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Leaching of residues of certain pesticides from black tea to brew
Food Chemistry 113 (2009) 522–525
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Leaching of residues of certain pesticides from black tea to brew Natarajan Manikandan b, Subbiah Seenivasan a, Muthukumar Navaneetha Krishna Ganapathy a, Narayanan Nair Muraleedharan a,*, Rajagopal Selvasundaram c a
UPASI Tea Research Foundation, Tea Research Institute, Nirar Dam BPO, Valparai 642 127, India Residue Analytical Section, Analytical R&D Department, Advinus Therapeutics Pvt. Limited, 21&22, Phase II, Peenya Industrial Area, Bangalore 560 058, India c M/s. Chemtura Chemicals India Pvt. Limited, A Chemtura Company, Unit No. 701, Business Point 349, Western Express Highway, Mumbai 400 069, India b
a r t i c l e
i n f o
Article history: Received 8 October 2007 Received in revised form 20 June 2008 Accepted 28 July 2008
Keywords: Black tea Pesticide residues Tea brew Transfer
a b s t r a c t Tea is the most commonly consumed beverage in the world. It is prepared after infusing processed black tea in hot water. During the process of brewing, along with flavour and aroma, the residues of plant protection chemicals may also be transferred into the tea brew or infusion. The leaching of certain pesticides, such as ethion, endosulfan, dicofol, chlorpyrifos, deltamethrin, hexaconazole, fenpropathrin, propargite, quinalphos and lambdacyhalothrin from powdered black tea into the brew was studied. The rate of transfer of the pesticide residue from black tea to the hot brew was largely influenced by physicochemical parameters, such as water solubility and octanol-water partition coefficient. Tea brews prepared from untreated black tea samples were fortified with standard solutions of the respective pesticides, extracted and analysed using GC and HPLC by following standardised methods. Results revealed that the rate of leaching of residues of these pesticides into the tea brew was low due to their low solubilities in aqueous medium and high octanol-water partitioning coefficients. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction A concern for human health and environmental safety demands foods free of pesticide residues and heavy metals and tea is no exception to this. However, it is not feasible to keep the tea plant completely free from synthetic pesticides as the plant is prone to attack by an array of pests and diseases throughout the year, depending on the climatic conditions of the tea-growing region. Tea fields are also invaded by several weeds. Hence, to augment the productivity of tea and to protect the plants from pests and diseases, it is necessary to adopt plant protection measures using synthetic chemicals. The plant protection chemicals applied in the tea fields find their way into the tea shoots and into the processed black tea. The maximum residue limit (MRL) of any pesticide on the commodity is decided on the basis of data generated through supervised field trials, carried out under good agricultural practices (GAP). Processed black tea is not consumed as such and it is only the hot water extract or the tea brew that is consumed. Obviously, tea brew is the potential route for human exposure to pesticide residues present in the black tea. The real impact lies in how far the residues in the black tea are leached into the tea brew and not on the mere presence of residues in the black tea (Barooah,
* Corresponding author. Tel.: +91 4253 235301, +91 4253 235303; fax: +91 4253 235302. E-mail addresses: [email protected] (N. Manikandan), vasan1981@ rediffmail.com (N. Muraleedharan), [email protected] (R. Selvasundaram). 0308-8146/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2008.07.094
Kalita, Collier, & Barbora, 1994; Kumar, Tewary, Ravindranath, & Shanker, 2004 and Shanker, Vipin, & Tewary, 2003). Therefore, it is necessary to study the leaching of residues into the tea brew, which will provide additional data for fixing realistic MRLs of pesticides in tea. 2. Materials and methods 2.1. Materials Analytical standards of ethion, dicofol, endosulfan, quinalphos, chlorpyrifos, deltamethrin, hexaconazole, fenpropathrin, propargite and lambdacyhalothrin were procured from Dr. Ehrenstorfer, Germany and the commercial formulations of pesticides were supplied by the manufacturers. Stock solutions (1000 lg/ml) were prepared in appropriate solvents and the working standard solutions were prepared from the stock solutions by serial dilutions. All the solvents and chemicals were of Analytical Grade and HPLC grade from E. Merck and SD Fine Chemicals. 2.2. Preparation of black tea Separate field trials were conducted in wet and dry seasons in UPASI Tea Experimental Farm (UPASI TEF), situated in Valparai, Tamil Nadu, at an elevation of 1150 m above mean sea level in South India. For each field trial, commercial formulations of
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N. Manikandan et al. / Food Chemistry 113 (2009) 522–525
ethion, endosulfan, dicofol, chlorpyrifos, deltamethrin, hexaconazole, fenpropathrin, propargite, quinalphos and lambdacyhalothrin were mixed appropriately with water as by the recommendations of UPASI Tea Research Institute (Muraleedharan, Hudson, & Durairaj, 2007) and sprayed on tea leaves using a hand-operated knapsack sprayer. After application, the green leaves, comprising three leaves and a bud, were plucked on the ‘0’, 7th, 10th and 14th days and shoots were processed in a miniature manufacturing unit, adopting the CTC (Crush, Tear and Curl) method to produce black tea (Ramasamy, 1993). 2.3. Residue analysis in black tea Black tea samples obtained from the field experiments were subjected to residue analysis using the standard procedures for ethion (IS 11773: 2003), dicofol (IS 14629: 1999), endosulfan (IS 12611: 1989), quinalphos (IS 14437: 1997), chlorpyrifos (IS 12365: 1988), fenpropathrin, deltamethrin and lambda cyhalothrin (JAOAC, 1999), hexaconazole (Manikandan, Karthika, Muraleedharan, Seenivasan, & Selvasundaram, 2006) and propargite (Selvasundaram, 2002) .
