solution in laminar air flow chamber. The treated soil (20 g) along with untreated control sample were transferred to the tubes and then brought to field capacity moisture level using double distilled water. The mouth of the tubes was closed with cotton plugs. All operations involving sterile soil were carried out under aseptic conditions in laminar air flow chamber to avoid any microbial contamination. Dissipation of kresoxim-methyl in sterile soil was compared with its dissipation in non-sterile soil incubated at the same temperature and moisture levels. The effect of soil type on persistence of kresoxim-methyl was studied in Delhi (Inceptisol) and Kerala (Ultisol) soil at 1 lg g 1 fortification level. The treated soil (20 g) along with untreated control samples were transferred to beakers and then brought to field capacity moisture level (20% for Inceptisol and 24% for the Ultisol). Effect of light on dissipation of kresoxim-methyl was studied in Delhi soil at 1 lg g 1 fortification level by exposing the samples to Xenon-light. The treated soil (20 g) along with untreated control sample were transferred to beakers and then brought to field capacity moisture level. The samples (20 g treated soil), along with untreated control samples, were exposed for 6 h d 1 to Xenon-light (wavelength > 400 nm, 35 W, 10 A, 12 V) fitted in BOD incubator which was maintained at 25 ± 1 °C temperature and about 70% relative humidity. Quantum flux of the Xenon-light source was recorded as 100 lmol m 2 s 1 with a point quantum sensor. One

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10

mAU

a

5 0 -5

Detector response (mAU)

0.0 10

1.0

2.0

3.0

4.0

5.0

6.0

7.0

min

1.0

2.0

3.0

4.0

5.0

6.0

7.0

min

1.0

2.0

3.0

4.0

5.0

6.0

7.0

min

mAU

b

5 0 -5 0.0 10

mAU

c

5 0 -5

0.0

Retention time (min) Fig. 2. HPLC chromatograms of (a) standard mixture of kresoxim-methyl (Rt 4.52 min) and acid metabolite (Rt 1.83 min), (b) untreated soil and (c) fortified soil.

set of sample was kept under dark conditions in incubator at above temperature and moisture condition. Effect of atmospheric CO2 on dissipation of kresoxim-methyl was studied at 1 lg g 1 fortification level in Delhi soil under field capacity moisture conditions in Open Top Chambers (OTC) installed at IARI, New Delhi. Fortified samples were kept in OTC maintained at ambient (385 ± 20 lL L 1) and elevated CO2 level (550 ± 15 lL L 1) throughout the study period. All the beakers of above treatments were weighed and placed in temperature and humidity controlled incubators maintained at 25 ± 1 °C temperature and 70% relative humidity along with untreated control samples unless stated otherwise. Constant weight of the beakers was maintained throughout the experiment by replenishing the lost water every alternate day. Samples in triplicate, i.e. three beakers per treatment were withdrawn along with control at different time intervals (0, 1, 3, 5, 10, 20, 30, 60, and 90 d after application) and processed. Residues were quantified on the basis of dry weight of the soil as per the standardized procedure.

2.6. Kinetic study and statistical analysis The data obtained from persistence experiment was subjected to first order kinetics. (oC/C = [email protected]), where ‘C’ is the concentration and ‘t’ is the time. The half-life of dissipation was calculated from the value of k by the formula (T1/2 = 0.693/k). Statistical analysis of the data was done along similar line as in Sebai et al. (2010). Two-way analysis of variance was performed on the data sets using SAS 9.3 software and significant effects (p < 0.05) were noted. Further, Duncan’s Multiple Range Test (DMRT) was done for pair-wise comparison of treatments and the effects which are significantly different were represented by different alphabets. The treatments which get same letter grouping are at par and the treatment pairs getting different letter grouping are significantly different. Least significant difference (lsd) or Critical Difference (C.D.) was also worked out as it gives a threshold value for pair-wise difference. Three C.D. have been

provided for comparing the treatment effects, day effects and their interaction. All other analyses were done using Microsoft Excel 2007. 3. Results 3.1. Degradation under different moisture condition Effect of moisture on dissipation of kresoxim-methyl was studied under air-dry, field capacity and submerged conditions in Inceptisol at 1 lg g 1 fortification level (Fig. 3a). Under air-dry condition, residues of the parent molecule, kresoxim-methyl, were detected up to 20 d. Percent dissipation of 43.6%, 77.7% and 94.7% was recorded for the parent molecule on 3, 5 and 10 d. The total residues (kresoxim-methyl + acid metabolite) persisted beyond 90 d with the mean initial deposits of 1.06 lg g 1. Residues declined with time and were 0.78, 0.57 and 0.28 lg g 1 on 10, 30 and 90 d. The mean half-life values for parent and total residue calculated from first order dissipation kinetics were 2.2 and 51 d (Table 2). Under field capacity moisture regime, parent molecule kresoxim-methyl dissipated beyond detectable level within 5 d. Initial deposits of 1.09 lg g 1 declined to 0.47 and 0.10 lg g 1 on 1 and 3 d amounting to the loss of 57.3% and 90.9%. Total residues of kresoxim-methyl (kresoximmethyl + acid metabolite) persisted beyond 90 d. Residues declined with time and were 0.69, 0.52 and 0.14 lg g 1 on 10, 30 and 90 d respectively. The half-life values of parent and total residues calculated from first order dissipation kinetics were 0.9 and 33.8 d (Table 2). Under submerged conditions, more than 98% dissipation of parent molecule was recorded on 3rd day. The total residues (kresoxim-methyl + acid metabolite) declined with time and were 0.62, 0.38 and 0.01 lg g 1 on 5, 10 and 30 d respectively. The halflife values of parent and total residues calculated from first order dissipation kinetics were 0.5 and 5.2 d (Table 2). 3.2. Degradation under different organic matter Effect of organic matter on dissipation of kresoxim-methyl was studied under field capacity condition in Inceptisol (Fig. 3b). In nor-

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Air dry Field capacity Submerged

1.2 1.0

(a)

(b)

Sludge amended soil 1.0

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2 0.0

0.0 0

20

40

60

Non sterile Sterile

1.2

Concentration (µg g-1)

Normal soil

1.2

80

0

100

(c)

20

1.0

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

0.0

60

80

Inceptisol Ultisol

1.2

1.0

40

100

(d)

0.0 0

20

40

60

Dark Xenon light

1.2

80

100

(e)

0

20

1.0

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

60

80

100

(f)

Ambient (385 µL L¯¹)

1.2

1.0

40

Elevated (550 µL L¯¹)

0.0

0.0 0

20

40

60

80

100

0

20

40

60

80

100

Days Fig. 3. Dissipation of total residues (kresoxim-methyl + acid metabolite) in soil under (a) moisture regime, (b) organic matter, (c) soil sterilization, (d) soil type, (e) light exposure and (f) atmospheric CO2 level.

mal soil and 5% sludge amended soil, residues of kresoxim-methyl were detected up to 5 and 3 d respectively whereas total residues persisted beyond 90 d. About 72.3% and 53.3% dissipation was recorded in sludge amended and normal soil in 30 d. The half-lives of kresoxim-methyl and total residues calculated from first order dissipation kinetics were 0.7 and 21.2 d in sludge amended soil and 0.9 and 33.8 d in normal soil (Table 2).

total residues persisted beyond 90 d. Initial deposits of 1.08 lg g 1 declined with time and were 0.81, 0.61 and 0.32 lg g 1 on 10, 30 and 90 d respectively. Only 70.7% of kresoxim-methyl residues were dissipated in 90 d compared to 87.5% dissipation recorded in non-sterilized soil. The half-life values of kresoxim-methyl and total residues calculated from first order dissipation kinetics were 2.7 and 56.7 d and 0.9 and 33.8 d in sterile and non-sterile soil (Table 2).

