Title of Invention

A METHOD FOR DETECTING THE PRESENCE OF DITHIOCARBAMATE FUNGICIDES THIRAM AND ZIRAM IN FRESH FRUITS AND VEGETABLES

Abstract The present invention relates to the development of a simple, quick and cost-effective detection kit for two dithiocarbamate fungicides, thiram and ziram in fruits and vegetables. The residues are extracted by swabbing fruit and vegetables surface thoroughly with cotton swab soaked in ethyl acetate and transferring it to a stoppered graduated vial containing ethyl acetate by dipping and pressing several times. On spotting 20 µl extract, the chemically soaked filter paper strip of size 5 cm x 1 cm develops yellow and pink spots for thiram and ziram, respectively.
Full Text The invention relates to a simple, rapid and cost-effective method for, detection of two important dithiocarbamate fungicides- thiram {(IUPAC: bis (dimethylthiocarbamoyl) disulfide} and ziram {(IUPAC: zinc bis (dimethyIdithiocarbamate)} in fresh fruits and vegetables.
PRIOR ART
Thiram and ziram are two important protective dithiocarbamate fungicides used worldwide on a range of crops, mainly due to their high efficiency in controlling plant fungal diseases. Their formulations are registered for use in many countries. Considering the presence of residues of dithiocarbamates including thiram and ziram in fruits and vegetables in may countries and their toxicological significance, there is a need for development of simple, rapid, reliable and cost-effective detection kit for monitoring thiram and ziram residues in fruits and vegetables. Apart from a couple of enzyme immunoassays, the prior art search of the published literatures, both patents as well research papers, has revealed non-availability of such a kit. Since 1964 there have been many non-selective determination technique for total dithiocarbamates,
which fail to distinguish among the member compounds in the dithiocarbamate group.
Cullen (1964) {Anal Chem 36:221-224} originally described a spectrophotometric method for estimating dithiocarbamates based on determination of cupric complex formed with the CS2 evolved from the acid decomposition of the dithiocarbamates in presence of SnCl2 as a reducing agent. This method has been used and modified by several authors. Verma et al. (1984) {Analyst 109:649-650} used of tetra-acetonitrile-copper (I) perchlorate for the spectrophotometric determination of thiram in grain. A flow injection-FTIR determination of thiram and ziram with LODs of 400 and 785 mg, respectively, has also been reported.
Dithiocarbamate residues can also be quantified by GLC head-space determination of the CS2 evolved on the basis of the method described by Cullen (1964) {Jianren et al. (1989) Sci Agric Sin 22: 76-80; Hill (1992) Mod Meth Pesti Anal. Emerg Strat Pesti Anal, CRC Press, London, pp. 213-231; Friedricks et al. (1995) Z Lebensm Unters Forsch 201:69-73; Harrington et al. (1998) Central Science Laboratory: York, UK, pp. 1-34}. Microwave-assisted extraction with GC-flame photometric analysis having LOD 0.005 ppm for thiram and ziram on peaches and recoveries 80-100% for dithiocarbamates has been reported {Vryzas et al. (2002) J Agric Food Chem 50(8):2220-2226}.
Reversed phase HPLC methods have been proposed for thiram and other dithiocarbamates in food using various detection methodologies (Gustafsson et al. (1983) J Agric Food Chem 31:461-463; Irth et al. (1986) J Chromatogr 370 (3):439-447; Miles and Moye (1988) Anal Chem 60(3): 220-226; Silva et al. (1999) J Liq Chromatogr Relat Technol 22:463-475; van Lishaut and Schwack (2000) J AOAC Int 83:720-727}.
All these analysis techniques are based on costly instrumentation, and deficient in distinguishing among the dithiocarbamates. Rolland et al (1992h){Med Fac Landbouww Univ Gent 57/3b, 1255-1260} described an HPLC method for thiram residues in crops that measured thiram as an intact molecule and distinguished it from other dithiocarbamates.
