Persistence Behaviour of Fungicide Tebuconazole in a ViticultureApplication

Chiranjit Kundu • Arnab Goon • Anjan Bhattacharyya

Received: 15 February 2013 / Accepted: 1 February 2014 / Published online: 13 February 2014

� Springer Science+Business Media New York 2014

Abstract Dissipation pattern and risk assessment of te-

buconazole in grapes was studied following two applica-

tion rates (250 and 500 mL ha-1) under tropical humid

climatic condition of West Bengal during 2009–2010.

Residues of tebuconazole were confirmed by liquid chro-

matography–mass spectrometry. The average recoveries

were found 87.53 % and 89.67 % for grapes and cropped

soil respectively. Following the first order kinetics the

fungicide dissipates in grapes with a half-life (T1/2) value

ranges between 2.62 and 2.86 days irrespective of seasons

and doses. No residues of tebuconazole were detected in

harvest grapes and soil samples which refers that, tebuco-

nazole does not possess any background contamination

property in grapes. So it may be concluded from the study

that tebuconazole does not possess any toxicological

property when applied at the recommended dose.

Keywords Persistence � Grapes � Fungicide �Tebuconazole

Grapes (Vitis vinifera L.) belong to the world’s largest fruit

crops with a global production of around 69 million tons in

2006 (FAOSTAT 2007). Grapes contain large amounts of

phytochemicals including anthocyanins and resveratrol,

which offer health benefits (Pezzuto 2008). Grapes how-

ever can suffer high yield losses due to fungal diseases.

Powdery mildew of grapes is caused by obligate parasitic

fungus Uncinula necator (Schw.) Burr. is a serious prob-

lem in most of the grape growing areas of west Bengal.

Powdery mildew is a highly destructive disease of grape-

vines in all grape-growing areas causing major losses if the

disease is not controlled during favourable weather.

Tebuconazole, a triazole fungicide [(RS)-1-(4-chloro-

phenyl)-4,4-dimethyl-3-(1H-1,2,4-triazol-1-ylmethyl)-pen-

tan-3-ol. Ratio (1:1)] (Fig. 1) used as a seed dressing

chemical is effective against various smut and bunt dis-

eases of cereals. Used as a spray, it controls numerous

pathogens in various crops: rust sp., powdery mildew of

grapes (Source-FAO). Tebuconazole when applied in the

form of fungicide accumulated in surface layers of soil and

became toxic to susceptible plants (Source-FAO). Also it is

rapidly absorbed by vegetative parts of the plants with

translocation principally acropetally. So, in order to strike

better balance of benefits of agriculture out of pesticides

against risks to human, environment and food, it is essen-

tial to study the residual fate and dissipation behaviour of

the applied fungicide tebuconazole. The objective of the

present work was to study the dissipation and the fate of

tebuconazole residue in/on grapes grown in different sea-

sons under tropical humid climatic condition of West

Bengal.

Materials and Methods

A two season field study was conducted at Malancha Farm,

Village – Siyan, District-Birbhum during March 2009–June

2009 (1st season) and March 2010–June 2010 (2nd season)

on grapes (variety – Madhu Angur). The formulation te-

buconazole 25.9 % EC was applied with the help of knap-

sack sprayer equipped with WFN 62 nozzle @ 250 mL ha-1

(T1) and @ 500 mL ha-1 (T2) in Randomized Block

Designed (RBD) plots and maintained untreated control (T3)

plots. Spraying of fungicide was done thrice at 7 days

C. Kundu � A. Goon � A. Bhattacharyya (&)

Pesticide Residue Laboratory, Department of Agricultural

Chemicals, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur,

Nadia 741252, West Bengal, India

e-mail: anjan_84@rediffmail.com

123

Bull Environ Contam Toxicol (2014) 92:415–419

DOI 10.1007/s00128-014-1223-8

interval for both the seasons. For persistence study, grapes

sample was collected at 0, 1, 3, 7, 10, 15 days after 3rd

application of the fungicide in both seasons. During both

seasons grapes and cropped soil samples were also collected

at the time of harvest following standard sampling proce-

dures. Grapes (0.5 kg) and field soil (0.5 kg) samples were

collected from five to seven random locations on each

sampling day from the treatment plots. Samples from

untreated control plots were also collected in the same way.