3.3. Degradation in sterile soil 3.4. Degradation under different soil type Effect of micro-organisms on dissipation of kresoxim-methyl was studied in sterilized Inceptisol at 1 lg g 1 fortification level under field capacity moisture condition (Fig. 3c). In sterile soil, the residues of kresoxim-methyl persisted only for 5 d whereas

Dissipation behavior of kresoxim-methyl was studied under Delhi (Inceptisol) and Kerala (Ultisol) soil at field capacity moisture condition (Fig. 3d). The residues of kresoxim-methyl were detected

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Table 2 Kinetic parameters of kresoxim-methyl and total residues under different moisture, soil type, organic matter, micro-organism, light and atmospheric CO2 level in soil. Treatment

Parameter Rate constant

R2

Regression equation

Kresoxim-methyl Moisture Dry Field capacity Submerged

0.304 0.347 1.393

0.99 0.99 0.99

y= y= y=

0.1324x + 0.0691 0.3446x + 0.0284 0.6048x 0.0732

2.2 0.9 0.5

Organic matter Normal Sludge amendment

0.347 0.934

0.99 0.99

y= y=

0.3446x + 0.0284 0.4059x + 0.0063

0.9 0.7

Micro-organism Sterile Non sterile

0.259 0.347

0.95 0.99

y= y=

0.1125x + 0.1197 0.3446x + 0.0284

2.7 0.9

Soil type Kerala (Ultisol) Delhi (Inceptisol)

0.460 0.347

0.89 0.99

y= y=

0.1999x + 0.1247 0.3446x + 0.0284

1.5 0.9

Light Dark Xenon-light

0.347 1.322

0.99 0.93

y= y=

0.3446x + 0.0284 0.5742x 0.1955

0.9 0.5

Atmospheric CO2 level (lL L 1) 385 550

3.489 3.645

1 1

y= y=

1.5152x + 0.0235 1.5828x + 0.0372

0.2 0.2

0.94 0.98 0.97

y= y= y=

0.0059x 0.0089x 0.0583x

0.0142 0.026 0.0478

51.0 33.8 5.2

Total residue (Kresoxim-methyl + acid metabolite) Moisture Dry 0.013 Field capacity 0.021 Submerged 0.134

Half life (days)

Organic matter Normal Sludge amendment Micro-organism Sterile Non sterile

0.021 0.032

0.98 0.99

y= y=

0.0089x 0.0142x

0.026 0.0584

33.8 21.2

0.012 0.021

0.94 0.98

y= y=

0.0053x 0.0089x

0.0089 0.026

56.8 33.8

Soil type Kerala (Ultisol) Delhi (Inceptisol)

0.015 0.021

0.97 0.98

y= y=

0.0069x 0.0089x

0.0298 0.026

43.6 33.8

Light Dark Xenon-light

0.021 0.087

0.98 0.98

y= y=

0.0089x 0.0378x

0.026 0.0503

33.8 8.0

Atmospheric CO2 level (lL L 1) 385 550

0.108 0.177

0.96 0.96

y= y=

0.0472x 0.0173 0.0768x + 0.0638

6.4 3.9

up to 3 and 5 d in Inceptisol and Ultisol soil. Percent dissipation of parent molecule was 57.3% and 90.9% in Inceptisol and 19.4% and 49.3% in Ultisol in 1 and 3 d respectively. Results revealed that in both Inceptisol and Ultisol total residues (kresoxim-methyl + acid metabolite) declined with time and were 0.69, 0.52, 0.31 and 0.14 lg g 1 and 0.70, 0.56, 0.40 and 0.22 lg g 1 on 10, 30, 60 and 90 d respectively. The half-life values of kresoxim-methyl and total residues calculated from first order dissipation kinetics were 0.9 and 33.8 d in Inceptisol and 1.5 and 43.6 d in Ultisol respectively suggesting faster dissipation in Inceptisol (Table 2). 3.5. Degradation under different light exposure Effect of light on dissipation of kresoxim-methyl was studied at 1 lg g 1 in Delhi soil under field capacity conditions (Fig. 3e). The total residues persisted for 30 d in Xenon-light treatment and beyond 90 d in dark conditions. Residue values were 0.49, 0.36 and 0.17 lg g 1 on 5, 10 and 20 d in light exposure treatment. The half-life values of kresoxim-methyl and total residues calculated from first order dissipation kinetics were 0.5 and 8.0 d in Xenon-light treatment and 0.9 and 33.8 d in dark treatment (Table 2).