Specific competitive inhibition enzyme-linked immunosorbent assays (ELISA) for thiram having LOD 0.03 ug/ml {Gueguen et al. (2000) J Agric Food Chem 48(10):4492-4499} and 0.04 ug/ml {Queffelec et al. (2001) J Agric Food Chem 49(4): 1675-1680} and little cross-reaction with other dithiocarbamates have been developed. These immunoassays are also expensive.
All the above analytical techniques are handicapped by high cost, sophisticated instrumentation, laborious extraction procedures, nonspecific detection and above all, difficulty to use or practise under the actual field conditions.
Prior art search for the detection of pesticides or fungicides other than dithiocarbamate fungicides thiram and ziram revealed a couple of such kits or techniques based on immunoassays, radiobiochemistry, bioluminescence, enzyme assays, other bioassays, etc. Pullen et al. (U.S. Pat. No. 5,723,306, March 1998) described an easy-to-use monoclonal antibodies based detection kit for pyrethrins, pyrethroids and their metabolites. Charm et al. (U.S. Pat. No. 5,200,311, April 6, 1993) described radiobiochemical pesticides determination method, which employed insect tissues containing specific binders or enzymes sensitive to various groups of pesticides. The pesticides compete with the radioactive tracers for the binders or the enzymes in the insect tissues. The extent of binding or enzyme activity may be inversely proportional to the amount of pesticide in the sample under standardized conditions. A
highly specific and sensitive immunoassay technique for detection of dichlorobenzonitrile, dichlorbenil, chlorthiamid, dichlorobenzthiamide and dichlorobenzamide has been described by Jackobsen et al. (U.S. Pat. No. 6,635,434, October 21, 2003). Fitzpatrick et al. (U.S. Pat. No. 5,654,178, August 5, 1997) disclosed immunoassay methods and a kit for detection and quantification of fungicide tetrachloroisophthalonitrile or its metabolite. An immunological technique utilising a compound useful for forming immunoconjugates used in the detection of organophosphorous pesticides has been described by Jones et al. (U.S. Pat. No. 5,677,432, October 14, 1997). This technique detects total organophosphorous pesticides without distinguishing the member compounds. Stanker et al. (U.S. Pat. No. 5,334,528, August 2, 1994) described methods for making specific monoclonal antibodies useful for detection of cyclodiene insecticides in foods and environmental samples. This method is appropriate only for laboratory analysis, not as a kit, for detection of cyclodiene group of organochlorine pesticides only. Zomer et al. (U.S. Pat. No. 5,283,180, February 1, 1994; U.S. Pat. No. 5,374,535, December 20, 1994)) described a bioluminescence method as well as a kit for determination of organophosphate and carbamate pesticides, altering or reducing the activities of insect brain preparation which are inversely correlated with pesticide concentration in a test sample. The substrate (6-substituted D-luciferin ester) used produces D-luciferin, which on subsequent reaction, produces bioluminescence, which can be measured and compared with a control in order to determine the concentration of a pesticide in the test sample. This detection technique as well as the kit could not distinguish individual pesticide in both organophosphate and carbamate pesticide groups. Chemicals, which alter or reduce the activities of the insect brain preparation, were also detected, giving a misleading result. According to Dodson et al. (U.S. Pat. No. 5,932,436, August 3, 1999), endocrine-disrupting pesticides in an aqueous sample can be detected by Daphnia reproductive bioassay. In

