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Investigation in tea on fate of fenazaquin residue and its transfer in brew
Food and Chemical Toxicology 44 (2006) 596–600 www.elsevier.com/locate/foodchemtox
Investigation in tea on fate of fenazaquin residue and its transfer in brew q Vipin Kumar, Dhananjay Kumar Tewary, Srigiripuram Desikachar Ravindranath, Adarsh Shanker
*
Institute of Himalayan Bioresource Technology, Council of Scientific and Industrial Research, Palampur 176 061, HP, India
Abstract Fenazaquin is a non-systemic acaricide/insecticide used widely in controlling mites and other related pests in fruits, vegetables and tea. The objective of this research was to investigate the disappearance trend in tea of fenazaquin residue level and its transfer in brew. Fenazaquin was applied on a tea crop at two rates, 125 and 250 g AI/ha in wet and dry seasons under field conditions. Samples (green shoots, made tea and its brew) were analyzed for fenazaquin and quantification was by high performance liquid chromatography using a UV detector. The residue dissipated faster in the wet season than in the dry season. Seven days after the treatment (normal round of plucking) the residues observed in the green shoots at the two rates were 2.17, 3.07 mg/kg and 2.04, 2.84 mg/kg in the wet and dry seasons, respectively. However, the degradation rale in both seasons followed first-order kinetics. Half-lives in green shoots were in range 1.43–1.70 and 2.10–2.21 days and in made tea 1.59–1.73 and 1.87–1.94 days for wet and dry seasons, respectively. During processing of green shoots to made tea considerable loss (42–70%) of residue was observed. The transfer of residue from made tea brew was in the range 3–22%. In brew residue were below 0.02 mg/l after 5 days of application at both the rates in either of the seasons. The estimated intake with brew (normal consumption of 10 cup/day/adult) thus would be below the acceptable daily intake for fenazaquin (0.005 mg/kg-body weight). To avoid health hazards due to the toxic effect of residues in brew, a waiting period for plucking the tea shoots after fenazaquin application of more than 5 days for both the seasons at recommended rate (125 g AI/ha) may be suggested and considered quite safe. 2005 Elsevier Ltd. All rights reserved. Keywords: Fenazaquin; Residue; Tea; Brew; Transfer
1. Introduction The tea crop is subject to attack from a wide range of insects and mite pests which besides causing crop loss deteriorate the quality of the processed tea. As has been the Abbreviations: AI, Active ingredient; ADI, Acceptable daily intake; EC, Emulsion concentrate; FAO, Food and Agriculture Organization; HPLC, High performance liquid chromatography; MRL, Maximum residue limit; ND, Not detected; US EPA, Environmental Protection Agency or United States; WHO, World Health Organization. q DOI of original article: 10.1016/j.fct.2003.10.004, 10.1016/j.fct.2005. 10.009. q IHBT Communication No. 0326. * Corresponding author. Tel.: +91 1894 230454; fax: +91 1894 230433. E-mail address: [email protected] (A. Shanker). 0278-6915/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2005.10.010
case with most large-scale agricultural ventures, the chemical control of pests dominates the tea growing environment as the single most widely used pest control strategy in almost all tea growing countries. One of the major disadvantages of pesticide use is that residue may remain in tea and may be transferred in infusion (brew) in amounts above maximum residue limits (MRLs). This could pose health hazards to consumers. This problem is being viewed seriously by international organizations (US EPA, Codex Alimenterious Commission, WHO and FAO of the United Nations). In the past few years there has been a continuous search for acaricide/insecticides with broad spectrum activity and minimum residual problems. Fenazaquin (IUPAC name: 4-tert-butylphenethyl quinazolin-4-yl ether), is a white to tan crystalline solid,
V. Kumar et al. / Food and Chemical Toxicology 44 (2006) 596–600
H2C
H2 C
O
CH3 CH3 CH3
N N Fig. 1. Structure of fenazaquin.
belonging to the quinazoline group (Fig. 1) of pesticides, for which the acceptable daily intake (ADI) was established at the level of 0.005 mg/kg body weight/day (Dow Elanco, 1993). It is marketed mainly in two formulation types, 200 g/l aqueous suspension concentrate and 100 g/l emulsion concentrate under various trade names, e.g. Magister, Matador, Totem, Demitam and Magus. Fenazaquin is a broad spectrum, non-systemic acaricidal compound, effective in controlling phytophagus mites infesting a variety of crops namely fruits and vegetables (Solomon et al., 1993) and tea (Shanker et al., 2001). It acts as an electron transport inhibitor, acting at Complex I of the mitochondrial respiratory chain (Hollingworth et al., 1992). This specific acaricide/insecticide has generally no effect on beneficial insects including predaceous mites (Hollingworth et al., 1992) and thus offers a desirable reason for its use in developing new strategies of integrated pest management in tea. Residue levels of many pesticides in tea and in its infusion have been reported (Chen et al., 1987; Chen and Wan, 1988; Muraleedharan, 1994; Bhattacharya et al., 1995; Jaggi et al., 2001). Residual studies of fenazaquin on some food commodities are available (Dow Elanco, 1993). However, no studies have been found in the literature on the dissipation in tea of fenazaquin residue and its transfer in brew from made tea (processed dry tea leaves). The present study was therefore, undertaken to generate data in wet and dry seasons in tea on the persistence of fenazaquin (Magister) residue and its transfer from made tea to infusion in hot water. This would help to establish adequate monitoring of the residue of this newly introduced acaricide/insecticides and its judicious incorporation in pest management strategies in tea fields. 2. Materials and methods 2.1. Field trials Two field trials (wet and dry season) were carried out at the IHBT tea experimental farm at Banuri, Palampur (32N · 76E), India. A random block design was used, each block containing 100 bushes (10 · 10) of Camellia sinensis (L.) O. Kurtze. Each block was separated from one another by leaving two untreated rows as guard rows to prevent pesticide from spill over. Fenazaquin was sprayed at two rates 125 g AI/ha (recommended) and 250 g AI/ha (double the recommended) in four replications with hand operated Knapsack sprayer using a recommended formulation volume of 400 1/ha. In control blocks only water was sprayed. About 2 kg of the green shoots (two leaves and a bud) was harvested from each replicate of treated and control plots and brought to
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the laboratory each time at 0 (1 h after spraying), 1, 3, 5, 7, 9, 11, 14 and 21 days after the treatment.
2.2. Reagent and apparatus 2.2.1. Analytical standards and working solutions An analytical standard of fenazaquin (active substance) and its commercial grade formulation (Magister 10EC) were supplied by De-Nocil Crop Protection Ltd., India. A standard stock solution (1000 mg/l) was prepared in acetonitrile and the solutions required for preparing a standard curve (0, 0.1, 0.2, 0.4, 0.8 and 1.0 lg/ml) were prepared from the standard stock solution by serial dilutions. All the solvents and chemicals used were of Analytical Grade from E. Merck. 2.2.2. Apparatus A high performance liquid chromatograph, LaChrom–Merck, equipped with a LiChrospher 100 C18 (reverse phase) endcapped (5 lm) column and a UV detector was used. The mobile phase was acetonitrile– water (80:20v/v) with 1 ml/min flow rate. The column oven was kept at 30 C. The best detection was attained at wavelength of 218 nm. The volume of the injection was 10 ll. 2.2.3. Florisil column Glass columns (30 cm · 1.1 cm i.d.) with Teflon stopcocks were packed from the bottom with a glass wool plug and 2.5 cm of Merck brand activated Florisil (60–100 mesh) between the layers of anhydrous sodium sulfate.
2.3. Tea leaves and infusion The untreated control and treated green leaves from the field were processed in the laboratory’s mini manufacturing unit using conventional an orthodox tea manufacturing process. The manufacturing process, in brief, involved withering of shoots at ambient temperature for 15–20 h; rolling (twisting and rupturing the tissue to express the juice) using a piezy roller for about 30 min with pressure followed by fermentation (oxidation) for 1–2 h at 25–30 C and 95% r.h. and finally drying in a tea dryer using hot air at 100 ± 5 C to a final moisture content of 2–3%. The made tea was further subjected to an infusion process wherein 5 g of manufactured tea was infused in 150 ml of boiling water. After 3 min of brewing, the water extract was filtered through a (2-lm sieve) stainless steel filter, cooled and examined for residue transfer from made tea. The matrices used for residue determination were the green shoots, made tea, the infusion prepared and the spent leaves left in the stainless steel filter.
2.4. Extraction 2.4.1. Green leaves and made tea Samples of green tea leaves (25 g) and made tea (10 g) were extracted with 150 and 100 ml of dichloromethane, respectively by mechanical shaking for 2 h. Extracts were filtered through a Whatman No. 1 filter paper containing 2 g of anhydrous sodium sulfate (impregnated with dichloromethane). 2.4.2. Infusion and spent leaves After removing the spent leaves, infusion was cooled to room temperature and transferred to a separating funnel (500 ml). The pesticide was partitioned (extracted) into 100 ml dichloromethane twice. The organic layer was separated and collected in a 250-ml beaker. The spent leaves were dried between the folds of filter paper and residues were extracted following similar method mentioned for made tea.
2.5. Cleanup The extracts were concentrated to 5 ml under a gentle air stream and transferred to a florisil column pre-washed with 50 ml of n-hexane to
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remove the coextractives. The column was eluted with 20 ml n-hexane followed by 10 ml of n-hexane and ethyl acetate (99:1 v/v). The eluates were collected in a 50-ml beaker, evaporated to dryness and finally reconstituted in 2 ml acetonitrile for final detection and quantification.
2.8. Statistical analysis The experimental data were subjected to statistical analysis using Microsoft Excel program (Windows 2000).
3. Results 2.6. Detection and quantification Quantification was accomplished by using a standard curve prepared by diluting the stock solution in acetonitrile. Good linearity was achieved between the range 0.1 and 1.0 lg/ml with a correlation coefficient of 0.9998. The limit of detection was estimated to be 0.05 lg/ml of pesticide, based on signal to noise ratio 2:1. The column was conditioned by repeated injections (2–4) of standard and sample extracts until HPLC peaks were reproducible. Recoveries of the pesticide at different fortification levels, i.e. 0.5, 1, 2, 5 and 10 lg/ml were determined in five replicates from each matrix to validate and evaluate the accuracy of the method.
2.7. Calculation of percent transfer of residue Percentage transfer of pesticide from made tea to brew ami spent leaves were calculated by following formula: Transferð%Þ ¼
C S AS 100; C mt W mt
CS = pesticide concentration (mg/kg) in substrate (brew or spent leaves); AS = volume or weight (ml or g) of substrate, i.e. brew or spent leaves; Cmt = pesticide concentration in made tea (mg/kg); Wmt = weight of made tea taken for infusion (g). As mentioned in Section 2.3, 5 g made tea was used to make brew in 150 ml of boiling water.
Data on residue dissipation in green shoots, made tea, transfer of residue in brew and spent leaves are presented in Table 1. Initial residues deposits in green shoots at two rates, 125 and 250 g AI/ha, were 29.42, 56.57 mg/kg and 17.90, 33.37 mg/kg in the wet and dry seasons, respectively. In made tea, processed from the samples of green shoots collected at different time intervals, initial residue deposits were 58.94, 96.37 mg/kg and 31.19, 58.76 mg/kg during wet and dry seasons, respectively. At normal plucking round (seventh day after application of insecticides) 5.43– 7.37% and 6.18–7.31% of the residue (in wet season) and 8.51–11.40% and 6.08–7.79% of the residue (in dry season) remained in green and made tea, respectively. No measurable amount of fenazaquin residue was observed after 11th day in either of the seasons. From the half-life value (Table 1), it is evident that the initial level of residue in green shoots dropped by half in 34.32–40.80 h in the wet season while in the dry season it takes 50–52 h. Similarly half-lives in made tea were in the range 38.16–41.52 and 46–50 h in wet and dry seasons, respectively. This indicated faster residue dissipation in the wet season then in the dry season.