added to black tea, spent tea leaves and tea brew, prepared in triplicate. The samples were subjected to the analytical procedure described above (Tables 3 and 4). 2.6. Apparatus The gas chromatograph, Hewlett Packard 5890 series II, equipped with a nitrogen phosphorus detector (NPD) and electron capture detector (ECD), was used for the analysis of residues of ethion, dicofol, endosulfan, quinalphos, chlorpyrifos, deltamethrin, fenpropathrin, lambdacyhalothrin, propargite and hexaconazole. Capillary columns of HP-608 (PH ME Siloxane wide bore capillary-30 m 0.53 mm 0.5 lm) and DB-5 columns were used for the GC. A high performance liquid chromatograph (HPLC), Agilent 1100, equipped with diode array detector (DAD) was used for the analysis of residues of propargite (Table 1). An HPLC column, Zorbax RX C18 (4.6 mm ID; 250 mm length; 5 lm particle size; stainless steel) was used for propargite residue analysis. 3. Results and discussion 3.1. General
2.4. Brewing process and residue analysis in tea brew and spent tea leaves The black tea manufactured in the miniature CTC unit was subjected to the infusion process. Hot water for preparing infusion was prepared by using a stainless steel electric kettle. Two grammes of black tea samples were weighed; to that, 100 ml of boiling water were added, which allowed brewing at boiling temperature for 6 min (ISO 3103, 1990). After the brewing process, the infusion was filtered through a stainless steel cartridge and cooled to room temperature. The spent tea leaves left after infusion were spread in a folded filter paper and air-dried. The infusion and the air-dried spent leaves were separately analysed for residues of the respective pesticides. The tea infusion was transferred to a separating funnel and subjected to partitioning with hexane (3 times 100 ml). The hexane layer was allowed to pass through anhydrous sodium sulphate. The combined hexane layer was collected in a 500 ml round bottom flask and concentrated to dryness using rotary vacuum evaporator at 45 °C. The residue was then reconstituted and quantified using GC and HPLC maintained by the conditions described in Table 1. 2.5. Recovery study To evaluate the recovery of pesticide residues in black tea, tea brew and spent leaves, 1 ml of the pesticide standard solution of the respective pesticide (100 lg/ml) prepared in acetone, was
Pesticides were identified from their respective retention times and confirmed by comparison with authentic standards. For partitioning with aqueous tea brew extract, hexane was found to be a suitable solvent, which is reflected in the recovery studies (Table 4). Since the pesticides selected for the studies are completely miscible with organic solvents, particularly in hexane, the recovery was selectively good in hexane. The reproducibility of the recovery results, as indicated by the standard deviations, suggested that extraction and cleanup procedure were reliable enough for the analysis of tea brew. 3.2. Residues of organophosphorous pesticides in tea brew The analytical data on the residues of chlorpyrifos, ethion and quinalphos, in black tea, tea brew and spent tea are given in Table 5. From the data it appears that only few pesticides were able to transfer into the brew, which could be attributed to their low solubility in water. Earlier studies had shown that 2–52% of residues of organophosphorous pesticides were transferred into tea brew (Nagayama et al., 1989). The Kow values for ethion, chlorpyrifos and quinalphos are, in decreasing order, 19054.6, 16255.5 and 1208, respectively. Larger the Kow values allow less transfer into the water. Among the three organophosphorus pesticides, quinalphos has the lowest Kow value, followed by chlorpyrifos and quinalphos. The percentages of transfer of residues from made tea to brew were 2.5 for ethion, 9.12 for chlorpyrifos and 9.20
Table 1 Instrumental parameters of GC and HPLC used for analytical determination Compound
Dicofol Endosulfan Deltamethrin Fenpropathrin k-Cyhalothrin Chlorpyrifos Ethion Quinalphos Hexaconaozle Propargite
Carrier gas/Mobile phase
N2 N2 N2 N2 N2 N2 N2 N2 N2 Methanol:Water
Flow rate (ml/min)
12 18 5 5 5 12 12 12 5 1.5
Temperature of (°C)
Detector
Oven
Injector
Detector
190 200 210 210 210 190 225 210 225 40
200 250 180 180 180 250 240 240 220 –
220 270 300 300 300 250 260 250 250 (k) 225 nm
GC–ECD GC–ECD GC–ECD GC–ECD GC–ECD GC–NPD GC–NPD GC–NPD GC–NPD HPLC–DAD
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for quinalphos, in our study. The solubilities of ethion, chlorpyrifos and quinalphos were found to be 2.2 and 20 mg/l. As the solubility increases, the transfer of residues from black tea into infusion also increased positively. 3.3. Residues of organochlorine compounds in tea brew The data on the residues of organochlorine pesticides, such as dicofol and endosulfan, in made tea, tea infusion and spent tea leaves are given in Table 5. When dicofol was sprayed, at 185 g/ ha, the initial deposit of residues on made tea was 91.4 ppm, whereas the residues were not detectable in the brew. Most of the residues remained in the spent tea. Since the solubility of dicofol was meager in water (0.8 mg/l) most of the residues were retained in the spent tea leaves and not transferred into the brew during the infusion process. In the case of endosulfan, the initial deposit on black tea was lower than that of dicofol but a very small amount leached into the brew. Since endosulfan exists as alpha and beta isomers, at a 2:1 ratio and they were converted into endosulfan sulphate (cyclic sulphate) after spraying onto the plant, only 2% of the total endosulfan residues were transferred into the tea brews. Though the solubility of endosulfan is low (0.3 mg/l), the presence of the water soluble metabolite, endosulfan sulphate, in the residues are responsible for the transfer of residues, up to 2%, into the tea infusion. This could be attributed to the moderate solubility of endosulfan in water (Chen and Haibin, 1988). The results of our study confirmed the earlier finding reported by Jaggi, Sood, Kumar, Ravindranath, and Shanker (2001). 3.4. Residues of synthetic pyrethroids in tea brew The residues of the synthetic pyrethroid insecticides deltamethrin, fenpropathrin and lambdacyhalothrin were not detectable in the tea brew since their water solubility was extremely low (0.002, 0.0141 and 0.005 mg/l) (Table 2). The Kow values for these
Table 2 Water-solubility and calculated Kow of pesticides under study Pesticides
Solubility (mg/l)
Kow
Chlorpyrifos Dicofol Deltamethrin Ethion Endosulfan Fenpropathrin Hexaconazole k-Cyhalothrin Propargite Quinalphos
2.0 0.8 0.002 2 0.3 0.0141 17 0.005 0.215 22.0
16,256 43,954 34,506 19,055 54,954–61,659 10,00,000 7943.3 100,00,000 501,187 1208
Source: The Pesticide Manual (2003). (Part of data in table reproduced from Jaggi et al. (2001)).
Table 3 Recovery of pesticides in black tea and spent tea leaves Chemical
Added conc. (lg)
Recovered (lg)
% Recoverya ± SD
Dicofol Endosulfan Deltamethrin Fenpropathrin k-Cyhalothrin Chlorpyrifos Ethion Quinalphos Hexaconazole Propargite
1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
0.897 0.910 0.940 0.903 0.857 0.910 0.927 0.961 0.940 0.911
89.7 ± 4.5 91.0 ± 5.3 94.0 ± 6.3 90.3 ± 5.6 85.7 ± 5.9 91.0 ± 4.2 92.7 ± 5.6 96.1 ± 3.7 94.0 ± 5.2 91.1 ± 5.4
a
Means of triplicate analyses.
Table 4 Recovery of chemicals from tea brew Chemical
Added conc. (lg)
Recovered (lg)
% Recoverya
% RSD ± SD
Dicofol Endosulfan Deltamethrin Fenpropathrin k-Cyhalothrin Chlorpyrifos Ethion Quinalphos Hexaconaozle Propargite
1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
0.917 0.923 0.920 0.923 0.897 0.920 0.920 0.933 0.930 0.917
91.7 ± 2.1 92.3 ± 1.3 92.0 ± 3.3 92.3 ± 2.6 89.7 ± 1.3 92.0 ± 2.2 92.0 ± 2.2 93.3 ± 1.7 93.0 ± 2.2 91.7 ± 0.5
2.2 1.4 3.6 2.8 1.4 2.4 2.4 1.8 2.3 0.5
a
Means of triplicate analyses.
Table 5 Residues of organophosphorus, organochlorine, synthetic pyrethroids and triazole fungicide in black tea, spent tea leaves and their transfer into tea brew Compounds/ group
Day after spraying
Residues in mg/kg
% Transfer
Black tea
Tea brew
Spent tea
14.3 0.71 0.07 0.04
1.30 0.04 ND ND
12.2 0.53 ND ND
9.12 – – –
0 7 10 14
61.9 3.10 0.52 0.22
1.30 0.07 ND ND
54.9 2.57 0.43 0.15
2.50 2.25 – –
0 7 10 14
15.0 0.07 0.03 ND
1.38 ND ND ND
13.5 ND ND ND
9.20 – – –
91.4 15.6 4.21 1.18
ND ND ND ND
90.3 14.9 4.11 1.02
– – – –
0 7 10 14
41.3 2.92 0.92 0.61
0.69 0.06 ND ND
40.1 2.25 0.79 0.48
1.67 2.05 – –
Synthetic pyrethroids Deltamethrin 0 7 @ 5.04 g ha 1 10 14
0.55 0.18 0.09 ND
ND ND ND ND
0.46 0.14 ND ND
– – – –
Organophosphorus compounds Chlorpyrifos 0 7 @ 150 g ha 1 10 14 Ethion @ 375 g ha
1
Quinalphos @ 125 g ha
1
Organochlorine compounds Dicofol 0 7 @ 185 g ha 1 10 14 Endosulfan @ 350 g ha
1
Fenpropathrin @ 60 g ha 1
0 7 10 14
2.74 0.17 ND ND
ND ND ND ND
2.63 0.14 ND ND
– – – –
k-Cyhalothrin @ 12.5 g ha 1
0 7 10 14
1.26 0.57 0.18 0.09
ND ND ND ND
1.15 0.43 0.11 ND
– – – –
0 7 10 14
142 ND ND ND
ND ND ND ND
135 ND ND ND
– – – –
0 7 10 14
1.98 0.49 0.20 0.08
ND ND ND ND
1.85 0.34 0.14 ND
– – – –
Other compounds Propargite @ 570 g ha 1
Hexaconazole @ 10 g ha 1
ND, non-detectable.
N. Manikandan et al. / Food Chemistry 113 (2009) 522–525
pesticides are also very high (34,506, 10,00,000 and 100,00,000), which supported the findings. Most of these synthetic pyrethroid pesticides are present in the spent tea leaves and are not transferred into the tea infusion during the infusion process. 3.5. Others The leaching behaviour of a few other pesticides, such as propargite and hexaconazole, were studied and results are given in Table 5. In the case of propargite, although the black tea contains significantly higher amounts of residues, in view of the Kow values, they are not transferred into the tea liquor. The residues of hexaconazole in the brew were very low and not quantifiable (below the detection limit <0.05 ppm). 4. Conclusion This present study revealed that the extent of leaching of residues from processed black tea to the brew (infusion) largely depends upon the solubility in water and Kow value. Transfer of pesticides into tea infusion was positively correlated with water solubility and negatively correlated with Kow. The residues of pesticides with low Kow and high water solubility leached more into the tea brew while the residues of pesticides with high log Kow and low water solubility were not transferred into the tea infusion. The latter were bound to their matrices (spent tea leaves). On the basis of the above findings it can be concluded that the persistence of pesticides in infusions/transfer behaviour may be strongly predictable from the physicochemical properties, such as water solubility and octanol–water partition coefficient. The rates of leaching were found to be low, in spite of the high initial deposit in the made tea, indicating the hydrophobic nature of the chemicals studied. 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 when fixing a realistic MRL for pesticides in black tea. Acknowledgement The authors are grateful to the Tea Board, Government of India, for the financial assistance under the X five year plans.
525
References Barooah, A. K., Kalita, J. N., Collier, P. D., & Barbora, B. C. (1994). Residues of pesticides in made tea and hot water brew. In Proceedings of the 32nd tocklai conference. Kolkata: TRA. Chen, Z. M., & Haibin, W. (1988). Factors affecting residues of pesticides in tea. Pesticide Science, 23, 109–118. IS 11773. (2003). Indian Standard. Methods for determination of ethion residues in food commodities (I rev.). Bureau of Indian Standards, Manak Bhavan, New Delhi. IS 14629. (1999). Indian Standard. Method for determination of dicofol residues in agricultural and food commodities. Bureau of Indian Standards, Manak Bhavan, New Delhi. IS 12611. (1989). Pesticides – Determination of residues in foods and water (Reaffirmed 1999) – Endosulfan. Bureau of Indian Standards, Manak Bhavan, New Delhi. IS 14437. (1997). Indian Standard. Method for determination of quinalphos residues in agricultural and food commodities. Bureau of Indian Standards, Manak Bhavan, New Delhi. IS 12365. (1988). Indian Standard. Method for determination of chlorpyrifos residues in food commodities (Reaffirmed 1999). Bureau of Indian Standards, Manak Bhavan, New Delhi. 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. Journal of Agricultural and Food Chemistry, 49, 5479–5483. 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 and Chemical Toxicology, 42, 423–428. Nagayama, T., Maki, T., Kan, K., Iida, M., Tamura, Y., & Nishima, T. (1989). Residues of organophosphorus pesticides in commercial tea and their leaching into tea. Nippon Noyaku Gakkaishi, 14, 39–45. Manikandan, K. N., Karthika, C., Muraleedharan, N., Seenivasan, S., & Selvasundaram, R. (2006). Studies on the residues of copper and hexaconazole during their combined application and their subsequent transfer into tea infusion. Journal of Plantation Crops, 34(3), 405–409. Muraleedharan, N., Hudson, J. B., & Durairaj, J. (2007). Guidelines on tea culture in South India (18th ed.). Coonoor, India: United Planters’ Association of Southern India. Pang, G. F., Cao, Y. Z., Fan, C. L., Zhang, J. J., & Li, X. M. (1999). Multi residue gas chromatographic method for determining synthetic pyrethroid residues in agricultural products: Collaborative study. Journal of AOAC International, 80(1), 186–211. Ramasamy, V. (1993). CTC tea manufacture. Coonoor: United Planters’ Association of Southern India. Shanker, A., Vipin, Kumar, & Tewary, D. K. (2003). Fate of pesticide residues on tea from leaf to cup. International Journal of Tea Science, 2(4), 18–26. Selvasundaram, R. (2002). Entomology. Annual report of UPASI tea research institute, Nirar Dam BPO, Valparai 642 127, Coimbatore District, India, pp. 31–32. The Pesticide Manual, (2003), 8th ed. UK: British Crop Protection Council.
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Leaching of residues of certain pesticides from black tea to brew Natarajan Manikandan b, Subbiah Seenivasan a, Muthukumar Navaneetha Krishna Ganapathy a, Narayanan Nair Muraleedharan a,*, Rajagopal Selvasundaram c a
UPASI Tea Research Foundation, Tea Research Institute, Nirar Dam BPO, Valparai 642 127, India Residue Analytical Section, Analytical R&D Department, Advinus Therapeutics Pvt. Limited, 21&22, Phase II, Peenya Industrial Area, Bangalore 560 058, India c M/s. Chemtura Chemicals India Pvt. Limited, A Chemtura Company, Unit No. 701, Business Point 349, Western Express Highway, Mumbai 400 069, India b
a r t i c l e
i n f o
Article history: Received 8 October 2007 Received in revised form 20 June 2008 Accepted 28 July 2008
Keywords: Black tea Pesticide residues Tea brew Transfer
a b s t r a c t Tea is the most commonly consumed beverage in the world. It is prepared after infusing processed black tea in hot water. During the process of brewing, along with flavour and aroma, the residues of plant protection chemicals may also be transferred into the tea brew or infusion. The leaching of certain pesticides, such as ethion, endosulfan, dicofol, chlorpyrifos, deltamethrin, hexaconazole, fenpropathrin, propargite, quinalphos and lambdacyhalothrin from powdered black tea into the brew was studied. The rate of transfer of the pesticide residue from black tea to the hot brew was largely influenced by physicochemical parameters, such as water solubility and octanol-water partition coefficient. Tea brews prepared from untreated black tea samples were fortified with standard solutions of the respective pesticides, extracted and analysed using GC and HPLC by following standardised methods. Results revealed that the rate of leaching of residues of these pesticides into the tea brew was low due to their low solubilities in aqueous medium and high octanol-water partitioning coefficients. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction A concern for human health and environmental safety demands foods free of pesticide residues and heavy metals and tea is no exception to this. However, it is not feasible to keep the tea plant completely free from synthetic pesticides as the plant is prone to attack by an array of pests and diseases throughout the year, depending on the climatic conditions of the tea-growing region. Tea fields are also invaded by several weeds. Hence, to augment the productivity of tea and to protect the plants from pests and diseases, it is necessary to adopt plant protection measures using synthetic chemicals. The plant protection chemicals applied in the tea fields find their way into the tea shoots and into the processed black tea. The maximum residue limit (MRL) of any pesticide on the commodity is decided on the basis of data generated through supervised field trials, carried out under good agricultural practices (GAP). Processed black tea is not consumed as such and it is only the hot water extract or the tea brew that is consumed. Obviously, tea brew is the potential route for human exposure to pesticide residues present in the black tea. The real impact lies in how far the residues in the black tea are leached into the tea brew and not on the mere presence of residues in the black tea (Barooah,
* Corresponding author. Tel.: +91 4253 235301, +91 4253 235303; fax: +91 4253 235302. E-mail addresses: [email protected] (N. Manikandan), vasan1981@ rediffmail.com (N. Muraleedharan), [email protected] (R. Selvasundaram). 0308-8146/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2008.07.094
Kalita, Collier, & Barbora, 1994; Kumar, Tewary, Ravindranath, & Shanker, 2004 and Shanker, Vipin, & Tewary, 2003). Therefore, it is necessary to study the leaching of residues into the tea brew, which will provide additional data for fixing realistic MRLs of pesticides in tea. 2. Materials and methods 2.1. Materials Analytical standards of ethion, dicofol, endosulfan, quinalphos, chlorpyrifos, deltamethrin, hexaconazole, fenpropathrin, propargite and lambdacyhalothrin were procured from Dr. Ehrenstorfer, Germany and the commercial formulations of pesticides were supplied by the manufacturers. Stock solutions (1000 lg/ml) were prepared in appropriate solvents and the working standard solutions were prepared from the stock solutions by serial dilutions. All the solvents and chemicals were of Analytical Grade and HPLC grade from E. Merck and SD Fine Chemicals. 2.2. Preparation of black tea Separate field trials were conducted in wet and dry seasons in UPASI Tea Experimental Farm (UPASI TEF), situated in Valparai, Tamil Nadu, at an elevation of 1150 m above mean sea level in South India. For each field trial, commercial formulations of
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N. Manikandan et al. / Food Chemistry 113 (2009) 522–525
ethion, endosulfan, dicofol, chlorpyrifos, deltamethrin, hexaconazole, fenpropathrin, propargite, quinalphos and lambdacyhalothrin were mixed appropriately with water as by the recommendations of UPASI Tea Research Institute (Muraleedharan, Hudson, & Durairaj, 2007) and sprayed on tea leaves using a hand-operated knapsack sprayer. After application, the green leaves, comprising three leaves and a bud, were plucked on the ‘0’, 7th, 10th and 14th days and shoots were processed in a miniature manufacturing unit, adopting the CTC (Crush, Tear and Curl) method to produce black tea (Ramasamy, 1993). 2.3. Residue analysis in black tea Black tea samples obtained from the field experiments were subjected to residue analysis using the standard procedures for ethion (IS 11773: 2003), dicofol (IS 14629: 1999), endosulfan (IS 12611: 1989), quinalphos (IS 14437: 1997), chlorpyrifos (IS 12365: 1988), fenpropathrin, deltamethrin and lambda cyhalothrin (JAOAC, 1999), hexaconazole (Manikandan, Karthika, Muraleedharan, Seenivasan, & Selvasundaram, 2006) and propargite (Selvasundaram, 2002) .
added to black tea, spent tea leaves and tea brew, prepared in triplicate. The samples were subjected to the analytical procedure described above (Tables 3 and 4). 2.6. Apparatus The gas chromatograph, Hewlett Packard 5890 series II, equipped with a nitrogen phosphorus detector (NPD) and electron capture detector (ECD), was used for the analysis of residues of ethion, dicofol, endosulfan, quinalphos, chlorpyrifos, deltamethrin, fenpropathrin, lambdacyhalothrin, propargite and hexaconazole. Capillary columns of HP-608 (PH ME Siloxane wide bore capillary-30 m 0.53 mm 0.5 lm) and DB-5 columns were used for the GC. A high performance liquid chromatograph (HPLC), Agilent 1100, equipped with diode array detector (DAD) was used for the analysis of residues of propargite (Table 1). An HPLC column, Zorbax RX C18 (4.6 mm ID; 250 mm length; 5 lm particle size; stainless steel) was used for propargite residue analysis. 3. Results and discussion 3.1. General
2.4. Brewing process and residue analysis in tea brew and spent tea leaves The black tea manufactured in the miniature CTC unit was subjected to the infusion process. Hot water for preparing infusion was prepared by using a stainless steel electric kettle. Two grammes of black tea samples were weighed; to that, 100 ml of boiling water were added, which allowed brewing at boiling temperature for 6 min (ISO 3103, 1990). After the brewing process, the infusion was filtered through a stainless steel cartridge and cooled to room temperature. The spent tea leaves left after infusion were spread in a folded filter paper and air-dried. The infusion and the air-dried spent leaves were separately analysed for residues of the respective pesticides. The tea infusion was transferred to a separating funnel and subjected to partitioning with hexane (3 times 100 ml). The hexane layer was allowed to pass through anhydrous sodium sulphate. The combined hexane layer was collected in a 500 ml round bottom flask and concentrated to dryness using rotary vacuum evaporator at 45 °C. The residue was then reconstituted and quantified using GC and HPLC maintained by the conditions described in Table 1. 2.5. Recovery study To evaluate the recovery of pesticide residues in black tea, tea brew and spent leaves, 1 ml of the pesticide standard solution of the respective pesticide (100 lg/ml) prepared in acetone, was
Pesticides were identified from their respective retention times and confirmed by comparison with authentic standards. For partitioning with aqueous tea brew extract, hexane was found to be a suitable solvent, which is reflected in the recovery studies (Table 4). Since the pesticides selected for the studies are completely miscible with organic solvents, particularly in hexane, the recovery was selectively good in hexane. The reproducibility of the recovery results, as indicated by the standard deviations, suggested that extraction and cleanup procedure were reliable enough for the analysis of tea brew. 3.2. Residues of organophosphorous pesticides in tea brew The analytical data on the residues of chlorpyrifos, ethion and quinalphos, in black tea, tea brew and spent tea are given in Table 5. From the data it appears that only few pesticides were able to transfer into the brew, which could be attributed to their low solubility in water. Earlier studies had shown that 2–52% of residues of organophosphorous pesticides were transferred into tea brew (Nagayama et al., 1989). The Kow values for ethion, chlorpyrifos and quinalphos are, in decreasing order, 19054.6, 16255.5 and 1208, respectively. Larger the Kow values allow less transfer into the water. Among the three organophosphorus pesticides, quinalphos has the lowest Kow value, followed by chlorpyrifos and quinalphos. The percentages of transfer of residues from made tea to brew were 2.5 for ethion, 9.12 for chlorpyrifos and 9.20
Table 1 Instrumental parameters of GC and HPLC used for analytical determination Compound
Dicofol Endosulfan Deltamethrin Fenpropathrin k-Cyhalothrin Chlorpyrifos Ethion Quinalphos Hexaconaozle Propargite
Carrier gas/Mobile phase
N2 N2 N2 N2 N2 N2 N2 N2 N2 Methanol:Water
Flow rate (ml/min)
12 18 5 5 5 12 12 12 5 1.5
Temperature of (°C)
Detector
Oven
Injector
Detector
190 200 210 210 210 190 225 210 225 40
200 250 180 180 180 250 240 240 220 –
220 270 300 300 300 250 260 250 250 (k) 225 nm
GC–ECD GC–ECD GC–ECD GC–ECD GC–ECD GC–NPD GC–NPD GC–NPD GC–NPD HPLC–DAD
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for quinalphos, in our study. The solubilities of ethion, chlorpyrifos and quinalphos were found to be 2.2 and 20 mg/l. As the solubility increases, the transfer of residues from black tea into infusion also increased positively. 3.3. Residues of organochlorine compounds in tea brew The data on the residues of organochlorine pesticides, such as dicofol and endosulfan, in made tea, tea infusion and spent tea leaves are given in Table 5. When dicofol was sprayed, at 185 g/ ha, the initial deposit of residues on made tea was 91.4 ppm, whereas the residues were not detectable in the brew. Most of the residues remained in the spent tea. Since the solubility of dicofol was meager in water (0.8 mg/l) most of the residues were retained in the spent tea leaves and not transferred into the brew during the infusion process. In the case of endosulfan, the initial deposit on black tea was lower than that of dicofol but a very small amount leached into the brew. Since endosulfan exists as alpha and beta isomers, at a 2:1 ratio and they were converted into endosulfan sulphate (cyclic sulphate) after spraying onto the plant, only 2% of the total endosulfan residues were transferred into the tea brews. Though the solubility of endosulfan is low (0.3 mg/l), the presence of the water soluble metabolite, endosulfan sulphate, in the residues are responsible for the transfer of residues, up to 2%, into the tea infusion. This could be attributed to the moderate solubility of endosulfan in water (Chen and Haibin, 1988). The results of our study confirmed the earlier finding reported by Jaggi, Sood, Kumar, Ravindranath, and Shanker (2001). 3.4. Residues of synthetic pyrethroids in tea brew The residues of the synthetic pyrethroid insecticides deltamethrin, fenpropathrin and lambdacyhalothrin were not detectable in the tea brew since their water solubility was extremely low (0.002, 0.0141 and 0.005 mg/l) (Table 2). The Kow values for these
Table 2 Water-solubility and calculated Kow of pesticides under study Pesticides
Solubility (mg/l)
Kow
Chlorpyrifos Dicofol Deltamethrin Ethion Endosulfan Fenpropathrin Hexaconazole k-Cyhalothrin Propargite Quinalphos
2.0 0.8 0.002 2 0.3 0.0141 17 0.005 0.215 22.0
16,256 43,954 34,506 19,055 54,954–61,659 10,00,000 7943.3 100,00,000 501,187 1208
Source: The Pesticide Manual (2003). (Part of data in table reproduced from Jaggi et al. (2001)).
Table 3 Recovery of pesticides in black tea and spent tea leaves Chemical
Added conc. (lg)
Recovered (lg)
% Recoverya ± SD
Dicofol Endosulfan Deltamethrin Fenpropathrin k-Cyhalothrin Chlorpyrifos Ethion Quinalphos Hexaconazole Propargite
1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
0.897 0.910 0.940 0.903 0.857 0.910 0.927 0.961 0.940 0.911
89.7 ± 4.5 91.0 ± 5.3 94.0 ± 6.3 90.3 ± 5.6 85.7 ± 5.9 91.0 ± 4.2 92.7 ± 5.6 96.1 ± 3.7 94.0 ± 5.2 91.1 ± 5.4
a
Means of triplicate analyses.
Table 4 Recovery of chemicals from tea brew Chemical
Added conc. (lg)
Recovered (lg)
% Recoverya
% RSD ± SD
Dicofol Endosulfan Deltamethrin Fenpropathrin k-Cyhalothrin Chlorpyrifos Ethion Quinalphos Hexaconaozle Propargite
1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
0.917 0.923 0.920 0.923 0.897 0.920 0.920 0.933 0.930 0.917
91.7 ± 2.1 92.3 ± 1.3 92.0 ± 3.3 92.3 ± 2.6 89.7 ± 1.3 92.0 ± 2.2 92.0 ± 2.2 93.3 ± 1.7 93.0 ± 2.2 91.7 ± 0.5
2.2 1.4 3.6 2.8 1.4 2.4 2.4 1.8 2.3 0.5
a
Means of triplicate analyses.
Table 5 Residues of organophosphorus, organochlorine, synthetic pyrethroids and triazole fungicide in black tea, spent tea leaves and their transfer into tea brew Compounds/ group
Day after spraying
Residues in mg/kg
% Transfer
Black tea
Tea brew
Spent tea
14.3 0.71 0.07 0.04
1.30 0.04 ND ND
12.2 0.53 ND ND
9.12 – – –
0 7 10 14
61.9 3.10 0.52 0.22
1.30 0.07 ND ND
54.9 2.57 0.43 0.15
2.50 2.25 – –
0 7 10 14
15.0 0.07 0.03 ND
1.38 ND ND ND
13.5 ND ND ND
9.20 – – –
91.4 15.6 4.21 1.18
ND ND ND ND
90.3 14.9 4.11 1.02
– – – –
0 7 10 14
41.3 2.92 0.92 0.61
0.69 0.06 ND ND
40.1 2.25 0.79 0.48
1.67 2.05 – –
Synthetic pyrethroids Deltamethrin 0 7 @ 5.04 g ha 1 10 14
0.55 0.18 0.09 ND
ND ND ND ND
0.46 0.14 ND ND
– – – –
Organophosphorus compounds Chlorpyrifos 0 7 @ 150 g ha 1 10 14 Ethion @ 375 g ha
1
Quinalphos @ 125 g ha
1
Organochlorine compounds Dicofol 0 7 @ 185 g ha 1 10 14 Endosulfan @ 350 g ha
1
Fenpropathrin @ 60 g ha 1
0 7 10 14
2.74 0.17 ND ND
ND ND ND ND
2.63 0.14 ND ND
– – – –
k-Cyhalothrin @ 12.5 g ha 1
0 7 10 14
1.26 0.57 0.18 0.09
ND ND ND ND
1.15 0.43 0.11 ND
– – – –
0 7 10 14
142 ND ND ND
ND ND ND ND
135 ND ND ND
– – – –
0 7 10 14
1.98 0.49 0.20 0.08
ND ND ND ND
1.85 0.34 0.14 ND
– – – –
Other compounds Propargite @ 570 g ha 1
Hexaconazole @ 10 g ha 1
ND, non-detectable.
N. Manikandan et al. / Food Chemistry 113 (2009) 522–525
pesticides are also very high (34,506, 10,00,000 and 100,00,000), which supported the findings. Most of these synthetic pyrethroid pesticides are present in the spent tea leaves and are not transferred into the tea infusion during the infusion process. 3.5. Others The leaching behaviour of a few other pesticides, such as propargite and hexaconazole, were studied and results are given in Table 5. In the case of propargite, although the black tea contains significantly higher amounts of residues, in view of the Kow values, they are not transferred into the tea liquor. The residues of hexaconazole in the brew were very low and not quantifiable (below the detection limit <0.05 ppm). 4. Conclusion This present study revealed that the extent of leaching of residues from processed black tea to the brew (infusion) largely depends upon the solubility in water and Kow value. Transfer of pesticides into tea infusion was positively correlated with water solubility and negatively correlated with Kow. The residues of pesticides with low Kow and high water solubility leached more into the tea brew while the residues of pesticides with high log Kow and low water solubility were not transferred into the tea infusion. The latter were bound to their matrices (spent tea leaves). On the basis of the above findings it can be concluded that the persistence of pesticides in infusions/transfer behaviour may be strongly predictable from the physicochemical properties, such as water solubility and octanol–water partition coefficient. The rates of leaching were found to be low, in spite of the high initial deposit in the made tea, indicating the hydrophobic nature of the chemicals studied. 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 when fixing a realistic MRL for pesticides in black tea. Acknowledgement The authors are grateful to the Tea Board, Government of India, for the financial assistance under the X five year plans.
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