this bioassay technique, only endocrine-disrupting pesticides may be detected without distinguishing the individual pesticides. Moreover, endocrine disruptors other than the endocrine-disrupting pesticides may also give false positive detection. Goodson et al. (U.S. Pat. No. 4,324,858, April 13, 1982) described a simple kit for detection of cholinesterase-inhibiting environmental pollutants such as organophosphorous pesticides and nerve agents. Gelman et al. (U.S. Pat. No. 3,049,411, August 14, 1962) disclosed a cholinesterase inhibition based kit for detection of organophosphorous pesticides. These two analysis techniques, specific to the organophosphorous group failed to distinguish among the member compounds in the group. Moreover carbamates group of pesticides and other cholinesterase-inhibitors could give false positive detection result. Jayaraman et al. (2004) {J Food Sci Technol 41(1), 14-18} developed a horse serum-acetylcholinesterase based spectrophotometric technique for detection and quantification of organophosphorous pesticides. But this is a laboratory technique requiring analytical instrument like spectrophotometer, frozen as well as ambient temperature controlled atmosphere and long analysis time. This analysis technique, specific to the organophosphorous group failed to distinguish among the member compounds in the group. Moreover carbamates group of pesticides and other cholinesterase-inhibitors could give false positive detection result. Grow (U.S. Pat. No. 4,411,989, October 25, 1983) disclosed methods and devices for detection of enzyme inhibitors like pesticides. In this technique a comparison of infrared absorption spectra of uncomplexed cholinesterase enzyme with that of the cholinesterase-pesticide complex detected the presence of the pesticide. This was also a laboratory technique requiring analytical instrument like spectrophotometer, temperature controlled atmosphere and long analysis time. Cholinesterase enzyme-inhibitors other than the pesticides may give false positive result in detection.
OBJECT OF THE INVENTION
An object of this invention is to propose a simple, rapid and cost-effective detection method for dithiocarbamate fungicides thiram and ziram in fresh fruits and vegetables.
Another object of this invention is to propose a simple, rapid and cost-effective detection method capable of distinguishing between two dithiocarbamate fungicides, thiram and ziram during detection, when present together in fruits and vegetables samples.
Still another object of this invention is to propose a simple, rapid and cost-effective detection method which is efficient.
Further objects and advantages of this invention will be more apparent from the ensuring description.
DISCRIPTION OF THE INVENTION
According to this invention there is provided a method for detecting the presence of dithiocarbamate fungicides thiram and ziram in fresh fruits and vegetables comprising in the steps of; dipping of cotton swabs in ethyl acetate; surface swabbkigs of fruits to be subjected to detection with the presoaked cotton swab; dipping the swabs into ethyl acetate to prepare an ethyl acetate extract; preparing a test reagent solution of dithizone or diphenyithiocarbazone in mixture of acetone-n-propanol; soaking flitter paper in said test reagent solution; spotting the extract on the soaked flitter paper strip with the presence of thiram and ziram providing yellow and pink spots respectively.
The process consists in the follow ings steps :
1. Cotton swabs consisting of a one-sided cotton head and a piastk stem of for example 34 inches length, used for swabbing are dipped in ethyl acetate in a glass breaker for 3-4 minutes with occasional stirring and pressing against the inner wall of the breaker to make these free from any chemical or fungicide, which may interfere with the detection process.
2. Whole fruits or vegetables (weighing preferably between 50-100 g) lice tomato, potato, brinjal, capsicum, chilli, okra, beans, cucumber, apple pear, orange, lemon, mango, guava, banana, etc., to be analysed are thoroughly surface-swabbed with the help of the cotton swab presoaked with ethyl acetate. The entire fruit or vegetable surface is to be swabbed, preferably two times with two swabs.
3. The swabs are then dipped in for example 750 µl ethyl acetate taken in a stoppered graduated PVC vial and pressed against the inner wall of the vial several times, so that the residues completely come into ethyl acetate.
4. After two times swabbing, the ethyl acetate extract volume is made-up for example 1ml with ethyl acetate.
5. The extraction process without any purification or clean up has shown about 76-90% recovery of the residue, depending on the type of fruit or vegetable.
6. The test reagent, 0.075% dithizone or diphenylthiocarbazone (C6H5.N:N.CS.NH.NH.C6H5) is prepared freshly in 10ml mixture of acetone-n-propanol (1:1) and kept in test tube.
7. A strip of for example size 5cm X1 cm from Whatman No. 1 filter paper is soaked for a moment in the test reagent solution and air-dried for 1-2 min.
8. With the help of micropipette, for example 20 µl extract is spotted on the soaked filter paper strip.
9. If present at a level of 2 ug in the extract (25-50 ppb in samples depending on a weight of the fruit or vegetable in the range of 50-100g), thiram and ziram give yelbw and pink spots, respectively.
Most of the spectrophotometric, colorimetric and chromatographic methods based on CS2 evolution from any member of dithiocarbamates group cant distinguish the member compounds. But, the developed kit for which claim, specially detects thiram or ziram separately.
In Blasco et al. (2004) method {J Chromatogr A 1028(2):267-276} for simultaneous determination of dithiocarbamates by LC-MS, thiram cant be recovered from high acid or sour fruits. The detection kit development can very well detect the same in sour fruits and vegetables like citrus fruits, tomato, etc., also as it involves only surface extraction.
According to Heise et al. (2000) {Fresenius J Anal Chem 366(8):851-856}, thiram concentration in aqueous solution decreases by 50-75% within 20 min in presence of the cut pieces of fruits and vegetables during extraction due to increased surface area. In the present invention, degradation of thiram during extraction is minimized, since extraction is done using ethyl acetate, without involving any aqueous phase, from surface without cutting or grinding the samples and in a very short time 2-3 min.
The present invention detects parent compounds thiram or ziram as then-respective residues, not degradation products or metabolites.
Limit of detection for the kit is 25-50 ppb, depending on the weight of fruit or vegetables sample in 50-100 g range and taking into account 80% residue extraction recovery.
The detection kit developed is very rapid, requiring only about 40 min to complete the analysis, whereas most other methods take long time of 1 or 2 hour.
unlike most of the methods whether spectrophotometric, coburimetric, chromatographic or immunoassays, the detection kit development does not require any sophisticated and costly instrument and chemicals, skilled hands, labourious extraction and analysis schedule.
Simple and rapid extraction procedure requiring 5 min without any clean up or filtration step gives 76-90% recovery of the residues depending on the type of fruit and vegetable.
Working example of process and product thereof:
A 50 g tomato, artificially spiked with thiram and ziram dissolved in water by smearing on entire surface with the help of micropipette, was analysed by the developed detection kit, as shown in the flow chart.
FLOW CHART
Spiking of a whole tomato of 50 g weight with 3 ppm each of thiram and ziram dissolved in water by smearing on entire surface with the
help of a micropipette.