Table 1 Fenazaquin residue in tea and its brewa Time interval (days)
Residue (mg kg1) Green leaves
Brew*
Made tea
Spent leaves
Trt. 1
Trt. 2
Trt. 1
Trt. 2
Trt. 1
Trt. 2
Trt. 1
Trt. 2
Wet season 0 1 3 5 7 9 11 14 21 t1/2
29.42 ± 2.08 12.02 ± 0.74 6.86 ± 0.42 3.87 ± 0.39 2.17 ± 0.56 0.62 ± 0.19 0.02 ± 0.02 ND ND 1.70
56.57 ± 3.35 20.83 ± 7.30 10.91 ± 0.78 5.58 ± 0.57 3.07 ± 0.57 1.08 ± 0.13 0.04 ± 0.04 ND ND 1.43
58.94 ± 2.12 22.04 ± 5.52 12.71 ± 0.88 6.64 ± 1.59 3.64 ± 0.31 1.23 ± 0.04 0.00 ± 0.01 ND ND 1.73
96.37 ± 8.56 31.48 ± 6.80 16.06 ± 1.47 10.54 ± 1.04 7.04 ± 0.37 1.91 ± 0.57 0.01 ± 0.01 ND ND 1.59
0.34 0.14 0.09 0.02 0.01 ND ND ND ND –
0.72 0.17 0.07 0.04 0.02 ND ND ND ND –
27.51 (46.67) 9.67 (43.90) 4.25 (33.47) 1.21 (18.27) 0.38 (10.54) 0.12 (9.76) ND ND ND –
36.00 (37.36) 15.62 (49.63) 6.96 (43.35) 2.31 (21.95) 0.70 (9.90) 0.21 (11.01) ND ND ND –
Dry season 0 1 3 5 7 9 11 14 21 t1/2
17.90 ± 1.42 10.83 ± 0.83 7.19 ± 0.52 4.36 ± 0.34 2.02 ± 0.09 1.00 ± 0.11 0.23 ± 0.03 ND ND 2.21
33.37 ± 2.54 23.82 ± 1.52 13.05 ± 0.54 6.26 ± 0.25 2.84 ± 0.17 1.34 ± 0.08 0.55 ± 0.08 ND ND 2.10
31.19 ± 1.98 16.84 ± 0.99 10.59 ± 0.93 5.77 ± 0.74 2.43 ± 0.15 1.12 ± 0.04 0.26 ± 0.02 ND ND 1.87
58.76 ± 2.03 41.93 ± 1.62 22.05 ± 1.03 8.87 ± 0.80 3.57 ± 0.23 1.34 ± 0.04 0.55 ± 0.03 ND ND 1.94
0.153 0.060 0.030 0.013 ND ND ND ND ND –
12.76 (40.91) 8.78 (52.13) 4.42 (41.72) 2.42 (41.97) 0.51 (21.02) 0.04 (3.57) ND ND ND –
24.70 (42.03) 17.49 (41.71) 7.11 (32.24) 3.38 (38.11) 0.78 (21.83) 0.21 (15.63) 0.05 (9.04) ND ND –
(17.27) (19.38) (20.46) (7.53) (8.25)
(14.71) (10.69) (8.50) (6.76)
(22.40) (16.42) (12.45) (10.44) (8.52)
0.337 0.207 0.087 0.020 0.003 ND ND ND ND –
(17.19) (14.79) (11.79) (6.76) (2.80)
Trt. 1—treatment No. 1 (spray at 125 g AI/ha); Trt. 2—treatment No. 2 (spray at 250 g AI/ha); ND—not detected (limit of detection, 0.05 mg/ml); – not applicable; t1/2—half-life in days; values in parentheses: percent transfer of residues from made tea. a In untreated control samples sprayed only with water, at all time intervals, no residue of fenazaquin was detected.
Loss (%) of residue in made tea
V. Kumar et al. / Food and Chemical Toxicology 44 (2006) 596–600 y = 0.0187x2 - 1.3524x + 66.928 R2 = 0.8387
80 70 60 50 40 30 20 10 0 0
5
10
15
20
25
30
35
Conc. (mg/kg) of residue in green tea
Transfer (%) of residue in brew
Fig. 2. Loss of fenazaquin residue during tea manufacturing.
y = -0.0013x2 + 0.2806x + 6.2838 R2 = 0.9204
25 20 15 10 5 0 0
10
20
30
40
50
60
70
Conc. (mg/kg) in made tea
Fig. 3. Transfer of fenazaquin residue in brew.
Considerable loss of residue was observed during the processing of green shoots to made tea. The loss of residue decreases as residue concentration in made tea increases (Fig. 2). During infusion only partial transfer (3–22%) of fenazaquin in brew has been observed (Table 1). Transfer of residue increased with an increase in concentration of residue in-made tea (Fig. 3). The maximum residue in brew (0.72 mg/l) was detected in the 0 day sample (wet season) of made tea with 96.37 mg/kg residue deposit. No measurable residue was found in brew when made from the samples harvested and processed after 7 and 5 days of treatment with recommended dose in wet and dry season, respectively. The range of pesticide remaining with spent leaves was 3.57–52.13%. However, a relationship with made tea residue concentration and seasonal variations was not observed. 4. Discussion The rapid degradation of fenazaquin and its metabolites in plants (citrus, pome and apple) has been reported (Dow Elanco, 1993). In tea field, besides the effect of some physical and chemical factors like photo, thermal, pH and moisture (Cosby et al., 1972; Miller and Zepp, 1983; Chen et al., 1987; Agnihothrudu and Muraleedharan, 1990; Miller and Donaldson, 1994), growth dilution factor (Chen and Wan, 1988; Agnihothrudu and Muraleedharan, 1990; Bisen and
599
Ghosh Hajara, 2000) might have played significant role and rendered fenazaquin residue unavailable in the short period of time in both wet (half-lives, 1.43–1.7 days) and dry seasons (half-lives, 2.10–2.21 days). At normal plucking round (seventh day after treatment) at both the doses P98% residues in wet season and P88% residues in dry seasons disappeared in green shoots and made tea. This difference in disappearance of fenazaquin residues in tea in different seasons may be attributed lo the agro-climatic conditions (Bhattacharya et al., 1995; Maniknandan et al., 2001). The ether linkage between quinazoline and ethyl phenyl components of fenazaquin molecule is believed to be the most photo liable portion (Hatton et al., 1992). During dry periods the overexposure of sunlight might cause considerable degradation of fenazaquin residue in tea shoots. The growth dilution factor is important in reducing the residue levels in tea crop as the foliage and the shoots on which pesticides are applied are in different stages of growth. The weight of these immature shoots increases over a period of 6–14 days, depending on the plucking interval. The immature buds by the time attain the size of pluckable shoots; the residue of applied pesticides on them will undergo a growth dilution. During processing of green shoots, leaves are dried and decreased a weight by a factor of 3.33. Theoretically, residues in made tea should be higher by the same factor but results show (Table 1) that the residue deposit in made tea is less (647%) than that of the value of theoretically calculated residue level. However, it is also evident (Fig. 2) that loss of residue in made tea increased with decrease in concentration of residues in green shoots. The present finding supports the studies reporting loss of many pesticides during processing (Chen and Wan, 1988). The loss of pesticides during processing might be due to three factors i.e. evaporation, degradation and codistillation (Cabras et al., 1998). The transfer rate of the pesticide residue in brew depends on the water solubility (Wan et al., 1991; Nagayama, 1996), partition coefficient (Tsumura-Hasegawa et al., 1992; Jaggi et al., 2001) and low vapour pressure (Chen and Wan, 1988). Fenazaquin has low solubility in water (102 lg/l) but its fairly reasonable organic matter adsorption capability and partitioning coefficient (Kow = 321,000) allows its transfer to brew in a considerable proportion. Loss of fenazaquin in brew due to fugacity would be low as the molecule has low vapour pressure (3.4 · 106 Pa at 25 C) as is solubility. However, the acidic nature of brew may favour its rapid degradation (hydrolysis) at elevated temperature (Dow Elanco, 1993). Considerable loss of fenazaquin residue from brew may also be due to adsorption to the organic component of the brew, i.e. leaves. The collective role of all these factors might be accountable for only partial transfer of residue in brew. The residue remained on spent leaf did not show any trend. It is evident (Table 1) that made tea processed from green shoots collected after the fifth day in either of the seasons are likely to have a residue level up to 3.64 mg/kg at
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recommended dose. As tea is subjected to infusion prior to consumption, it is the residue transferred in brew which actually contributes to the dietary intake. Considering a maximum of 10 cup of tea (150 ml per cup) consumption by an individual, the actual intake of residue on the basis of residue data in brew, is only 0.015 mg of fenazaquin when brew is prepared from the made tea processed from the samples treated at recommended rate and harvested after 5 days of the treatment in either of the seasons. The estimated intake with brew (maximum consumption of 10 cup/day/adult) thus would be below the acceptable daily intake for fenazaquin (0.005 mg/kg-body weight). The MRLs in tea in most of the cases are fixed on the basis of pesticides residue present in made tea (European Union, 2002). However, it is the actual residue of pesticides in brew that should be considered for setting up realistic MRLs. Fenazaquin like many of the pesticides listed for tea do not have yet firm limits assigned because of lack of data representing the actual residue contributing to the dietary intake. Therefore, for the time being importing countries are free to maintain or set their own tentative limits or to adopt stipulations fixed by an international organization such as Codex Alimenterious Commission, which sets standards for the World Health Organization and the Food and Agricultural Organization of the United Nations (Jaggi et al., 2001). In this context, data of the present research are very relevant and useful for setting up standards for pesticide safety limit considered in regulating problems with consumption of tea and its global trade. 5. Conclusion Tea is generally cultivated under identical agro-climatic conditions and for commercial cultivation the most common species of tea is C. sinensis. In the present experiment the same species of tea cultivated under common agroclimatic conditions was used. The results showed that fenazaquin residue in tea, prior to consumption in the form of brew, dissipates at three stages: (1) in green shoots residues disappear after 11 days of treatment in both seasons; (2) during processing to made tea with no residues in the sample from 11 to 14 days of wet and dry seasons, respectively and thus balancing the factors accountable for increased concentration of residue deposits in made tea from green shoots; and (3) during infusion in brew with residue free samples after 7 days in both the seasons, respectively. The acceptable daily intake for fenazaquin is 0.005 mg/kg body weight (Dow Elanco, 1993). As tea is submitted to infusion/prior to consumption, a waiting period for more than 5 days for both the seasons at the recommended rate (125 g AI/ha) may be suggested and considered quite safe to avoid health hazards due to the toxic effect of residues in brew. Thus the present findings are useful in monitoring the fenazaquin residue in tea.
References Agnihothrudu, V., Muraleedharan, N., 1990. Pesticide residues in tea. Planters Chronicle 85, 125–127. Bhattacharya, A., Chowdhury, A., Somchowdhury, A.K., Pallarl, A.K., Roy, U.S., 1995. Studies on residues, persistence and pre-harvest interval of cythion, durmet and ripcord in made tea of Darjeeling. Peslology 21 (2), 28–36. Bisen, J.S., Ghosh Hajara, N., 2000. Persistence and degradation of some insecticides in Darjeeling tea. Journal of Plantation Crop 28 (2), 123– 131. Cabras, P., Angioni, A., Garau, V.L., Menelli, E.V., Cabitza, F., Pala, M., 1998. Pesticide residue in raisin processing. Journal of Agriculture and Food Chemistry 46, 1309–2311. Chen, Z.M., Wan, H.B., 1988. Factors affecting residues of pesticides in tea. Pesticide Science 23, 109–118. Chen, Z.M., Wan, H.B., Wang, Y., Xue, Y., Xia, H., 1987. Fate of pesticides in the ecosystem of tea garden. In: Proceedings of the International Symposium on Tea Quality-Human Health. Tea Research Institute, Chinese Academy of Agriculture Sciences, Hangzhou, China, pp. 146–149 (4–9 November). Cosby, P.G., Moilanon, K.W., Nakagawa, M., Wong, A.S., 1972. Environmental Toxicology of Pesticides. Academic Press, New York. Dow Elanco, 1993. Fenazaquin—A Profile. Department of Regulatory Toxicology and Environmental Affairs, Agriculture Products Research and Development, Oxfordshire, UK. European Union, 2002. Appendix I EU/German Maximum Residue Limits on Tea, Appendices, 16 December. Hatton, C.J., Babcock, J.M., Schoonover, J.R., Dripps, J.E., 1992. Fenazaquin, a new acaricide/insecticide. In: Proceedings of the International Cong. Ent., p. 535. Hollingworth, R.M., Ahammadsahip, K.I., Gadelhak, G.G., Mclughin, J.L., 1992. Complex I of mitochondrial respiratory chain: a target for pesticide development by both man and nature. In: Proceedings of the American Chemical Society, Abs. No. 156. Jaggi, S., Sood, C., Kumar, V., Ravindranath, S.D., Shanker, A., 2001. Leaching of pesticides in tea brew. Journal of Agriculture and Food Chemistry 49, 5479–5483. Manikandan, K.N., Muraleedharan, N., Selvasundaram, R., 2001. Residues and persistence of chlorpyrifos in processed black tea. Journal of Plantation Crops 29 (3), 35–37. Miller, G.C., Donaldson, S.G., 1994. Factor affecting photolysis of organic compounds on soils. In: Helz, G.R., Zepp, R.G., Crosby, D.G. (Eds.), Aquatic and Surface Photochemistry. Lewis Publishers, Boca Raton, FL, pp. 97–109. Miller, G.C., Zepp, R.G., 1983. Extrapolating photolysis rate from the laboratory to the environment. Residue Review 85, 89–110. Muraleedharan, N., 1994. Pesticide residues in tea: problems and prospective. Planters Chronicle 9, 371–375. Nagayama, T., 1996. Behavior of residual organ phosphorus pesticides in foodstuffs during leaching or cooking. Journal of Agriculture and Food Chemistry 44, 2388–2393. Shanker, A., Jasrotia, P., Kumar, A., Jaggi, S., Kurnar, V., Sood, C., 2001. Bioefficacy of new milicide: fenazaquin. Pestology 25 (6), 57–60. Solomon, M.G., Fitzgerald, J.D., Ridout, M.S., 1993. Fenazaquin, a selective acaricide for use in IPM in the UK. Crop Protection 12, 255– 258. Tsumura-Hasegawa, Y., Tonogai, Y., Nakamura, Y., Ito, T., 1992. Residues of post harvest application of pesticides in citrus fruits after storage and processing into lemon marmalade. Shokuhin Eisegaku Zasshi 33, 258–266. Wan, H., Xia, H., Chen, Z., 1991. Extraction of pesticide residue in tea by water during the infusion process. Food Additives and Contaminations 8, 497–500.
Investigation in tea on fate of fenazaquin residue and its transfer in brew q Vipin Kumar, Dhananjay Kumar Tewary, Srigiripuram Desikachar Ravindranath, Adarsh Shanker
*
Institute of Himalayan Bioresource Technology, Council of Scientific and Industrial Research, Palampur 176 061, HP, India
Abstract Fenazaquin is a non-systemic acaricide/insecticide used widely in controlling mites and other related pests in fruits, vegetables and tea. The objective of this research was to investigate the disappearance trend in tea of fenazaquin residue level and its transfer in brew. Fenazaquin was applied on a tea crop at two rates, 125 and 250 g AI/ha in wet and dry seasons under field conditions. Samples (green shoots, made tea and its brew) were analyzed for fenazaquin and quantification was by high performance liquid chromatography using a UV detector. The residue dissipated faster in the wet season than in the dry season. Seven days after the treatment (normal round of plucking) the residues observed in the green shoots at the two rates were 2.17, 3.07 mg/kg and 2.04, 2.84 mg/kg in the wet and dry seasons, respectively. However, the degradation rale in both seasons followed first-order kinetics. Half-lives in green shoots were in range 1.43–1.70 and 2.10–2.21 days and in made tea 1.59–1.73 and 1.87–1.94 days for wet and dry seasons, respectively. During processing of green shoots to made tea considerable loss (42–70%) of residue was observed. The transfer of residue from made tea brew was in the range 3–22%. In brew residue were below 0.02 mg/l after 5 days of application at both the rates in either of the seasons. The estimated intake with brew (normal consumption of 10 cup/day/adult) thus would be below the acceptable daily intake for fenazaquin (0.005 mg/kg-body weight). To avoid health hazards due to the toxic effect of residues in brew, a waiting period for plucking the tea shoots after fenazaquin application of more than 5 days for both the seasons at recommended rate (125 g AI/ha) may be suggested and considered quite safe. 2005 Elsevier Ltd. All rights reserved. Keywords: Fenazaquin; Residue; Tea; Brew; Transfer
1. Introduction The tea crop is subject to attack from a wide range of insects and mite pests which besides causing crop loss deteriorate the quality of the processed tea. As has been the Abbreviations: AI, Active ingredient; ADI, Acceptable daily intake; EC, Emulsion concentrate; FAO, Food and Agriculture Organization; HPLC, High performance liquid chromatography; MRL, Maximum residue limit; ND, Not detected; US EPA, Environmental Protection Agency or United States; WHO, World Health Organization. q DOI of original article: 10.1016/j.fct.2003.10.004, 10.1016/j.fct.2005. 10.009. q IHBT Communication No. 0326. * Corresponding author. Tel.: +91 1894 230454; fax: +91 1894 230433. E-mail address: [email protected] (A. Shanker). 0278-6915/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2005.10.010
case with most large-scale agricultural ventures, the chemical control of pests dominates the tea growing environment as the single most widely used pest control strategy in almost all tea growing countries. One of the major disadvantages of pesticide use is that residue may remain in tea and may be transferred in infusion (brew) in amounts above maximum residue limits (MRLs). This could pose health hazards to consumers. This problem is being viewed seriously by international organizations (US EPA, Codex Alimenterious Commission, WHO and FAO of the United Nations). In the past few years there has been a continuous search for acaricide/insecticides with broad spectrum activity and minimum residual problems. Fenazaquin (IUPAC name: 4-tert-butylphenethyl quinazolin-4-yl ether), is a white to tan crystalline solid,
V. Kumar et al. / Food and Chemical Toxicology 44 (2006) 596–600
H2C
H2 C
O
CH3 CH3 CH3
N N Fig. 1. Structure of fenazaquin.
belonging to the quinazoline group (Fig. 1) of pesticides, for which the acceptable daily intake (ADI) was established at the level of 0.005 mg/kg body weight/day (Dow Elanco, 1993). It is marketed mainly in two formulation types, 200 g/l aqueous suspension concentrate and 100 g/l emulsion concentrate under various trade names, e.g. Magister, Matador, Totem, Demitam and Magus. Fenazaquin is a broad spectrum, non-systemic acaricidal compound, effective in controlling phytophagus mites infesting a variety of crops namely fruits and vegetables (Solomon et al., 1993) and tea (Shanker et al., 2001). It acts as an electron transport inhibitor, acting at Complex I of the mitochondrial respiratory chain (Hollingworth et al., 1992). This specific acaricide/insecticide has generally no effect on beneficial insects including predaceous mites (Hollingworth et al., 1992) and thus offers a desirable reason for its use in developing new strategies of integrated pest management in tea. Residue levels of many pesticides in tea and in its infusion have been reported (Chen et al., 1987; Chen and Wan, 1988; Muraleedharan, 1994; Bhattacharya et al., 1995; Jaggi et al., 2001). Residual studies of fenazaquin on some food commodities are available (Dow Elanco, 1993). However, no studies have been found in the literature on the dissipation in tea of fenazaquin residue and its transfer in brew from made tea (processed dry tea leaves). The present study was therefore, undertaken to generate data in wet and dry seasons in tea on the persistence of fenazaquin (Magister) residue and its transfer from made tea to infusion in hot water. This would help to establish adequate monitoring of the residue of this newly introduced acaricide/insecticides and its judicious incorporation in pest management strategies in tea fields. 2. Materials and methods 2.1. Field trials Two field trials (wet and dry season) were carried out at the IHBT tea experimental farm at Banuri, Palampur (32N · 76E), India. A random block design was used, each block containing 100 bushes (10 · 10) of Camellia sinensis (L.) O. Kurtze. Each block was separated from one another by leaving two untreated rows as guard rows to prevent pesticide from spill over. Fenazaquin was sprayed at two rates 125 g AI/ha (recommended) and 250 g AI/ha (double the recommended) in four replications with hand operated Knapsack sprayer using a recommended formulation volume of 400 1/ha. In control blocks only water was sprayed. About 2 kg of the green shoots (two leaves and a bud) was harvested from each replicate of treated and control plots and brought to
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the laboratory each time at 0 (1 h after spraying), 1, 3, 5, 7, 9, 11, 14 and 21 days after the treatment.
2.2. Reagent and apparatus 2.2.1. Analytical standards and working solutions An analytical standard of fenazaquin (active substance) and its commercial grade formulation (Magister 10EC) were supplied by De-Nocil Crop Protection Ltd., India. A standard stock solution (1000 mg/l) was prepared in acetonitrile and the solutions required for preparing a standard curve (0, 0.1, 0.2, 0.4, 0.8 and 1.0 lg/ml) were prepared from the standard stock solution by serial dilutions. All the solvents and chemicals used were of Analytical Grade from E. Merck. 2.2.2. Apparatus A high performance liquid chromatograph, LaChrom–Merck, equipped with a LiChrospher 100 C18 (reverse phase) endcapped (5 lm) column and a UV detector was used. The mobile phase was acetonitrile– water (80:20v/v) with 1 ml/min flow rate. The column oven was kept at 30 C. The best detection was attained at wavelength of 218 nm. The volume of the injection was 10 ll. 2.2.3. Florisil column Glass columns (30 cm · 1.1 cm i.d.) with Teflon stopcocks were packed from the bottom with a glass wool plug and 2.5 cm of Merck brand activated Florisil (60–100 mesh) between the layers of anhydrous sodium sulfate.
2.3. Tea leaves and infusion The untreated control and treated green leaves from the field were processed in the laboratory’s mini manufacturing unit using conventional an orthodox tea manufacturing process. The manufacturing process, in brief, involved withering of shoots at ambient temperature for 15–20 h; rolling (twisting and rupturing the tissue to express the juice) using a piezy roller for about 30 min with pressure followed by fermentation (oxidation) for 1–2 h at 25–30 C and 95% r.h. and finally drying in a tea dryer using hot air at 100 ± 5 C to a final moisture content of 2–3%. The made tea was further subjected to an infusion process wherein 5 g of manufactured tea was infused in 150 ml of boiling water. After 3 min of brewing, the water extract was filtered through a (2-lm sieve) stainless steel filter, cooled and examined for residue transfer from made tea. The matrices used for residue determination were the green shoots, made tea, the infusion prepared and the spent leaves left in the stainless steel filter.
2.4. Extraction 2.4.1. Green leaves and made tea Samples of green tea leaves (25 g) and made tea (10 g) were extracted with 150 and 100 ml of dichloromethane, respectively by mechanical shaking for 2 h. Extracts were filtered through a Whatman No. 1 filter paper containing 2 g of anhydrous sodium sulfate (impregnated with dichloromethane). 2.4.2. Infusion and spent leaves After removing the spent leaves, infusion was cooled to room temperature and transferred to a separating funnel (500 ml). The pesticide was partitioned (extracted) into 100 ml dichloromethane twice. The organic layer was separated and collected in a 250-ml beaker. The spent leaves were dried between the folds of filter paper and residues were extracted following similar method mentioned for made tea.
2.5. Cleanup The extracts were concentrated to 5 ml under a gentle air stream and transferred to a florisil column pre-washed with 50 ml of n-hexane to
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remove the coextractives. The column was eluted with 20 ml n-hexane followed by 10 ml of n-hexane and ethyl acetate (99:1 v/v). The eluates were collected in a 50-ml beaker, evaporated to dryness and finally reconstituted in 2 ml acetonitrile for final detection and quantification.
2.8. Statistical analysis The experimental data were subjected to statistical analysis using Microsoft Excel program (Windows 2000).
3. Results 2.6. Detection and quantification Quantification was accomplished by using a standard curve prepared by diluting the stock solution in acetonitrile. Good linearity was achieved between the range 0.1 and 1.0 lg/ml with a correlation coefficient of 0.9998. The limit of detection was estimated to be 0.05 lg/ml of pesticide, based on signal to noise ratio 2:1. The column was conditioned by repeated injections (2–4) of standard and sample extracts until HPLC peaks were reproducible. Recoveries of the pesticide at different fortification levels, i.e. 0.5, 1, 2, 5 and 10 lg/ml were determined in five replicates from each matrix to validate and evaluate the accuracy of the method.
2.7. Calculation of percent transfer of residue Percentage transfer of pesticide from made tea to brew ami spent leaves were calculated by following formula: Transferð%Þ ¼
C S AS 100; C mt W mt
CS = pesticide concentration (mg/kg) in substrate (brew or spent leaves); AS = volume or weight (ml or g) of substrate, i.e. brew or spent leaves; Cmt = pesticide concentration in made tea (mg/kg); Wmt = weight of made tea taken for infusion (g). As mentioned in Section 2.3, 5 g made tea was used to make brew in 150 ml of boiling water.
Data on residue dissipation in green shoots, made tea, transfer of residue in brew and spent leaves are presented in Table 1. Initial residues deposits in green shoots at two rates, 125 and 250 g AI/ha, were 29.42, 56.57 mg/kg and 17.90, 33.37 mg/kg in the wet and dry seasons, respectively. In made tea, processed from the samples of green shoots collected at different time intervals, initial residue deposits were 58.94, 96.37 mg/kg and 31.19, 58.76 mg/kg during wet and dry seasons, respectively. At normal plucking round (seventh day after application of insecticides) 5.43– 7.37% and 6.18–7.31% of the residue (in wet season) and 8.51–11.40% and 6.08–7.79% of the residue (in dry season) remained in green and made tea, respectively. No measurable amount of fenazaquin residue was observed after 11th day in either of the seasons. From the half-life value (Table 1), it is evident that the initial level of residue in green shoots dropped by half in 34.32–40.80 h in the wet season while in the dry season it takes 50–52 h. Similarly half-lives in made tea were in the range 38.16–41.52 and 46–50 h in wet and dry seasons, respectively. This indicated faster residue dissipation in the wet season then in the dry season.
Table 1 Fenazaquin residue in tea and its brewa Time interval (days)
Residue (mg kg1) Green leaves
Brew*
Made tea
Spent leaves
Trt. 1
Trt. 2
Trt. 1
Trt. 2
Trt. 1
Trt. 2
Trt. 1
Trt. 2
Wet season 0 1 3 5 7 9 11 14 21 t1/2
29.42 ± 2.08 12.02 ± 0.74 6.86 ± 0.42 3.87 ± 0.39 2.17 ± 0.56 0.62 ± 0.19 0.02 ± 0.02 ND ND 1.70
56.57 ± 3.35 20.83 ± 7.30 10.91 ± 0.78 5.58 ± 0.57 3.07 ± 0.57 1.08 ± 0.13 0.04 ± 0.04 ND ND 1.43
58.94 ± 2.12 22.04 ± 5.52 12.71 ± 0.88 6.64 ± 1.59 3.64 ± 0.31 1.23 ± 0.04 0.00 ± 0.01 ND ND 1.73
96.37 ± 8.56 31.48 ± 6.80 16.06 ± 1.47 10.54 ± 1.04 7.04 ± 0.37 1.91 ± 0.57 0.01 ± 0.01 ND ND 1.59
0.34 0.14 0.09 0.02 0.01 ND ND ND ND –
0.72 0.17 0.07 0.04 0.02 ND ND ND ND –
27.51 (46.67) 9.67 (43.90) 4.25 (33.47) 1.21 (18.27) 0.38 (10.54) 0.12 (9.76) ND ND ND –
36.00 (37.36) 15.62 (49.63) 6.96 (43.35) 2.31 (21.95) 0.70 (9.90) 0.21 (11.01) ND ND ND –
Dry season 0 1 3 5 7 9 11 14 21 t1/2
17.90 ± 1.42 10.83 ± 0.83 7.19 ± 0.52 4.36 ± 0.34 2.02 ± 0.09 1.00 ± 0.11 0.23 ± 0.03 ND ND 2.21
33.37 ± 2.54 23.82 ± 1.52 13.05 ± 0.54 6.26 ± 0.25 2.84 ± 0.17 1.34 ± 0.08 0.55 ± 0.08 ND ND 2.10
31.19 ± 1.98 16.84 ± 0.99 10.59 ± 0.93 5.77 ± 0.74 2.43 ± 0.15 1.12 ± 0.04 0.26 ± 0.02 ND ND 1.87
58.76 ± 2.03 41.93 ± 1.62 22.05 ± 1.03 8.87 ± 0.80 3.57 ± 0.23 1.34 ± 0.04 0.55 ± 0.03 ND ND 1.94
0.153 0.060 0.030 0.013 ND ND ND ND ND –
12.76 (40.91) 8.78 (52.13) 4.42 (41.72) 2.42 (41.97) 0.51 (21.02) 0.04 (3.57) ND ND ND –
24.70 (42.03) 17.49 (41.71) 7.11 (32.24) 3.38 (38.11) 0.78 (21.83) 0.21 (15.63) 0.05 (9.04) ND ND –
(17.27) (19.38) (20.46) (7.53) (8.25)
(14.71) (10.69) (8.50) (6.76)
(22.40) (16.42) (12.45) (10.44) (8.52)
0.337 0.207 0.087 0.020 0.003 ND ND ND ND –
(17.19) (14.79) (11.79) (6.76) (2.80)
Trt. 1—treatment No. 1 (spray at 125 g AI/ha); Trt. 2—treatment No. 2 (spray at 250 g AI/ha); ND—not detected (limit of detection, 0.05 mg/ml); – not applicable; t1/2—half-life in days; values in parentheses: percent transfer of residues from made tea. a In untreated control samples sprayed only with water, at all time intervals, no residue of fenazaquin was detected.
Loss (%) of residue in made tea
V. Kumar et al. / Food and Chemical Toxicology 44 (2006) 596–600 y = 0.0187x2 - 1.3524x + 66.928 R2 = 0.8387
80 70 60 50 40 30 20 10 0 0
5
10
15
20
25
30
35
Conc. (mg/kg) of residue in green tea
Transfer (%) of residue in brew
Fig. 2. Loss of fenazaquin residue during tea manufacturing.
y = -0.0013x2 + 0.2806x + 6.2838 R2 = 0.9204
25 20 15 10 5 0 0
10
20
30
40
50
60
70
Conc. (mg/kg) in made tea
Fig. 3. Transfer of fenazaquin residue in brew.
Considerable loss of residue was observed during the processing of green shoots to made tea. The loss of residue decreases as residue concentration in made tea increases (Fig. 2). During infusion only partial transfer (3–22%) of fenazaquin in brew has been observed (Table 1). Transfer of residue increased with an increase in concentration of residue in-made tea (Fig. 3). The maximum residue in brew (0.72 mg/l) was detected in the 0 day sample (wet season) of made tea with 96.37 mg/kg residue deposit. No measurable residue was found in brew when made from the samples harvested and processed after 7 and 5 days of treatment with recommended dose in wet and dry season, respectively. The range of pesticide remaining with spent leaves was 3.57–52.13%. However, a relationship with made tea residue concentration and seasonal variations was not observed. 4. Discussion The rapid degradation of fenazaquin and its metabolites in plants (citrus, pome and apple) has been reported (Dow Elanco, 1993). In tea field, besides the effect of some physical and chemical factors like photo, thermal, pH and moisture (Cosby et al., 1972; Miller and Zepp, 1983; Chen et al., 1987; Agnihothrudu and Muraleedharan, 1990; Miller and Donaldson, 1994), growth dilution factor (Chen and Wan, 1988; Agnihothrudu and Muraleedharan, 1990; Bisen and
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Ghosh Hajara, 2000) might have played significant role and rendered fenazaquin residue unavailable in the short period of time in both wet (half-lives, 1.43–1.7 days) and dry seasons (half-lives, 2.10–2.21 days). At normal plucking round (seventh day after treatment) at both the doses P98% residues in wet season and P88% residues in dry seasons disappeared in green shoots and made tea. This difference in disappearance of fenazaquin residues in tea in different seasons may be attributed lo the agro-climatic conditions (Bhattacharya et al., 1995; Maniknandan et al., 2001). The ether linkage between quinazoline and ethyl phenyl components of fenazaquin molecule is believed to be the most photo liable portion (Hatton et al., 1992). During dry periods the overexposure of sunlight might cause considerable degradation of fenazaquin residue in tea shoots. The growth dilution factor is important in reducing the residue levels in tea crop as the foliage and the shoots on which pesticides are applied are in different stages of growth. The weight of these immature shoots increases over a period of 6–14 days, depending on the plucking interval. The immature buds by the time attain the size of pluckable shoots; the residue of applied pesticides on them will undergo a growth dilution. During processing of green shoots, leaves are dried and decreased a weight by a factor of 3.33. Theoretically, residues in made tea should be higher by the same factor but results show (Table 1) that the residue deposit in made tea is less (647%) than that of the value of theoretically calculated residue level. However, it is also evident (Fig. 2) that loss of residue in made tea increased with decrease in concentration of residues in green shoots. The present finding supports the studies reporting loss of many pesticides during processing (Chen and Wan, 1988). The loss of pesticides during processing might be due to three factors i.e. evaporation, degradation and codistillation (Cabras et al., 1998). The transfer rate of the pesticide residue in brew depends on the water solubility (Wan et al., 1991; Nagayama, 1996), partition coefficient (Tsumura-Hasegawa et al., 1992; Jaggi et al., 2001) and low vapour pressure (Chen and Wan, 1988). Fenazaquin has low solubility in water (102 lg/l) but its fairly reasonable organic matter adsorption capability and partitioning coefficient (Kow = 321,000) allows its transfer to brew in a considerable proportion. Loss of fenazaquin in brew due to fugacity would be low as the molecule has low vapour pressure (3.4 · 106 Pa at 25 C) as is solubility. However, the acidic nature of brew may favour its rapid degradation (hydrolysis) at elevated temperature (Dow Elanco, 1993). Considerable loss of fenazaquin residue from brew may also be due to adsorption to the organic component of the brew, i.e. leaves. The collective role of all these factors might be accountable for only partial transfer of residue in brew. The residue remained on spent leaf did not show any trend. It is evident (Table 1) that made tea processed from green shoots collected after the fifth day in either of the seasons are likely to have a residue level up to 3.64 mg/kg at
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recommended dose. As tea is subjected to infusion prior to consumption, it is the residue transferred in brew which actually contributes to the dietary intake. Considering a maximum of 10 cup of tea (150 ml per cup) consumption by an individual, the actual intake of residue on the basis of residue data in brew, is only 0.015 mg of fenazaquin when brew is prepared from the made tea processed from the samples treated at recommended rate and harvested after 5 days of the treatment in either of the seasons. The estimated intake with brew (maximum consumption of 10 cup/day/adult) thus would be below the acceptable daily intake for fenazaquin (0.005 mg/kg-body weight). The MRLs in tea in most of the cases are fixed on the basis of pesticides residue present in made tea (European Union, 2002). However, it is the actual residue of pesticides in brew that should be considered for setting up realistic MRLs. Fenazaquin like many of the pesticides listed for tea do not have yet firm limits assigned because of lack of data representing the actual residue contributing to the dietary intake. Therefore, for the time being importing countries are free to maintain or set their own tentative limits or to adopt stipulations fixed by an international organization such as Codex Alimenterious Commission, which sets standards for the World Health Organization and the Food and Agricultural Organization of the United Nations (Jaggi et al., 2001). In this context, data of the present research are very relevant and useful for setting up standards for pesticide safety limit considered in regulating problems with consumption of tea and its global trade. 5. Conclusion Tea is generally cultivated under identical agro-climatic conditions and for commercial cultivation the most common species of tea is C. sinensis. In the present experiment the same species of tea cultivated under common agroclimatic conditions was used. The results showed that fenazaquin residue in tea, prior to consumption in the form of brew, dissipates at three stages: (1) in green shoots residues disappear after 11 days of treatment in both seasons; (2) during processing to made tea with no residues in the sample from 11 to 14 days of wet and dry seasons, respectively and thus balancing the factors accountable for increased concentration of residue deposits in made tea from green shoots; and (3) during infusion in brew with residue free samples after 7 days in both the seasons, respectively. The acceptable daily intake for fenazaquin is 0.005 mg/kg body weight (Dow Elanco, 1993). As tea is submitted to infusion/prior to consumption, a waiting period for more than 5 days for both the seasons at the recommended rate (125 g AI/ha) may be suggested and considered quite safe to avoid health hazards due to the toxic effect of residues in brew. Thus the present findings are useful in monitoring the fenazaquin residue in tea.
References Agnihothrudu, V., Muraleedharan, N., 1990. Pesticide residues in tea. Planters Chronicle 85, 125–127. Bhattacharya, A., Chowdhury, A., Somchowdhury, A.K., Pallarl, A.K., Roy, U.S., 1995. Studies on residues, persistence and pre-harvest interval of cythion, durmet and ripcord in made tea of Darjeeling. Peslology 21 (2), 28–36. Bisen, J.S., Ghosh Hajara, N., 2000. Persistence and degradation of some insecticides in Darjeeling tea. Journal of Plantation Crop 28 (2), 123– 131. Cabras, P., Angioni, A., Garau, V.L., Menelli, E.V., Cabitza, F., Pala, M., 1998. Pesticide residue in raisin processing. Journal of Agriculture and Food Chemistry 46, 1309–2311. Chen, Z.M., Wan, H.B., 1988. Factors affecting residues of pesticides in tea. Pesticide Science 23, 109–118. Chen, Z.M., Wan, H.B., Wang, Y., Xue, Y., Xia, H., 1987. Fate of pesticides in the ecosystem of tea garden. In: Proceedings of the International Symposium on Tea Quality-Human Health. Tea Research Institute, Chinese Academy of Agriculture Sciences, Hangzhou, China, pp. 146–149 (4–9 November). Cosby, P.G., Moilanon, K.W., Nakagawa, M., Wong, A.S., 1972. Environmental Toxicology of Pesticides. Academic Press, New York. Dow Elanco, 1993. Fenazaquin—A Profile. Department of Regulatory Toxicology and Environmental Affairs, Agriculture Products Research and Development, Oxfordshire, UK. European Union, 2002. Appendix I EU/German Maximum Residue Limits on Tea, Appendices, 16 December. Hatton, C.J., Babcock, J.M., Schoonover, J.R., Dripps, J.E., 1992. Fenazaquin, a new acaricide/insecticide. In: Proceedings of the International Cong. Ent., p. 535. Hollingworth, R.M., Ahammadsahip, K.I., Gadelhak, G.G., Mclughin, J.L., 1992. Complex I of mitochondrial respiratory chain: a target for pesticide development by both man and nature. In: Proceedings of the American Chemical Society, Abs. No. 156. Jaggi, S., Sood, C., Kumar, V., Ravindranath, S.D., Shanker, A., 2001. Leaching of pesticides in tea brew. Journal of Agriculture and Food Chemistry 49, 5479–5483. Manikandan, K.N., Muraleedharan, N., Selvasundaram, R., 2001. Residues and persistence of chlorpyrifos in processed black tea. Journal of Plantation Crops 29 (3), 35–37. Miller, G.C., Donaldson, S.G., 1994. Factor affecting photolysis of organic compounds on soils. In: Helz, G.R., Zepp, R.G., Crosby, D.G. (Eds.), Aquatic and Surface Photochemistry. Lewis Publishers, Boca Raton, FL, pp. 97–109. Miller, G.C., Zepp, R.G., 1983. Extrapolating photolysis rate from the laboratory to the environment. Residue Review 85, 89–110. Muraleedharan, N., 1994. Pesticide residues in tea: problems and prospective. Planters Chronicle 9, 371–375. Nagayama, T., 1996. Behavior of residual organ phosphorus pesticides in foodstuffs during leaching or cooking. Journal of Agriculture and Food Chemistry 44, 2388–2393. Shanker, A., Jasrotia, P., Kumar, A., Jaggi, S., Kurnar, V., Sood, C., 2001. Bioefficacy of new milicide: fenazaquin. Pestology 25 (6), 57–60. Solomon, M.G., Fitzgerald, J.D., Ridout, M.S., 1993. Fenazaquin, a selective acaricide for use in IPM in the UK. Crop Protection 12, 255– 258. Tsumura-Hasegawa, Y., Tonogai, Y., Nakamura, Y., Ito, T., 1992. Residues of post harvest application of pesticides in citrus fruits after storage and processing into lemon marmalade. Shokuhin Eisegaku Zasshi 33, 258–266. Wan, H., Xia, H., Chen, Z., 1991. Extraction of pesticide residue in tea by water during the infusion process. Food Additives and Contaminations 8, 497–500.