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Residues of lambda-cyhalothrin in tea
Food and Chemical Toxicology 47 (2009) 502–505
Contents lists available at ScienceDirect
Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Residues of lambda-cyhalothrin in tea Subbiah Seenivasan, Narayanan Nair Muraleedharan * UPASI Tea Research Foundation, Tea Research Institute, Nirar Dam BPO, Valparai 642 127, India
a r t i c l e
i n f o
Article history: Received 20 August 2008 Accepted 15 December 2008
Keywords: Lambda-cyhalothrin Residues Tea Persistence Dissipation MRL
a b s t r a c t Field experiments were conducted at two places in Tamil Nadu (India) during dry season of 2006 to determine the residues of lambda-cyhalothrin in fresh green tea leaves and black tea. Residues were quantified at different harvest intervals of ‘0’ (3 h), 1st, 3rd, 5th, 7th, 10th and 14th day after insecticide application. Persistence, dissipation pattern, half-life value and safe harvest interval of the insecticide in tea were calculated. Residues of lambda-cyhalothrin dissipated exponentially after application at both the locations and reached below the European Union maximum residue limit (MRL) of 1 mg/kg on the 5th day. Lambda-cyhalothrin showed that like other insecticides it followed the first order dissipation kinetics. Half-life values varied from 2.8 to 3.5 days for lambda-cyhalothrin and a safety harvest interval of 5 days is suggested for tea at the recommended dosage. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction India is the largest producer of black tea and about 20% of its production is exported. Tea is attacked by several pests and diseases and it is imperative that plant protection chemicals are applied to reduce the severity of infestation. Synthetic pyrethroids display a broad spectrum of insecticidal activity coupled with low mammalian toxicity and they have comparatively low application rates for insect control, making them environmentally more acceptable than the older, more persistent and toxic organo-chlorine and organo-phosphorous insecticides. They are often used in rotation with other pesticides to delay development of resistance to insecticides (Ripley et al., 2001). Lambda-cyhalothrin is a third generation pyrethroid, with contact, residual and stomach-poisoning action coupled with repellent property. For several years now, it has been used to control a wide range of arthropod pests on field and plantation crops. Lambda-cyhalothrin (Kung Fu 2.5 EC) [S)-a-cyano-3-phenoxybenzyl-(Z)-(1R,3R)-3-(2-chloro-3,3,3-trifluoroprop-1-enyl)-2,1-3,3, 3-trifluoroprop-1-enyl)-2,1-3,3,3-trifluoroprop-1-enyl)-2,1-3,3,3trifluoroprop-1-enyl)-2,1-1-enyl)-2,2-dimethylcyclopropanecarboxylate)] is a new insecticide with an unique chemical configuration consisting of dimethyl cyclopropane carboxylate moiety. It has found a place in the list of chemicals recommended for the control of the tea stem boring beetle, called the shot hole borer, in south India. However, it has a very high activity against a wide range of chewing and sucking pests, particularly, Lepidop-
* Corresponding author. Tel.: +91 4253 235301/235303; fax: +91 4253 235302. E-mail address: [email protected] (N. Muraleedharan). 0278-6915/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2008.12.010
tera, Coleoptera and mites infesting fruits, cereals, maize, cotton, wheat, pulses, oilseeds and in public health as a vector control agent (Davey et al., 1992; Roberts et al., 1993; Dikshit et al., 2000; Mathirajan et al., 2000). A few reports are available on the degradation of certain commonly used pesticides and their residues in tea (Rajukkannu et al., 1981; Singh and Agnihotri, 1984; Manikandan et al., 2001, 2005, 2006; Kumar et al., 2004; Sood et al., 2004). However, there is no published information on the residues of lambda-cyhalothrin in green leaves and black tea, under the climatic conditions of south India. Hence, a study was undertaken to generate data on the residues, persistence and dissipation of lambda-cyhalothrin in green leaves and black tea when the tea crop was treated with lambda-cyhalothrin 2.5 EC @ 250, 500 and 1000 mL/ha. 2. Materials and methods 2.1. Field trials and experimental design The experiments were conducted in January, 2006 in tea fields at Valparai and Gudalur (Tamil Nadu, India) situated at an elevation of 1140 and 1150 m above MSL. Plots measuring 100 sq.m, containing tea plants of mixed cultivars with appropriate guard rows, were used for the study. Tea plants had been planted in double hedge, in triangular planting system at a spacing of 0.75 0.75 1.25 m under the shade tree, Grevillea robusta (6 6 m spacing). The tea plants were last pruned in April 2004 at a height of 60 cm above ground level. The treatments were lambda-cyhalothrin 2.5 EC @ 250, 500 and 1000 mL/ha and an untreated control. The insecticide was applied with hand operated knapsack sprayer, using a spray volume of 400 L/ha. Tea shoots consisting of three leaves and a bud were harvested on 0 (3 h), 1st, 3rd, 5th, 7th, 10th and 14th days after application of the chemical. The shoots harvested on the specific day after chemical application were processed in a miniature manufacturing unit. Harvested shoots were spread in a withering trough and allowed to wither with natural air, blown underneath for 16–18 h.
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S. Seenivasan, N. Muraleedharan / Food and Chemical Toxicology 47 (2009) 502–505 Withered leaves were passed through a rotorvane for crushing and mixing of leaves and juice. This was passed four times through a roller cut CTC (Crush, Tear and Curl) machine. The resulting cut ‘‘dhool” was spread over the fermentation trays at a thickness of about 2 cm, maintaining a relative humidity of 90–95% for 1 h. Fermented (enzymic oxidation) ‘‘dhool” was dried in a mini fluid bed drier to attain a final moisture content of 2.5–3.0%. Black tea samples thus obtained were analysed in a gas chromatograph (Hewlett-Packard 5890 series II) equipped with electron capture detector, following standard procedure. 2.2. Chemicals and reagents Pesticide standard, lambda-cyhalothrin was purchased from Dr.Ehrenstorfer, Augsburg, Germany. Hexane, acetone, acetonitrile, diethyl ether, sodium chloride and sodium sulphate were obtained from M/s. Merck, Mumbai, India; all were of chromatographic purity. Florisil (60–100 mesh) was obtained from M/s. Sigma–Aldrich fine chemicals, Bangalore, India. 2.3. Instrument and calibration Lambda-cyhalothrin technical standard of 99.7% purity (as per the certificate of analysis) was used for analysis. Details of the instruments employed and the conditions of operations for analysis of residues of lambda-cyhalothrin are given below. A Hewlett-Packard HP 5890 Series II GC with a electron capture detector was used for separation and quantitative analysis, and a HP-5 Cross linked 5% PH ME Siloxane capillary column 30 m length, 0.32 mm internal diameter and 0.25 l film thickness) was used for gas chromatographic determination. Nitrogen at a speed of 5 mL/min was used as the carrier gas. The temperature of oven, injector and detector was set at 210, 180 and 300 °C, respectively and 0.5 lL of sample was injected for detection. Quantification was accomplished by using a standard curve prepared by diluting the stock solution in hexane. Good linearity was achieved in the range 0.01– 1.0 lg/mL with a correlation coefficient of 0.9992. The limit of detection was estimated to be 0.005 lg/mL of lambda-cyhalothrin. The column was conditioned by three repeated injections of standard and sample extracts until GC peaks were reproducible. 2.4. Analysis 2.4.1. Extraction Twenty gram of black tea sample was extracted with 150 mL of acetonitrile:water (2:1, v/v) by keeping it in a mechanical shaker for 2 h. The contents were filtered and to the filtrate 200 mL of 4% NaCl and 60 mL of hexane were added. After partitioning, the hexane layer was passed through anhydrous sodium sulphate layer to a 500 mL round bottomed flask. 2.4.2. Clean up The extract was evaporated to dryness on a rotary vacuum evaporator and the residue was dissolved in 10 mL hexane and again transferred to 125 mL separating funnel. The round bottom flask was rinsed with 5 mL portions of hexane and the rinses were added to the separating funnel. About 30 mL acetonitrile–saturated with hexane was added to it and the acetonitrile layer was drained into a 250 mL round bottom flask containing anhydrous sodium sulphate. The acetonitrile extract was evaporated to dryness at 60 °C. The concentrated residue was dissolved in 5 mL hexane and cleaned up by adsorption column chromatography using 10 g of 5% deactivated florisil and 150 mL of 6% diethyl ether in hexane as eluting solvent. Prior to elution, the column was washed with 50 mL of hexane to remove the coextractives. The eluate collected, was concentrated at about 60 °C to dryness and diluted with 10 mL of hexane and injected into GLC as per the standard operating conditions (AOAC, 2005). 2.4.3. Preparation of acetonitrile saturated with hexane Three portions of acetonitrile were combined with one portion of hexane in a 125 mL separating funnel, gently shaken and then the lower part of acetonitrile layer was collected. This is called acetonitrile saturated with hexane.
3. Results and discussion 3.1. Recovery of lambda-cyhalothrin from black tea and green leaves The analytical method was validated for black tea and fresh green leaves prior to actual analysis. To validate the analytical method, recovery percentage was established by fortification of technical standard solutions of lambda-cyhalothrin from untreated control black tea and fresh green leaf samples. For determination of recovery percent of lambda-cyhalothrin from black tea samples, 20 g of control tea sample was fortified with 0.13958 mg/kg, (by adding 2 mL aliquot of 1.3958 mg standard lambda-cyhalothrin solution in hexane) replicated three times. Similarly, for green leaves, 10 g of control green leaves were fortified with 0.013958 mg/kg, (by adding 1 mL of 0.13598 mg of standard lambda-cyhalothrin solution in hexane) replicated three times separately. After mixing and allowing the solvent to evaporate, the samples were analysed for the concentration of lambda-cyhalothrin residues as described earlier. The recovery achieved was 96.3% at 0.13958 mg/kg level of fortification for black tea and 93.8% at 0.013958 mg/kg level of fortification for green leaves which was a clear validation of the procedure adopted for extraction and analysis of lambda-cyhalothrin residues from tea samples. Recoveries of the pesticide at different fortification levels, i.e. 0.08, 0.1, 0.2, 0.5 and 1.0 lg/mL were determined in three replicates from each matrix to validate different analysts and evaluate the accuracy of the method. 3.2. Persistence of lambda-cyhalothrin in green leaves and black tea The residues of lambda-cyhalothrin in green leaves and black tea at Gudalur and Valparai when applied @ 250, 500 and 1000 mL/ha during dry season at different harvest intervals are given in the Tables 1 and 2. The residue level of lambda-cyhalothrin in black tea when the formulation lambda-cyhalothrin (Kung Fu 2.5 EC) was applied @ 250, 500 and 1000 mL/ha during dry season exponentially dissipated after spraying and reached below the MRL of 1 mg/kg, prescribed by European Union, on 5th day after application at the recommended dosage of 500 mL/ha in Gudalur and Valparai. Based on the above data, half-life, the pre-harvest interval (PHI) after application of lambda-cyhalothrin in tea could be fixed as 5 days (Table 3). Studies on the infusion indicated that lambda-cyhalothrin residues did not leach into the tea brew (Table 4). Jayakrishnan et al. (2005) reported that application of lambdacyhalothrin at the doses (15 and 30 g ai/ha) that were effective in managing tomato fruit borer did not result in residue problem in fruits. Gu et al. (2008) studied the persistence and dissipation of synthetic pyrethroids in red soils from the Yangtze River delta area. Data are also available on the degradation and persistence of
Table 1 Residues of lambda-cyhalothrin in green leaves at Gudalur (G) and Valparai (V). Day
2.4.4. Lambda-cyhalothrin in green leaves The above methodology was adopted to determine the residues of lambdacyhalothrin in fresh green leaves also. Ten gram of fresh leaves (on fresh weight basis) were taken for the analysis and proceeded as described earlier. 2.4.5. Lambda-cyhalothrin residues in tea brew Two gram of made tea was infused in 100 mL of boiling water (ISO 3103 -1990). After 6 min of brewing, the water extract was filtered, cooled and partitioned with 100 mL of hexane. The organic phase was passed through anhydrous sodium sulphate. The extract was concentrated by evaporating in a rotary vacuum evaporator and diluted with 10 mL hexane and analysed for the residues of lambda-cyhalothrin. The spent leaves were dried between the folds of filter paper and residues were extracted following the method described above for black tea.
Control
Residues of lambda-cyhalothrin (mg/kg)a @ 250 ml
0 1 3 5 7 10 14
ND ND ND ND ND ND ND
@ 500 ml
@ 1000 ml
G
V
G
V
G
V
5.54 4.35 1.82 0.93 0.84 0.71 0.27
6.04 4.17 1.29 1.11 1.07 1.02
6.08 4.44 1.86 1.42 1.07 0.80 0.31
14.96 5.46 4.66 3.02 2.40 2.22 0.27
17.27 8.35 3.42 2.35 1.82 1.42 0.53
15.41 8.70 7.46 5.77 5.24 3.15 0.58
ND – non detectable; BDL – below detection limit (0.08 mg/kg). a Dry weight basis.
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S. Seenivasan, N. Muraleedharan / Food and Chemical Toxicology 47 (2009) 502–505
Table 2 Residues of lambda-cyhalothrin in black tea at Gudalur (G) and Valparai (V). Day
Residues of lambda-cyhalothrin (mg/kg)a
Control
@ 250 ml
0 1 3 5 7 10 14
ND ND ND ND ND ND ND
@ 500 ml
@ 1000 ml
G
V
G
V
G
V
1.26 1.13 1.06 0.82 0.57 0.18 0.09
1.33 1.16 1.12 0.80 0.69 0.49 0.09
3.46 1.70 1.52 0.98 0.70 0.22 0.10
3.98 2.09 1.31 0.87 0.69 0.24 0.10
7.38 5.62 2.08 1.85 0.78 0.58 0.51
5.02 2.02 1.98 1.65 1.60 0.84 0.15
ND – non detectable. a Dry weight basis.
Table 3 Kinetics of dissipation of lambda-cyhalothrin in black tea at Gudalur (G) and Valparai (V). Dosage (mL/ha) Gudalur 250 500 1000 Valparai 250 500 1000
Regression equationa
Correlation coefficient (r)
Half-life (days)
PHIb (days)
log y = 0.2132 + ( 0.0859) x log y = 0.4779 + ( 0.1054) x log y = 0.7190 + ( 0.0867) x
0.969 0.987 0.939
3.5 2.9 3.5
4.5
log y = 0.2289 + ( 0.0745) x log y = 0.5025 + ( 0.108) x log y = 0.6329 + ( 0.0882) x
0.927 0.992 0.927
4.0 2.8 3.4
4.7
a log y = a + bx; where, y = residue level in ppm; x – harvest interval in days and a, b – regression constants. b Based on the European Union MRL of 1 mg/kg.
Table 4 Residues of lambda-cyhalothrin in tea infusion. Day
0 1 3 5 7 10 14
Residues of lambda-cyhalothrin (mg/kg) Gudalur @ 500 ml/ha
Valparai @ 500 ml/ha
Tea brew
Spent tea
Tea brew
Spent tea
ND ND ND ND ND ND ND
1.17 1.01 0.91 0.62 0.51 0.32 0.03
ND ND ND ND ND ND ND
1.12 0.96 0.89 0.71 0.46 0.11 0.03
ND – non detectable; detection limit – 0.02 mg/kg.
synthetic pyrethroids in tropical soil and aquatic environment (Awasthi and Prakash, 1997). In tea fields, besides the effects of some physical and chemical factors like light, temperature, pH, moisture, degradation of insecticides, growth dilution might have played significant role and rendered lambda-cyhalothrin residue unavailable in short a period. Half-life of 3.2 days was reported by Chen and Wan (1997) for deltamethrin in tea. They further reported that the other pyrethroids such as cypermethrin and permethrin also had similar half lives in tea. Growth dilution is an important factor in reducing the residue levels in tea crop as the shoots on which pesticides are applied, are in different stages of growth (Agnihothrudu and Muraleedharan, 1990; Bisen and Ghosh Hajra, 2000; Chen and Wan, 1988). Loss of residues ranging from 40% to 45%, specifically of pyrethroid group of chemical due to growth dilution had also been reported (Xue and Chen, 1985). Withering of tea leaves was a major reason for total degradation. This might be due to evaporation during withering process. Chen and Wan
(1988) reported 30–60% of reduction in pesticide residues during processing, especially during drying. Higher the vapour pressure, the loss of pesticides will be more during the manufacturing process. Owing to their high thermal stability and low vapour pressure, pyrethroid pesticides were found to be more stable during drying than the other pesticides. In the present study we have quantified that approximately 45% of lambda-cyhalothrin present in the green leaves were lost during tea manufacturing process. The present findings support the studies reporting the loss of many pesticides during tea processing (Chen and Wan, 1988; Jaggi et al., 2001). 3.3. Transfer of residue from manufactured tea to infusion and spent leaves The quantity of lambda-cyhalothrin residues transferred from processed black tea to infusion was below the limit of detection. While 95% of the residues remained in the spent leaves and no significant relationship was observed between the transfer of lambda-cyhalothrin residue and the rate of application @ 500 mL/ha. Generally, only those pesticides with high water solubility are potentially transferred to the tea cup, in significant amounts. The rate of transfer of the pesticide residues to the infusion depends on its solubility in water (Nagayama et al., 1996) and partition coefficient (Jaggi et al., 2001). The residues of lambdacyhalothrin were not detectable in the tea brew since its water solubility is extremely low (0.005 mg/L). The Kow for these pesticides is also very high (Kow 1,00,00,000) and supported the findings (The Pesticide Manual, 2003). 4. Conclusions The transfer of lambda-cyhalothrin residues from made tea to infusion was not detectable. On the basis of the above findings it can be concluded that at or after the 0 day of application, there was no detectable transfer of residues to the infusion when applied at the recommended dosage (500 mL/ha). Thus, the consumption of tea infusion is safe, when the leaves are processed after the normal harvesting interval, after the spraying of lambda-cyhalothrin. It is confirmed that the actual consumption of pesticide residues is many times lower than what is actually present in the made tea since it is brewed before consumption. It is suggested that the data on leaching of residues into tea brew should be taken into account for fixing a realistic MRL for pesticides in black tea. The MRLs fixed in tea for lambda- cyhalothrin is 1 mg/kg but it is evident that black tea processed from green shoots collected after 7– 10 days, after spraying at recommended dose contained residues below MRL values. The waiting period based on MRL in black tea may be therefore fixed as 5 days for lambda-cyhalothrin. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgements The authors are grateful to the National Tea Research Foundation (NTRF), C/o. Tea Board, Govt. of India for the financial assistance for this work. References Agnihothrudu, V., Muraleedharan, N., 1990. Pesticide residues in tea. Planters’ Chron. 85, 125–127. AOAC, 2005. Official Method 998.01. Synthetic pyrethroids in agricultural products. Off. methods Anal. In: AOAC Int. 18th Eds., Maryland, USA, pp. 74–77. Awasthi, M.D., Prakash, N.B., 1997. Persistence of chlorpyrifos in soil under different moisture regimes. Pestic. Sci. 50, 1–4.
S. Seenivasan, N. Muraleedharan / Food and Chemical Toxicology 47 (2009) 502–505 Bisen, J.S., Ghosh Hajra, N., 2000. Residues and persistence of certain insecticides in Darjeeling tea. J. Plantn. Crops 28 (2), 123–131. Chen, Z.M., Wan, H., 1988. Factors affecting residues of pesticides in Tea. Pestic. Sci. 23, 109–118. Chen, Z.M., Wan, H., 1997. Degradation of pesticides on plant surfaces and its prediction. A case study on tea plant. Environ. Monitor. Assess. 44, 305–313. Davey, R.V., Ahrens, E.H., Goerge, J.E., 1992. Efficacy of cyhalothrin and lambda cyhalothrin against Boophilus microplus (Acarina: Ixodidae). J. Econ. Ent. 85, 2286–2290. Dikshit, A.K., Lal, O.P., Srivastava, Y.N., 2000. Persistence of pyrethroids and nicotinyl insecticides on okra fruits. Pestic. Res. J. 12, 227–231. Gu, X.Z., Zhang, G.Y., Chen, L., Dai, R.L., Yu, Y.C., 2008. Persistence and dissipation of synthetic pyrethroid pesticides in red soils from the Yangtze River Delta area. Environ. Geochem. Health 30, 67–77. ISO 3103: 1990. IS 6400: 1993. Indian Standard. Method for Preparation of Tea Infusion for Sensory Evaluation (I rev.). Bureau of Indian Standards, Manak Bhavan, New Delhi. Jaggi, S., Sood, C., Kumar, V., Ravindranath, S.D., Shanker, A., 2001. Leaching of pesticides in tea brew. J. Agric. Food Chem. 49, 5479–5483. Jayakrishnan, S., Dikshit, A.K., Singh, J.P., Pachauri, D.C., 2005. Dissipation of lambdacyhalothrin on tomato (Lycopersicon esculentum Mill.) and removal of its residues by different washing processes and steaming. Bull. Environ. Contam.Toxicol. 75, 324–328. Kumar, V., Tewary, D.K., Ravindranath, S.D., Shanker, A., 2004. Investigation in tea on fate of fenazaquin residue and its transfer in brew. Food Chem. Toxicol. 42, 423–428. Manikandan, K.N., Muraleedharan, N., Selvasundaram, R., 2001. Residues of deltamethrin in black CTC tea. Bull. UPASI Tea Res. Foundn. 54, 88–92.
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Manikandan, K.N., Muraleedharan, N., Selvasundaram, R., 2005. Degradation of quinalphos during the processing of black CTC tea. J. Plantn. Crops 33 (2), 146– 148. Manikandan, K.N., Smitha, S., Seenivasan, S., Muraleedharan, N., Selvasundaram, R., 2006. Fenazaquin residues in tea and its persistence in south Indian climatic conditions. J. Plantn. Crops 34 (3), 410–413. Mathirajan, V.G., Natarajan, K., Kuttalam, S., Chandrasekaran, S., Regupathy, A., 2000. Efficacy of lambda cyhalothrin (Karate 5 EC) against brinjal shoot and fruit borer (Leucinodes orbonalis Guen.). Pestic. Res. J 12, 117–119. Nagayama, T., Maki, T., Kan, K., Iida, M., Tamura, Y., Nishima, T., 1996. Residues of organophosphorus pesticides in commercial tea and their leaching into tea. Nippon Noyaku Gakk. 14, 39–45. Rajukkannu, K., Blasubramanian, M., Vasudevan, P., 1981. Residues of dicofol and tetradifon in tea leaves. J. Plantn. Crops 9 (2), 124–125. Ripley, B.D., Ritcey, G.M., Harris, C.R., Denomme, M.A., Brown, P.D., 2001. Pyrethroid insecticide residues on vegetable crops. Pest Manag. Sci. 57, 683– 687. Roberts, J.M.F., Hodgson, C.J., Jackai, L.E.N., Thottappilly, G., Singh, S., 1993. Interaction between two synthetic pyrethroids and the spread of two non persistent viruses in cowpea. Ann. Appl. Biol. 122, 57–67. Singh, R.P., Agnihotri, N.P., 1984. Residues of dicofol, endosulfan and Malathion on tea, Camellia sinensis (L.) O. Kuntz. J. Ent. Res. 8 (1), 14–16. Sood, C., Jaggi, S., Kumar, V., Ravindranath, S.D., Shanker, A., 2004. How manufacturing processes affect the level of pesticide residues in tea. J. Sci. Food Agric. 84, 2123–2127. The Pesticide Manual, 2003, eighth ed. British Crop Protection Council, UK. Xue, Y.Z., Chen, Z.M., 1985. Studies on the uptake, translocation, distribution and metabolism of carbofuran in the tea seedlings. Tea Sci. Res. J.
Contents lists available at ScienceDirect
Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Residues of lambda-cyhalothrin in tea Subbiah Seenivasan, Narayanan Nair Muraleedharan * UPASI Tea Research Foundation, Tea Research Institute, Nirar Dam BPO, Valparai 642 127, India
a r t i c l e
i n f o
Article history: Received 20 August 2008 Accepted 15 December 2008
Keywords: Lambda-cyhalothrin Residues Tea Persistence Dissipation MRL
a b s t r a c t Field experiments were conducted at two places in Tamil Nadu (India) during dry season of 2006 to determine the residues of lambda-cyhalothrin in fresh green tea leaves and black tea. Residues were quantified at different harvest intervals of ‘0’ (3 h), 1st, 3rd, 5th, 7th, 10th and 14th day after insecticide application. Persistence, dissipation pattern, half-life value and safe harvest interval of the insecticide in tea were calculated. Residues of lambda-cyhalothrin dissipated exponentially after application at both the locations and reached below the European Union maximum residue limit (MRL) of 1 mg/kg on the 5th day. Lambda-cyhalothrin showed that like other insecticides it followed the first order dissipation kinetics. Half-life values varied from 2.8 to 3.5 days for lambda-cyhalothrin and a safety harvest interval of 5 days is suggested for tea at the recommended dosage. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction India is the largest producer of black tea and about 20% of its production is exported. Tea is attacked by several pests and diseases and it is imperative that plant protection chemicals are applied to reduce the severity of infestation. Synthetic pyrethroids display a broad spectrum of insecticidal activity coupled with low mammalian toxicity and they have comparatively low application rates for insect control, making them environmentally more acceptable than the older, more persistent and toxic organo-chlorine and organo-phosphorous insecticides. They are often used in rotation with other pesticides to delay development of resistance to insecticides (Ripley et al., 2001). Lambda-cyhalothrin is a third generation pyrethroid, with contact, residual and stomach-poisoning action coupled with repellent property. For several years now, it has been used to control a wide range of arthropod pests on field and plantation crops. Lambda-cyhalothrin (Kung Fu 2.5 EC) [S)-a-cyano-3-phenoxybenzyl-(Z)-(1R,3R)-3-(2-chloro-3,3,3-trifluoroprop-1-enyl)-2,1-3,3, 3-trifluoroprop-1-enyl)-2,1-3,3,3-trifluoroprop-1-enyl)-2,1-3,3,3trifluoroprop-1-enyl)-2,1-1-enyl)-2,2-dimethylcyclopropanecarboxylate)] is a new insecticide with an unique chemical configuration consisting of dimethyl cyclopropane carboxylate moiety. It has found a place in the list of chemicals recommended for the control of the tea stem boring beetle, called the shot hole borer, in south India. However, it has a very high activity against a wide range of chewing and sucking pests, particularly, Lepidop-
* Corresponding author. Tel.: +91 4253 235301/235303; fax: +91 4253 235302. E-mail address: [email protected] (N. Muraleedharan). 0278-6915/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2008.12.010