3.6. Degradation under different level of atmospheric CO2 levels Effect of atmospheric CO2 level on dissipation of kresoximmethyl was studied at 1 lg g 1 in Delhi soil under field capacity conditions in open top chambers (Fig. 3f). Total residue values were 0.77, 0.57 and 0.06 lg g 1 on 1, 5 and 20 d under ambient CO2 condition, whereas under elevated atmospheric CO2 condition residues were 0.78, 0.51 and 0.03 on 1, 5 and 20 d respectively. In both the treatments, >90% dissipation of total residues were recorded in 20 d. Residues of kresoxim-methyl were not detected in soil after one day suggesting the rapid degradation of parent in open top chamber. The half-life value of kresoxim-methyl and total residues were 0.2 and 6.4 d at ambient CO2 level and 0.2 and 3.9 d under elevated CO2 level in open top chambers (Table 2). Replicated data obtained for different treatment were subjected to statistical analysis. The results of two way analysis of variance (ANOVA) performed for each of the parameters and significant difference observed for treatment effects, days and treatment  days interaction are presented in Table 3. The Critical Difference (C.D.) values for pair-wise comparison of treatments, days and treatment  days interactions and the treatment means along with Duncan’s letter grouping were also worked out (Table 3).

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A. Khandelwal et al. / Chemosphere 111 (2014) 209–217 Table 3 Results of (A) two-way ANOVA along with C.D. values and (B) treatment means with Duncan’s grouping. Parameters

EMS

F value

Critical Difference

A. Error Mean Square (EMS) and F values for different parameters along with C.D. values Moisture 0.000078 4570.70* Organic matter 0.000076 4237.33* Micro-organism 0.00012 1884.75* Soil type 0.00014 1684.80* Light 0.000078 5065.76* CO2 level 0.000081 5489.83* Parameters

Days

Treatment  Days

0.004843 0.004835 0.006179 0.006656 0.004887 0.006147

0.008388 0.010257 0.013107 0.01412 0.010368 0.010648

0.014528 0.014505 0.018536 0.019969 0.014662 0.015058

Treatment

Mean

Dry Field capacity Submerged

0.749362a 0.661506b 0.401154c

Organic matter

Normal Sludge amended

0.661506a 0.548510b

Micro-organism

Sterile Non sterile

0.773293a 0.661506b

Soil type

Ultisol Inceptisol

0.69822a 0.661506b

Light

Dark Xenon-light

0.661506a 0.396580b

CO2 level

Ambient Elevated

0.497074a 0.486800b

B. Treatment means with Duncan’s letter grouping Moisture

*

Treatment

All effects are significant at p < 0.0001; means with same superscript are not significantly different.