Samples (Grapes and soil sample) were air dried to facilitate

extraction. A valid representative (10 g) of grapes and soil

samples were prepared by quartering technique in the labo-

ratory and taken for final analysis separately.

The samples were blended using Polytron homogenizer.

Five gram (5 g) of the homogenized sample was taken in a

50 mL centrifuge tube and 10 mL (Ethyl Acetate:Cyclo-

hexane) mixture was added and subjected to vortex for

2 min. After that adding 5 g of activated Na2SO4, the sample

was again vortex for 3 min. Then the sample was centrifuged

for 15 min at 10,000 rpm and then 5 mL supernatant liquid

was taken in 10 mL centrifuge tube. Afterwards 25 mg

florisil and 25 mg PSA was added to it and vortex for 2 min

and the sample was again centrifuged for 10 min at

5,000 rpm. Then 3 mL supernatant liquid was collected

from it and evaporated to dryness in N2-Evaporator at 25 �C.

The residue was then reconstituted in 3 mL of [MeOH:H2O

(9:1, v/v) ? 5 mM CH3COONH4]. The sample was then

filtered through 0.2 lm membrane filter. The quantitative

analysis of tebuconazole was performed by liquid chroma-

tography–tandem mass spectrometry (LC–MS/MS) (Sta-

jnbaher and Zupancic Kralj 2003). The HPLC separation was

performed by injecting 5 lL via autosampler on a Symmetry

C18 (5 lm; 2.1 9 100 mm) column (Waters, USA) at the

flow rate of 0.2 mL/min. The mobile phase was composed of

(A) methanol/water 10/90 (v/v) with 5 mM ammonium

acetate and (B) methanol/water 90/10 (v/v) with 5 mM

ammonium acetate. The optimized MS instrument parame-

ters includes: capillary voltage, 1.00 kV; cone voltage,

35 V; source temperature, 120�C; desolvation temperature,

350�C; desolvation gas flow, 650 L/h nitrogen; cone gas

flow, 25 L/h argon; collision gas pressure to 3.5e-3 psi for

MS/MS. Quantification was performed by multiple reaction

monitoring with two mass transition (308.14 ? 69.6,

308.14 ? 125.1, and 308.14 ? 150.9). The limit of

detection (LOD) and limit of quantification (LOQ) for te-

buconazole were 0.005 and 0.01 lg/g respectively.

The calibration curve was constructed by plotting rele-

vant standard concentration versus absorption for tebuco-

nazole (Fig. 2).

Recovery studies were carried out for both tebuconazole

in order to establish the reliability of the analytical method

and to know the efficiency of extraction and clean up steps

employed for the present study, by fortifying the field soil

and plant (Grapes) samples with different levels of the

analytical standard solution. Results of recovery study are

shown in Table 1.

Results and Discussion

The recovery percentage of tebuconazole in grapes and

field soil were 87.53 % and 89.67 % respectively

(Table 1). Hence the methods can be adopted.

The results of field study of persistence of tebuconazole

in grapes has been summarized in Table 2 (For season-I)

and Table 3 (For season-II). It was found that the residues

Fig. 1 Structure of tebuconazole. Source The pesticide manual,

Tomlin (1997)

Fig. 2 Calibration curve of tebuconazole

Table 1 Recovery study of tebuconazole in different substrates of

grapes

Substrate Amount

fortified

(lg/g)

Amount

recovereda

(lg/g)

% Recovery Average %

recovery

Grapes sample 0.01 0.008 80.00 87.53

0.10 0.086 86.00

0.50 0.483 96.60

Field soil 0.01 0.008 80.00 89.67

0.10 0.092 92.00

0.50 0.485 97.00

a Average of three replicates

416 Bull Environ Contam Toxicol (2014) 92:415–419

123

gradually decreased with time following 1st order kinetics.

The initial deposits (2 h after spraying) of tebuconazole in

grapes were found 0.98 lg/g (T1) and 1.96 lg/g (T2) for

1st season and 0.95 lg/g (T1) and 1.81 lg/g (T2) for 2nd

season respectively. More than 50 % of the initial deposit

was dissipated within 3 days irrespective of any doses and

seasons. The dissipation patterns of the present study are in

well agreement with the earlier studies conducted in grapes

under semi-arid tropical climatic condition (Mohapatra

et al. 2010). Sandra et al. (1999) stated the same trend in

their studies in dissipation of propiconazole and tebuco-

nazole in peppermint crops. The initial deposit of tebuco-

nazole ranges from 0.95 to 1.96 lg/g and more than 50 %

of the residue was degraded in grapes samples within

3 days irrespective of any season and treatment. Chuan

(2009) stated the same trend in their studies in determi-

nation of tebuconazole residue in soil and apple (Fig. 3;

Table 4).

Risk Assessment Study of Tebuconazole

Risk assessment is crucial to the process of making decisions

about pesticides, both new and existing. New pesticides must

be evaluated before they can enter the market. Existing pes-

ticides must be re-evaluated periodically to ensure that they

continue to meet the appropriate safety standard. Generally a

pesticide remains in soil, water and plant for some time after

its application and before it breaks down. So, proper risk

assessment study is required accordingly so that a minimum

amount of pesticide is left in environment as well as the food

chain. Residues of pesticides may remain in treated products

and get into human food chain. These residues should not

exceed a limit above which they may pose risks to human

health. The concepts of Persistence, Maximum residue limits

(MRLs), Acceptable daily intake (ADI) for pesticides have

been devised to keep a check on the pesticide residues in food

chain and keep them within safe limits.

Table 2 Dissipation of

tebuconazole in grapes sample

for season-I

BDL below detectable limit

Days after application Treat-ment Residues in ppm. Dissipation (%)

R1 R2 R3 Mean ± S.D

0 T1 0.97 0.99 0.98 0.98 ± 0.008 –

1 0.59 0.61 0.62 0.61 ± 0.012 37.75

3 0.35 0.36 0.36 0.36 ± 0.004 63.27

7 0.11 0.12 0.12 0.12 ± 0.005 87.76

10 0.07 0.07 0.06 0.07 ± 0.004 92.86

15 BDL BDL BDL – –

0 T2 1.99 1.94 1.96 1.96 ± 0.021 –

1 1.18 1.20 1.19 1.19 ± 0.008 39.29

3 0.80 0.78 0.81 0.80 ± 0.012 59.18

7 0.28 0.29 0.29 0.29 ± 0.005 85.20

10 0.15 0.16 0.16 0.16 ± 0.004 91.84

15 BDL BDL BDL – –

Table 3 Dissipation of

tebuconazole in grapes sample

for season-II

BDL below detectable limit

Days after application Treat-ment Residues in ppm. Dissipation (%)

R1 R2 R3 Mean ± S.D

0 T1 0.99 0.96 0.92 0.95 ± 0.028 –

1 0.68 0.71 0.64 0.67 ± 0.028 29.47

3 0.37 0.41 0.39 0.39 ± 0.016 58.95

7 0.14 0.15 0.16 0.15 ± 0.008 84.21

10 0.07 0.06 0.06 0.06 ± 0.004 93.68

15 BDL BDL BDL – –

0 T2 1.82 1.76 1.86 1.81 ± 0.041 –

1 1.19 1.16 1.20 1.18 ± 0.017 34.80

3 0.76 0.71 0.79 0.75 ± 0.032 58.56

7 0.34 0.28 0.31 0.31 ± 0.024 82.87

10 0.17 0.11 0.15 0.14 ± 0.025 92.26

15 BDL BDL BDL – –

Bull Environ Contam Toxicol (2014) 92:415–419 417

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The persistence of any chemical is generally expressed

in terms of half-life (T1/2) or DT50 i.e. time for disap-

pearance of pesticide to 50 per cent of its initial concen-

tration. T1/2 values are often obtained by fitting first-order

kinetics to observed degradation patterns as

Ct ¼ Co � e �ktð Þ

where Ct is chemical concentration (mg kg-1) at time t (h),

C0 is initial concentration (mg kg-1), and k is the first order

rate constant (h-1) independent of Ct and C0. The T1/2 of

tebuconazole were calculated using Hoskins formula (Ho-

skins 1966). The half-life (T1/2) of tebuconazole was varied

between 2.67 and 2.86 days in 1st season and

2.62–2.84 days in 2nd season respectively. In the present

study no residues of tebuconazole were detected in harvest

grapes and soil samples irrespective of any doses and sea-

sons, which refers that tebuconazole does not possess any

background contamination property in grapes when applied

at the recommended dose. The harvest residue study is in

well agreement with the earlier studies conducted in grape

leaves, grape, berry and soil (Jyot et al. 2009).

The initial deposits (2 h after spraying) of tebuconazole

in grapes varies between 0.95 and 1.96 lg/g for both the

seasons which was found to be less than its Indian maxi-

mum residue limit (2 lg/g for table grapes and wine

grapes) value fixed as by Ministry of Health and Family

Welfare, Govt. of India (Anonymous 2004). The residues

dissipated gradually to 0.07–0.16 lg/g and 0.06–0.14 lg/g

(For both seasons respectively) by 10 days, and were

below the quantifiable limit of 0.01 lg/g at the time of

harvest (60 days after the last treatment). Soil at harvest

was free of any pesticide residues. Based on these obser-

vations, it may be concluded that residual tebuconazole

concentrations at the time of harvest will be below those

associated with toxicological risk when applied at the

recommended dose. Similar observations have been pre-

viously reported in studies investigating the persistence of

tebuconazole in agricultural applications (European Food

Safety Authority Journal 2009; Food and Drug Adminis-

tration of the United States 2003).

Acceptable daily intake (ADI) for tebuconazole has

been observed to be 0.03 mg kg-1 body weight per day

(SANCO database 2008). Accordingly, an adult of 60 kg

can tolerate an intake of 1,800 lg day-1 without any

appreciable exposure risks. A consumption of 200 g of test

substrate by an adult with initial deposit as observed at

double dose (1.96 and 1.81 lg/g for season-I and season-II

respectively) will lead to intake of only 360–400 lg day-1

(approx.) which is toxicologically acceptable and quite safe

from health point of view of the consumer.

References

Anonymous (2004) The prevention of food adulteration act: Ministry

of Health & Family Welfare, Gazette of India, extraordinary,

part II, section 3

Chuan LIU (2009) Determination of tebuconazole residue in soil and

apple. J Anhui Agric Sci 37:135–139

European Commission Health and Consumers Directorate General:

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171/08- revision 1. 9 Sept 2008

European Food Safety Authority Journal (2009) 7(10):1368

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Fig. 3 Linear plot of

dissipation of tebuconazole in

grapes in (a) season-I &

(b) season-II

Table 4 Results of statistical interpretation of residue data

Substrate Regression equation Half-life

(T1/2) (days)

Tebuconazole (season-I) y = 2.927 - 0.113x (T1)

y = 3.230 - 0.105x (T2)

2.67–2.86

Tebuconazole (season-II) y = 2.959 - 0.116x (T1)

y = 3.215 - 0.106x (T2)

2.62–2.84

418 Bull Environ Contam Toxicol (2014) 92:415–419

123

Jyot G, Arora P, Saha S, Singh B, Singh R (2009) Persistence of

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