tera, Coleoptera and mites infesting fruits, cereals, maize, cotton, wheat, pulses, oilseeds and in public health as a vector control agent (Davey et al., 1992; Roberts et al., 1993; Dikshit et al., 2000; Mathirajan et al., 2000). A few reports are available on the degradation of certain commonly used pesticides and their residues in tea (Rajukkannu et al., 1981; Singh and Agnihotri, 1984; Manikandan et al., 2001, 2005, 2006; Kumar et al., 2004; Sood et al., 2004). However, there is no published information on the residues of lambda-cyhalothrin in green leaves and black tea, under the climatic conditions of south India. Hence, a study was undertaken to generate data on the residues, persistence and dissipation of lambda-cyhalothrin in green leaves and black tea when the tea crop was treated with lambda-cyhalothrin 2.5 EC @ 250, 500 and 1000 mL/ha. 2. Materials and methods 2.1. Field trials and experimental design The experiments were conducted in January, 2006 in tea fields at Valparai and Gudalur (Tamil Nadu, India) situated at an elevation of 1140 and 1150 m above MSL. Plots measuring 100 sq.m, containing tea plants of mixed cultivars with appropriate guard rows, were used for the study. Tea plants had been planted in double hedge, in triangular planting system at a spacing of 0.75 0.75 1.25 m under the shade tree, Grevillea robusta (6 6 m spacing). The tea plants were last pruned in April 2004 at a height of 60 cm above ground level. The treatments were lambda-cyhalothrin 2.5 EC @ 250, 500 and 1000 mL/ha and an untreated control. The insecticide was applied with hand operated knapsack sprayer, using a spray volume of 400 L/ha. Tea shoots consisting of three leaves and a bud were harvested on 0 (3 h), 1st, 3rd, 5th, 7th, 10th and 14th days after application of the chemical. The shoots harvested on the specific day after chemical application were processed in a miniature manufacturing unit. Harvested shoots were spread in a withering trough and allowed to wither with natural air, blown underneath for 16–18 h.
503
S. Seenivasan, N. Muraleedharan / Food and Chemical Toxicology 47 (2009) 502–505 Withered leaves were passed through a rotorvane for crushing and mixing of leaves and juice. This was passed four times through a roller cut CTC (Crush, Tear and Curl) machine. The resulting cut ‘‘dhool” was spread over the fermentation trays at a thickness of about 2 cm, maintaining a relative humidity of 90–95% for 1 h. Fermented (enzymic oxidation) ‘‘dhool” was dried in a mini fluid bed drier to attain a final moisture content of 2.5–3.0%. Black tea samples thus obtained were analysed in a gas chromatograph (Hewlett-Packard 5890 series II) equipped with electron capture detector, following standard procedure. 2.2. Chemicals and reagents Pesticide standard, lambda-cyhalothrin was purchased from Dr.Ehrenstorfer, Augsburg, Germany. Hexane, acetone, acetonitrile, diethyl ether, sodium chloride and sodium sulphate were obtained from M/s. Merck, Mumbai, India; all were of chromatographic purity. Florisil (60–100 mesh) was obtained from M/s. Sigma–Aldrich fine chemicals, Bangalore, India. 2.3. Instrument and calibration Lambda-cyhalothrin technical standard of 99.7% purity (as per the certificate of analysis) was used for analysis. Details of the instruments employed and the conditions of operations for analysis of residues of lambda-cyhalothrin are given below. A Hewlett-Packard HP 5890 Series II GC with a electron capture detector was used for separation and quantitative analysis, and a HP-5 Cross linked 5% PH ME Siloxane capillary column 30 m length, 0.32 mm internal diameter and 0.25 l film thickness) was used for gas chromatographic determination. Nitrogen at a speed of 5 mL/min was used as the carrier gas. The temperature of oven, injector and detector was set at 210, 180 and 300 °C, respectively and 0.5 lL of sample was injected for detection. Quantification was accomplished by using a standard curve prepared by diluting the stock solution in hexane. Good linearity was achieved in the range 0.01– 1.0 lg/mL with a correlation coefficient of 0.9992. The limit of detection was estimated to be 0.005 lg/mL of lambda-cyhalothrin. The column was conditioned by three repeated injections of standard and sample extracts until GC peaks were reproducible. 2.4. Analysis 2.4.1. Extraction Twenty gram of black tea sample was extracted with 150 mL of acetonitrile:water (2:1, v/v) by keeping it in a mechanical shaker for 2 h. The contents were filtered and to the filtrate 200 mL of 4% NaCl and 60 mL of hexane were added. After partitioning, the hexane layer was passed through anhydrous sodium sulphate layer to a 500 mL round bottomed flask. 2.4.2. Clean up The extract was evaporated to dryness on a rotary vacuum evaporator and the residue was dissolved in 10 mL hexane and again transferred to 125 mL separating funnel. The round bottom flask was rinsed with 5 mL portions of hexane and the rinses were added to the separating funnel. About 30 mL acetonitrile–saturated with hexane was added to it and the acetonitrile layer was drained into a 250 mL round bottom flask containing anhydrous sodium sulphate. The acetonitrile extract was evaporated to dryness at 60 °C. The concentrated residue was dissolved in 5 mL hexane and cleaned up by adsorption column chromatography using 10 g of 5% deactivated florisil and 150 mL of 6% diethyl ether in hexane as eluting solvent. Prior to elution, the column was washed with 50 mL of hexane to remove the coextractives. The eluate collected, was concentrated at about 60 °C to dryness and diluted with 10 mL of hexane and injected into GLC as per the standard operating conditions (AOAC, 2005). 2.4.3. Preparation of acetonitrile saturated with hexane Three portions of acetonitrile were combined with one portion of hexane in a 125 mL separating funnel, gently shaken and then the lower part of acetonitrile layer was collected. This is called acetonitrile saturated with hexane.
3. Results and discussion 3.1. Recovery of lambda-cyhalothrin from black tea and green leaves The analytical method was validated for black tea and fresh green leaves prior to actual analysis. To validate the analytical method, recovery percentage was established by fortification of technical standard solutions of lambda-cyhalothrin from untreated control black tea and fresh green leaf samples. For determination of recovery percent of lambda-cyhalothrin from black tea samples, 20 g of control tea sample was fortified with 0.13958 mg/kg, (by adding 2 mL aliquot of 1.3958 mg standard lambda-cyhalothrin solution in hexane) replicated three times. Similarly, for green leaves, 10 g of control green leaves were fortified with 0.013958 mg/kg, (by adding 1 mL of 0.13598 mg of standard lambda-cyhalothrin solution in hexane) replicated three times separately. After mixing and allowing the solvent to evaporate, the samples were analysed for the concentration of lambda-cyhalothrin residues as described earlier. The recovery achieved was 96.3% at 0.13958 mg/kg level of fortification for black tea and 93.8% at 0.013958 mg/kg level of fortification for green leaves which was a clear validation of the procedure adopted for extraction and analysis of lambda-cyhalothrin residues from tea samples. Recoveries of the pesticide at different fortification levels, i.e. 0.08, 0.1, 0.2, 0.5 and 1.0 lg/mL were determined in three replicates from each matrix to validate different analysts and evaluate the accuracy of the method. 3.2. Persistence of lambda-cyhalothrin in green leaves and black tea The residues of lambda-cyhalothrin in green leaves and black tea at Gudalur and Valparai when applied @ 250, 500 and 1000 mL/ha during dry season at different harvest intervals are given in the Tables 1 and 2. The residue level of lambda-cyhalothrin in black tea when the formulation lambda-cyhalothrin (Kung Fu 2.5 EC) was applied @ 250, 500 and 1000 mL/ha during dry season exponentially dissipated after spraying and reached below the MRL of 1 mg/kg, prescribed by European Union, on 5th day after application at the recommended dosage of 500 mL/ha in Gudalur and Valparai. Based on the above data, half-life, the pre-harvest interval (PHI) after application of lambda-cyhalothrin in tea could be fixed as 5 days (Table 3). Studies on the infusion indicated that lambda-cyhalothrin residues did not leach into the tea brew (Table 4). Jayakrishnan et al. (2005) reported that application of lambdacyhalothrin at the doses (15 and 30 g ai/ha) that were effective in managing tomato fruit borer did not result in residue problem in fruits. Gu et al. (2008) studied the persistence and dissipation of synthetic pyrethroids in red soils from the Yangtze River delta area. Data are also available on the degradation and persistence of
Table 1 Residues of lambda-cyhalothrin in green leaves at Gudalur (G) and Valparai (V). Day
2.4.4. Lambda-cyhalothrin in green leaves The above methodology was adopted to determine the residues of lambdacyhalothrin in fresh green leaves also. Ten gram of fresh leaves (on fresh weight basis) were taken for the analysis and proceeded as described earlier. 2.4.5. Lambda-cyhalothrin residues in tea brew Two gram of made tea was infused in 100 mL of boiling water (ISO 3103 -1990). After 6 min of brewing, the water extract was filtered, cooled and partitioned with 100 mL of hexane. The organic phase was passed through anhydrous sodium sulphate. The extract was concentrated by evaporating in a rotary vacuum evaporator and diluted with 10 mL hexane and analysed for the residues of lambda-cyhalothrin. The spent leaves were dried between the folds of filter paper and residues were extracted following the method described above for black tea.
Control
Residues of lambda-cyhalothrin (mg/kg)a @ 250 ml
0 1 3 5 7 10 14
ND ND ND ND ND ND ND
@ 500 ml
@ 1000 ml
G
V
G
V
G
V
5.54 4.35 1.82 0.93 0.84 0.71 0.27
6.04 4.17 1.29 1.11 1.07 1.02
6.08 4.44 1.86 1.42 1.07 0.80 0.31
14.96 5.46 4.66 3.02 2.40 2.22 0.27
17.27 8.35 3.42 2.35 1.82 1.42 0.53
15.41 8.70 7.46 5.77 5.24 3.15 0.58
ND – non detectable; BDL – below detection limit (0.08 mg/kg). a Dry weight basis.
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Table 2 Residues of lambda-cyhalothrin in black tea at Gudalur (G) and Valparai (V). Day
Residues of lambda-cyhalothrin (mg/kg)a
Control
@ 250 ml
0 1 3 5 7 10 14
ND ND ND ND ND ND ND
@ 500 ml
@ 1000 ml
G
V
G
V
G
V
1.26 1.13 1.06 0.82 0.57 0.18 0.09
1.33 1.16 1.12 0.80 0.69 0.49 0.09
3.46 1.70 1.52 0.98 0.70 0.22 0.10
3.98 2.09 1.31 0.87 0.69 0.24 0.10
7.38 5.62 2.08 1.85 0.78 0.58 0.51
5.02 2.02 1.98 1.65 1.60 0.84 0.15
ND – non detectable. a Dry weight basis.
Table 3 Kinetics of dissipation of lambda-cyhalothrin in black tea at Gudalur (G) and Valparai (V). Dosage (mL/ha) Gudalur 250 500 1000 Valparai 250 500 1000
Regression equationa
Correlation coefficient (r)
Half-life (days)
PHIb (days)
log y = 0.2132 + ( 0.0859) x log y = 0.4779 + ( 0.1054) x log y = 0.7190 + ( 0.0867) x
0.969 0.987 0.939
3.5 2.9 3.5
4.5
log y = 0.2289 + ( 0.0745) x log y = 0.5025 + ( 0.108) x log y = 0.6329 + ( 0.0882) x
0.927 0.992 0.927
4.0 2.8 3.4
4.7
a log y = a + bx; where, y = residue level in ppm; x – harvest interval in days and a, b – regression constants. b Based on the European Union MRL of 1 mg/kg.
Table 4 Residues of lambda-cyhalothrin in tea infusion. Day
0 1 3 5 7 10 14
Residues of lambda-cyhalothrin (mg/kg) Gudalur @ 500 ml/ha
Valparai @ 500 ml/ha
Tea brew
Spent tea
Tea brew
Spent tea
ND ND ND ND ND ND ND
1.17 1.01 0.91 0.62 0.51 0.32 0.03
ND ND ND ND ND ND ND
1.12 0.96 0.89 0.71 0.46 0.11 0.03
ND – non detectable; detection limit – 0.02 mg/kg.
synthetic pyrethroids in tropical soil and aquatic environment (Awasthi and Prakash, 1997). In tea fields, besides the effects of some physical and chemical factors like light, temperature, pH, moisture, degradation of insecticides, growth dilution might have played significant role and rendered lambda-cyhalothrin residue unavailable in short a period. Half-life of 3.2 days was reported by Chen and Wan (1997) for deltamethrin in tea. They further reported that the other pyrethroids such as cypermethrin and permethrin also had similar half lives in tea. Growth dilution is an important factor in reducing the residue levels in tea crop as the shoots on which pesticides are applied, are in different stages of growth (Agnihothrudu and Muraleedharan, 1990; Bisen and Ghosh Hajra, 2000; Chen and Wan, 1988). Loss of residues ranging from 40% to 45%, specifically of pyrethroid group of chemical due to growth dilution had also been reported (Xue and Chen, 1985). Withering of tea leaves was a major reason for total degradation. This might be due to evaporation during withering process. Chen and Wan
(1988) reported 30–60% of reduction in pesticide residues during processing, especially during drying. Higher the vapour pressure, the loss of pesticides will be more during the manufacturing process. Owing to their high thermal stability and low vapour pressure, pyrethroid pesticides were found to be more stable during drying than the other pesticides. In the present study we have quantified that approximately 45% of lambda-cyhalothrin present in the green leaves were lost during tea manufacturing process. The present findings support the studies reporting the loss of many pesticides during tea processing (Chen and Wan, 1988; Jaggi et al., 2001). 3.3. Transfer of residue from manufactured tea to infusion and spent leaves The quantity of lambda-cyhalothrin residues transferred from processed black tea to infusion was below the limit of detection. While 95% of the residues remained in the spent leaves and no significant relationship was observed between the transfer of lambda-cyhalothrin residue and the rate of application @ 500 mL/ha. Generally, only those pesticides with high water solubility are potentially transferred to the tea cup, in significant amounts. The rate of transfer of the pesticide residues to the infusion depends on its solubility in water (Nagayama et al., 1996) and partition coefficient (Jaggi et al., 2001). The residues of lambdacyhalothrin were not detectable in the tea brew since its water solubility is extremely low (0.005 mg/L). The Kow for these pesticides is also very high (Kow 1,00,00,000) and supported the findings (The Pesticide Manual, 2003). 4. Conclusions The transfer of lambda-cyhalothrin residues from made tea to infusion was not detectable. On the basis of the above findings it can be concluded that at or after the 0 day of application, there was no detectable transfer of residues to the infusion when applied at the recommended dosage (500 mL/ha). Thus, the consumption of tea infusion is safe, when the leaves are processed after the normal harvesting interval, after the spraying of lambda-cyhalothrin. It is confirmed that the actual consumption of pesticide residues is many times lower than what is actually present in the made tea since it is brewed before consumption. It is suggested that the data on leaching of residues into tea brew should be taken into account for fixing a realistic MRL for pesticides in black tea. The MRLs fixed in tea for lambda- cyhalothrin is 1 mg/kg but it is evident that black tea processed from green shoots collected after 7– 10 days, after spraying at recommended dose contained residues below MRL values. The waiting period based on MRL in black tea may be therefore fixed as 5 days for lambda-cyhalothrin. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgements The authors are grateful to the National Tea Research Foundation (NTRF), C/o. Tea Board, Govt. of India for the financial assistance for this work. References Agnihothrudu, V., Muraleedharan, N., 1990. Pesticide residues in tea. Planters’ Chron. 85, 125–127. AOAC, 2005. Official Method 998.01. Synthetic pyrethroids in agricultural products. Off. methods Anal. In: AOAC Int. 18th Eds., Maryland, USA, pp. 74–77. Awasthi, M.D., Prakash, N.B., 1997. Persistence of chlorpyrifos in soil under different moisture regimes. Pestic. Sci. 50, 1–4.
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