3.7. Degradation product formed in different conditions Under different treatments, kresoxim-methyl acid metabolite was detected as a major degradation product. In dry soil, degradation of kresoxim-methyl was slow as compared to field capacity and submerged conditions. Under air dry conditions, amount of acid metabolite formed was 0.02, 0.72 and 0.28 lg g 1 on 0, 10 and 90 d. Under field capacity condition, amount of acid metabolite formed was 0.03, 0.74 and 0.14 on 0, 5 and 90 d, whereas in submerged soil 0.21, 0.63 and 0.01 lg g 1 of acid metabolite was present on 0, 3 and 30 d. On different days, 2.0–69.7%, 2.5–70.7% and 26.9–81.3% of the initial deposits of kresoxim-methyl were accounted for acid metabolite under air, field capacity and submerged conditions respectively (Fig. 4a). In 5% sludge amended Inceptisol soil, 0 day samples showed the presence of 0.04 lg g 1 of acid metabolite. The concentration of acid metabolite increased to 0.72 lg g 1 on 3rd day and thereafter showed declining trend. About 4–70% of kresoxim methyl was accounted for acid metabolite on different days in sludge amended Inceptisol (Fig. 4b). Under sterile condition, maximum amount of acid metabolite i.e. 0.73 lg g 1 was recorded on 10th day and on different days 0–68.2% of kresoxim-methyl was accounted for the acid metabolite (Fig. 4c). In Inceptisol and Ultisol, the amount of acid metabolite formed were 0.03, 0.74 and 0.14 lg g 1 and 0, 0.73 and 0.22 lg g 1 on 0, 5 and 90 d respectively amounting to the loss of 2.5–70.7% and 0–67.9% of kresoxim-methyl (Fig. 4d). On Xenon-light exposure, the amounts of acid metabolite formed were 0.03, 0.80, 0.01 lg g 1 on 0, 1 and 30 d whereas under dark condition 0.03, 0.77, 0.14 lg g 1 of acid metabolite residues were recorded on 0, 3 and 90 d. About 2.7–82.6% of kresoximmethyl was accounted for acid metabolite on different days following Xenon-light exposure (Fig. 4e). Under elevated atmospheric CO2 level, 0.03, 0.74 and 0.06 lg g 1 of acid metabolite were formed on 0, 1 and 20 d

respectively. About 3.4–68.6% of initial deposits of kresoximmethyl were observed as acid metabolite under elevated atmospheric CO2 conditions (Fig. 4f). 4. Discussion 4.1. Effect of moisture level The results suggest that soil moisture play an important role in dissipation of kresoxim-methyl residues from soil. The total residues of kresoxim-methyl dissipated with slower rate in dry soil conditions as compared to wet conditions. Longer persistence under air dry condition may be due to low microbial activity in dry soil. Slow dissipation of another strobilurin fungicide pyraclostrobin under air dry condition in comparison to wet conditions has been reported by Reddy et al. (2013). Among wet conditions, dissipation was faster under submerged condition as compared to field capacity. It seems that microbes present in the partial anaerobic conditions under submerged moisture are more efficient in degrading residues of kresoxim-methyl and its metabolite. Faster dissipation of other members of strobilurin group like azoxystrobin and pyraclostrobin under submerged condition in comparison to field capacity moisture have been reported in literature (Ghosh and Singh, 2009; Reddy et al., 2013). 4.2. Effect of organic matter Amending the soil with organic sludge fastens the rate of dissipation of kresoxim-methyl. Faster dissipation in sludge amended soil could be explained on the basis of its high organic matter content. The increased organic matter content might be responsible for the increase in soil microbial population which in turn might have increased the dissipation rate of kresoxim-methyl. Reddy et al. (2013) have also reported faster dissipation of pyraclostrobin (a strobilurin fungicide) in sludge amended soil (T1/2 9.2 d) in

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Fig. 4. Formation of acid metabolite in soil under (a) moisture regime, (b) organic matter, (c) soil sterilization, (d) soil type, (e) light exposure and (f) atmospheric CO2 level.

comparison to normal soil. Ghosh and Singh (2009) have also observed faster dissipation of azoxystrobin in 5% sludge fortified soil. 4.3. Effect of sterile soil The slower dissipation of kresoxim-methyl in sterile soil suggests that microbial population play an important role in dissipation of kresoxim-methyl residues in soil. Slow dissipation of pyraclostrobin in sterile soil than non-sterile soil has been reported (Reddy et al., 2013). Similar results have been reported for different class of pesticides like metaflumizone (Chatterjee et al., 2013) metolachlor (Rice et al., 2002) and metsulfuron-methyl (Yutai et al., 1999).