Dipping of two cotton swabs in ethyl acetate for a few minutes with
occasional stirring.

Thorough swabbing of the entire tomato surface twice with the help of
the cotton swabs presoaked in ethyl acetate.

Dipping the swabs in 750 µl ethyl acetate in a stoppered 2 ml
graduated PVC vial and pressing against the inner wall of the vial
several times to extract the residues into ethyl acetate appropriately.
Making the ethyl acetate extract volume up to 1 ml with ethyl acetate.

Dipping the filter paper strip for a moment to soak in the test reagent and leaving it for air-drying for 1-2 min.

Spotting of 20 µl extract on the filter paper with micropipette and appearance of yellow or pink colour on the spot in a few seconds.










WE CLAIM
1. A method for detecting the presence of dithiocarbamate fungicides thiram
and ziram in fresh fruits and vegetables comprising in the steps of:
a) dipping of cotton swabs in ethyl acetate,
b) surface swabbings of fruits to be subjected to detection with the presoaked cotton swab,
c) dipping the swabs into ethyl acetate to prepare an ethyl acetate extract,
d) preparing a test reagent solution of dithizone or diphenylthiocarbazone in mixture of acetone-n-propanol,
e) soaking flitter paper in said test reagent solution,
f) spotting the extract on the soaked flitter paper strip with the presence of thiram and ziram providing yellow and pink spots respectively.

2. A process as claimed in claim 1 wherein in step (a) wherein the cotton swabs are dipped in ethyl acetate and made free from impurities.
3. A process as claimed in claim 1 wherein in step (b) the fruit or vegetable is preferably swabbed twice.
4. A process as claimed in claim 1 wherein the reagent mixture comprising dithizone or diphenylthiocarbazone is prepared in a mixture of acetone-a-propanol preferably in the ratio of 1:1.
5. A process for detecting the presence of dithiocarbamates substantially as herein described.

Documents:

354-del-2006-Abstract-(02-11-2012).pdf

354-del-2006-abstract.pdf

354-del-2006-Claims-(02-11-2012).pdf

354-del-2006-claims.pdf

354-del-2006-Correspondence-Others-(02-11-2012).pdf

354-del-2006-correspondence-others.pdf

354-del-2006-correspondence-po.pdf

354-del-2006-description (complete).pdf

354-del-2006-form-1.pdf

354-del-2006-form-18.pdf

354-del-2006-Form-2-(02-11-2012).pdf

354-del-2006-form-2.pdf

354-del-2006-form-3.pdf

354-del-2006-GPA-(02-11-2012).pdf


Patent Number 257297
Indian Patent Application Number 354/DEL/2006
PG Journal Number 39/2013
Publication Date 27-Sep-2013
Grant Date 21-Sep-2013
Date of Filing 07-Feb-2006
Name of Patentee DIRECTOR GENERAL DEFENCE RESEARCH & DEVELOPMENT ORGANISATION
Applicant Address MINISTRY OF DEFENCE, GOVT. OF INDIA, WEST BLOCK-VIII, WING-1, SECTOR-1, RK PURAM, NEW DELHI 110066,INDIA
Inventors:
# Inventor's Name Inventor's Address
1 HIMANISH DAS DEFENCE RESEARCH & DEVELOPMENT LABORATORY, SIDDHARTHANAGAR, MYSORE - 570011, INDIA
2 MAHADEVA NAIKA DEFENCE RESEARCH & DEVELOPMENT LABORATORY, SIDDHARTHANAGAR, MYSORE - 570011, INDIA
3 SWAMINATHA N JAYARAMAN DEFENCE RESEARCH & DEVELOPMENT LABORATORY, SIDDHARTHANAGAR, MYSORE - 570011, INDIA
4 AMARINDER SINGH BAWA, DEFENCE RESEARCH & DEVELOPMENT LABORATORY, SIDDHARTHANAGAR, MYSORE - 570011, INDIA
PCT International Classification Number A01N
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA