1 Background
Tea is a beverage endowed with numerous beneficial health effects and it is imperative that this drink is kept free of toxic contaminants like pesticide residues and heavy metals. It is necessary to apply pesticides, synthetic fertilizers and micro nutrients to augment the productivity of tea gardens and to protect the plants from pests and diseases. Synthetic pyrethroids display a broad spectrum of insecticidal/acaricidal activity coupled with low mammalian toxicity and they have comparatively low application rates for pest 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).
 
Bifenthrin[IUPACname2-methylbiphenyl-3-ylm-ethyl(Z)-(1RS)-cis-3-(2-chloro-3,3,3-trifluoroprop-1-enyl)-2,2-dimethyl cyclopropanecarboxylate] is an off-white to pale tan waxy solid faint, slightly sweet smell, belonging to pyrethroids chemical class, for which the Acceptable Daily Intake was established at the level of 0.015 mg kg-1 of body weight (Royal Society of Chemistry, 1994). It is non-systemic insecticide and acaricide which kills pests on contact or on its ingestion along with plant substrate. There are a number of highly desirable physical characteristics for bifenthrin such as low water solubility (0.1 mg l-1), high octanol-water coefficient (Koc 2,30,000) and  stability over a range 5-9.5 pH.

As tea is subjected to infusion prior to consumption, residues of pesticides in tea and its transfer in brew must be monitored prior to permitting the consumption by human beings. A few reports are available on the degradation of certain commonly used pesticides and their residues in tea (Rajukkannu et al., 1981; Singh & Agnihotri, 1984; Manikandan et al., 2001, 2005 & 2006; Kumar et al., 2004; Sood et al., 2004; Tewary et al., 2005; Seenivasan and Muraleedharan, 2009). However, there is no published information on the residues of bifenthrin in black tea, under the climatic conditions of south India. Hence, a study was undertaken to generate data on the residues, persistence and dissipation of bifenthrin in black tea when the tea crop was treated with bifenthrin 8 SC @ 1000 and 2000 ml ha-1.

2 Results and discussion
2.1 Recovery of bifenthrin from black tea
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 bifenthrin from black tea samples, control tea sample of 20 g was fortified with 0.944 mg l-1 (by adding 2 ml aliquot of 9.44 mg l-1 standard bifenthrin solution in hexane) replicated six times separately. After mixing and allowing the solvent to evaporate, the samples were analyzed for the concentration of bifenthrin residue as described earlier. The recovery obtained was 94.93 % at 0.944 mg kg-1 level of fortification and a recovery of 91.70% at 0.0944 mg/kg level all of which show a clear validation to the procedure adopted for extraction and analysis of bifenthrin residues from tea samples.

Recoveries of the pesticide at different fortification levels, i.e. 0.01, 0.1, 0.2 and 0.5 µg ml-1 were determined in three replicates from each matrix to validate different analysts and evaluate the accuracy of the method.

2.2 Persistence of bifenthrin in black tea
The residues of bifenthrin in black tea at Gudalur and Valparai when applied @ 1000 and 2000 ml ha-1 during wet and dry season at different harvest intervals are given in the Table 2.The residue level of bifenthrin in black tea when the formulation bifenthrin (Brigade 8 SC) was applied @ 1000 and 2000 ml ha-1 during wet and dry seasons exponentially dissipated after spraying and reached below the MRL of 5 mg kg-1, prescribed by European Union, on 7th day after application at the recommended dosage of 1000 ml ha-1 in Gudalur and Valparai. Based on the above data, half life, the pre-harvest interval (PHI) after application of bifenthrin in tea could be fixed as 7 days (Table 3). Studies on the infusion indicated that bifenthrin residues did not leach into the tea brew (Table 4).

Linda & Hooper (2002) reported the degradation of bifenthrin on several substrates. 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 synthetic pyrethroids in tropical soil and aquatic environment (Awasthi, 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 bifenthrin residue unavailable in short a period. Half-life of 0.52 – 0.77 days was reported by Chen & Wan (1988) for bifenthrin 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 & Muraleedharan, 1990; Bisen & Ghosh Hajara, 2000; Chen & Wan, 1988). The weight of these immature shoots increase during growth, depending on the plucking interval. The immature buds, by the time they attain the size of pluckable shoots, the residues of applied pesticides on them will undergo a growth dilution. Loss of residues ranging from 40 to 45%, specifically of pyrethroid group of chemicals due to growth dilution had also been reported (Xue & Chen, 1992). A rainfall shortly after the application plays a significant role in washing of applied pesticides (Chen & Wan, 1997) and might be the major reason for faster dissipation of residues in wet season as observed in the present study (Table 1). The present findings support the dissipation behavior of bifenthrin residues in tea during wet and dry conditions (Tewary et al., 2005). Withering of tea leaves was a major reason for total degradation. This might be due to evaporation during withering process. Chen & Wan (1988) reported 30 to 60 per cent of reduction in pesticide residues during processing, especially during drying. After 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 (Jaggi et al., 2000).

2.3 Transfer of residue from manufactured tea to infusion and spent leaves¬
The quantity of bifenthrin residues transferred from processed black tea to infusion was below 5 %. While the residues remained in the spent leaves, the infusion was almost free from bifenthrin at the recommended rate of application @ 1000 ml ha-1. Generally, only those pesticides with high water solubility are potentially transferred to the tea infusion, in significant amounts. The rate of transfer of the pesticide residues to the infusion depends on its solubility in water (Nagayama, 1996) and partition coefficient (Jaggi et al., 2001). Bifenthrin has very low water solubility (0.1 mg l^-1), fairly reasonable octanol-water coefficient (Kow 2,30,000) and low vapour pressure (1.81 x 10-7 mm Hg at 250 ºC) supported the findings (The Pesticide Manual, 2003). These factors account only for the partial transfer of residue into brew.
 
3 Conclusions
The transfer of bifenthrin residues from made tea to infusion was extremely low. On the basis of the above findings it can be concluded that at on or after the 7th day of application, there was no detectable transfer of residues to the infusion when applied at the recommended dosage (1000 ml ha^-1). Thus, the consumption of tea infusion is safe, when the leaves are processed after the normal harvesting interval, after the spraying of bifenthrin. 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 bifenthrin is 5 mg kg-1 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 4 days for bifenthrin.
 
4 Materials and methods
4.1 Field trials and experimental design
The experiments were conducted tea fields at Valparai and Gudalur (Tamil Nadu, India) situated at an elevation of 1140 m and 1150m 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 0.75x0.75x1.25 m under the shade tree, Grevillea robusta (6x6 m spacing). The tea plants were last pruned at a height of 60 cm above ground level. The treatments were bifenthrin 8 SC @ 1000 and 2000 mL/ha and an untreated control. The acaricide was applied with hand operated knapsack sprayer, using a spray volume of 400 ml ha-1. Tea shoots consisting of three leaves and a bud were harvested on 0 (3 hr), 1st, 3rd, 5 th, 7 th, 10 th and 14 th 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 hrs. 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 one hr. 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 (Perkin Elmer Clarus 500 GC) equipped with electron capture detector, following standard procedure.

4.2 Chemicals and reagents
Pesticide standard, bifenthrin 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.

4.3 Instrument and calibration
Bifenthrin technical standard of 99.0% 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 bifenthrin are given below. A Perkin Elmer Clarus 500 GC with an electron capture detector was used for separation and quantitative analysis, and a DB-5 Cross linked 5% PH ME Siloxane capillary column 30 m length, 0.32 mm internal diameter and 0.25µ film thickness) was used for gas chromatographic determination. Nitrogen at a speed of 5 ml min-1 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 µl 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 0.003–1.0 µg ml-1 with a correlation coefficient of 0.9996. The limit of detection was estimated to be 0.003 µg ml-1 of bifenthrin. The column was conditioned by three repeated injections of standard and sample extracts until GC peaks were reproducible.
 
4.4 Analysis
4.4.1 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 two hrs. 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.

4.4.2 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 co-extractives. The collected elution was concentrated at about 60°C to dryness and dissolved with 10 ml of hexane and injected into GLC as per the standard operating conditions (AOAC, 2005).
 
4.4.3 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.

4.4.4 Bifenthrin residues in tea brew
2 g of made tea was infused in 100 ml of boiling water (ISO 3103 -1990). After 6 minutes of brewing, the water extract was filtered, cooled and partitioned with 100 ml of hexane. The organic phase was passed through anhydrous sodium sulfate. The extract was concentrated by evaporating in a rotary vacuum evaporator and dissolved with 10 ml hexane and analysed for the residues of bifenthrin. The spent leaves were dried between the folds of filter paper and residues were extracted following the method described above for black tea.

Authors Contributions
SS conducted the field trials, sample preparation, residue analysis and interpreted the results. NM approved the protocol and drafted the manuscript.

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. and Muraleedharan N., 1990, Pesticide residues in tea, Planters’ Chronicle, 85: 125-127.

AOAC 2005, Official Method 998.01. Synthetic pyrethroids in agricultural products, Official methods Analysis, AOAC International, (18th Eds), Maryland, USA.
Awasthi M.D. and Prakash N.B., 1997, Persistence of chlorpyrifos in soil under different moisture regimes, Pesticide Science, 50: 1-4.
http://dx.doi.org/10.1002/(SICI)1096-9063(199705)50:1%3C1::AID-PS549%3E3.0.CO;2-X

Bisen J.S. and Ghosh Hajra N., 2000, Residues and persistence of certain insecticides in Darjeeling tea, Journal of Plantation Crops, 28(2): 123-131.

Chen Z.M. and Wan H., 1988, Factors affecting residues of pesticides in Tea. Pesticide Science, 23: 109-118.
http://dx.doi.org/10.1002/ps.2780230204

Chen Z.M. and Wan H., 1997, Degradation of pesticides on plant surfaces and its prediction. A case study on tea plant, Environmental Monitoring and Assessment, 44: 305-313.

Gu X.Z., Zhang G.Y., Chen L., Dai R.L. and Yu Y.C., 2008, Persistence and dissipation of synthetic pyrethroid pesticides in red soils from the Yangtze River Delta area, Environmental Geochemistry and Health, 30: 67–77.
http://dx.doi.org/10.1007/s10653-007-9108-y

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. and Shanker A., 2001, Leaching of pesticides in tea brew, Journal of Agricultural and Food Chemistry, 49: 5479-5483.
http://dx.doi.org/10.1021/jf010436d

Kumar V., Tewary D.K., Ravindranath S.D. and Shanker A., 2004, Investigation in tea on fate of fenazaquin residue and its transfer in brew, Food and Chemical Toxicology, 42: 423–428.
http://dx.doi.org/10.1016/j.fct.2003.10.004

Manikandan K.N., Muraleedharan N. and Selvasundaram R., 2001, Residues of deltamethrin in black CTC tea, Bulletin of UPASI Tea Research Foundation, 54: 88-92.

Manikandan K.N., Muraleedharan N. and Selvasundaram R., 2005, Degradation of quinalphos during the processing of black CTC tea, Journal of Plantation Crops, 33(2): 146-148.

Manikandan K.N., Smitha S., Seenivasan S., Muraleedharan N. and Selvasundaram R., 2006, Fenazaquin residues in tea and its persistence in south Indian climatic conditions, Journal of Plantation Crops, 34(3): 410-413.

Nagayama T., Maki T., Kan K., Iida M., Tamura Y. and Nishima T., 1996, Residues of organophosphorus pesticides in commercial tea and their leaching into tea, Nippon  Noyaku Gakkaishi, 14: 39-45.

Rajukkannu K., Blasubramanian M. and Vasudevan P., 1981, Residues of dicofol and tetradifon in tea leaves, Journal of Plantation Crops, 9(2): 124-125.

Ripley B.D., Ritcey G.M., Harris C.R., Denomme M.A. and Brown P.D., 2001, Pyrethroid insecticide residues on vegetable crops, Pest Management Science, 57: 683-687.  
http://dx.doi.org/10.1002/ps.325

Royal Society of Chemistry, 1994, The agrochemicals handbook (3rd ed.), Information Systems, Survey, England: Unwin Brothers Ltd.
Seenivasan S. and Muraleedharan N., 2009, Residues of lambda-cyhalothrin in tea, Food and Chemical Toxicology, 47(2): 502-505.
 http://dx.doi.org/10.1016/j.fct.2008.12.010

Singh R.P. and Agnihotri N. P., 1984, Residues of dicofol, endosulfan and Malathion on tea, Camellia sinensis (L) O.Kuntz, Journal of Entomological Research, 8(1):14-16.

Sood C., Jaggi S., Kumar V., Ravindranath S.D. and Shanker A., 2004, How manufacturing processes affect the level of pesticide residues in tea, Journal of Food and Agricultural Chemistry, 84: 2123–2127.
http://dx.doi.org/10.1002/jsfa.1774

Tewary D.K., Kumar V., Ravindranath S.D. and Shanker A., 2005, Dissipation behavior of bifenthrin residues in tea and its brew, Food Control, 16(3): 231-237.  
http://dx.doi.org/10.1016/j.foodcont.2004.02.004

The Pesticide Manual, (2003), 8th ed. UK: British Crop Protection Council.
Xue Y.Z. and Chen Z.M., 1985, Studies on the uptake, translocation, distribution and metabolism of carbofuran in the tea seedlings. Tea Science Research Journal, 2, 76-85.