4.4. Effect of soil type Physico-chemical properties of the test soil significantly affect the dissipation behavior of pesticides. In different soils half life values of 0.55 to 3.11 d have been reported for kresoxim-methyl (EFSA, 2010). In the present study, Kerala soil (Laterite, Ultisol) has high organic carbon (1.23%) and clay content (34.2%) and low pH (4.9) in comparison to Delhi soil (Inceptisol) having moderate organic carbon (0.37%) and clay content (22.3%) and high pH (8.15). In literature, high Freundlich adsorption coefficient values (KF) for kresoxim-methyl and its acid metabolite have been reported in soils having high organic carbon content and low soil pH soil as compared to alkaline soils with low organic carbon content (EFSA, 2010). Probably the strong adsorption of the parent and

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the metabolite in organic carbon, clay rich and acidic pH containing Ultisol soil is responsible for the slow dissipation of total residues. Low bioavailability due to strong adsorption of fenamiphos in acidic soil has also been cited as a reason for slow dissipation of fenamiphos (Cáceres et al., 2008). 4.5. Effect of light Faster dissipation of kresoxim-methyl residues under Xenonlight exposure in comparison to dark condition revealed that photo-degradation plays an important role in kresoxim-methyl dissipation. As per EFSA (2010) report, soil photolysis studies of kresoxim-methyl in soils of different agro-ecological regions also showed faster dissipation under light (DT90 2.2 d) in comparison to dark condition (DT90 < 5 d). Faster dissipation of metaflumizone under Xenon light (T1/2 43.0 d) exposure in comparison to dark condition (T1/2 50.1 d) in soil has earlier been reported (Chatterjee et al., 2013). 4.6. Effect of CO2 level Kresoxim-methyl dissipated at a faster rate under elevated CO2 condition than under ambient CO2 condition. Chatterjee et al. (2013) have reported that microbial biomass carbon of the soil increased at elevated CO2 condition. The increase in microbial biomass carbon means increase in microbial population which might have caused the faster degradation of kresoxim-methyl at elevated CO2 level. Dissipation of another strobilurin fungicide azoxystrobin was also reported to be faster under elevated CO2 levels than ambient level in open top chambers (Manna et al., 2013). Williams et al. (1992) have also reported that increase in the CO2 level from 300 to 660 lg g 1 slightly increases atrazine loses. Statistical analysis performed on the data sets showed significant difference between different treatments (at 95% confidence level p < 0.0001) in two way analysis of variance (ANOVA). Different letter groupings obtained for various treatments in DMRT analysis further collaborate the results of ANOVA. Comparison of treatment mean values showed that with increase in soil moisture, organic matter, microbial population, light exposure and CO2 level, the dissipation rate of kresoxim-methyl in soil also increases.

5. Conclusion The results suggest that kresoxim-methyl alone has very low to moderate persistence but the total residues (kresoxim-methyl + acid metabolite) persist for a long period. In soil, kresoxim-methyl rapidly dissipates into its acid metabolite. Results revealed that various factors like soil moisture, organic carbon content, microbial activity, soil type, light exposure, atmospheric CO2 level significantly affect the dissipation of kresoxim-methyl from soil. A two way analysis of variance and DMRT analysis revealed significant difference among various treatments. Acknowledgments The authors are grateful to the Head, Division of Agricultural Chemicals, Division of Soil Science & Agricultural Chemistry and Division of Plant Physiology, IARI for all the required infrastructure; and Indian Council of Agricultural Research for financial help in the form of Junior Research Fellowship.

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Degradation of kresoxim-methyl in soil: impact of varying moisture, organic matter, soil sterilization, soil type, light and atmospheric CO2 level.

In the present investigation, persistence of kresoxim-methyl (a broad spectrum strobilurin fungicide) was studied in two different soil types of India...
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