Quantitative Estimation of Fungicide, Pesticide Residues from Pepper (Piper Nigrum L.) as Obtained in the Selected Area of Kodagu District, Karnataka.

Introduction

Pepper (Piper nigrum L.)  belongs to the family Piperaceae, the crop grow well in a temperature range between 25°-40°C  and rainfall around 1250 mm. In India, Karnataka, Kerala, Tamilnadu states are play a major role in the production of pepper. As per the 2013-14 report 21,250 tones of black pepper worth Rs. 94,002 lakhs products were exported from India to various countries.

Pepper is one of the important spiceused for flavor, and aroma of food and beverages, is produced from the still-green, unripe drupes of the pepper plant. Peppercontains volatile oil so it makes an important contribution to sensory qualities¹

In Indian ayurveda system the pepper are used as a medicine since 5000 years, as a diet and herbal remedies, it is a source of important drugs such as atropine, codeine, digoxin, morphine, quinine and vincristine².While emphasizing the body, mind and spirit in disease prevention and treatment³.

Black pepper is a king of spices, used for medicinal agent, preservative, and in perfumery⁴. Its contains major pungent alkaloid Piperine(1-peperoyl piperidine), exhibits⁵ pharmacological activities like antihypertensive and antiplatelets⁶,antioxidant, antitumor, anti- asthmatics⁷, antipyretic, analgesic, anti-inflammatory, anti-diarrheal,  antispasmodic, anxiolytic, antidepressants⁸hepato-protective⁹,immuno-modulatory, antibacterial, antifungal, anti-thyroids, anti-metastatic, antimutagenic, etc.

Piperine has been found to enhance the therapeutic efficacy of many drugs, vaccines and nutrients by increasing oral bioavailability by inhibiting various metabolizing enzymes¹⁰.The consumption of black pepper increases the orocecal transit time.¹¹and also decreased pressure in arteries, it inhibited high K⁺ pre contractions due to blockade Ca²⁺ channel¹², apart from that it having an anti diarrheal property hence in most of the developing countries local peoples, herbal industries, and herbal practioners formulate the peppercorn for diarrhea for all ages¹³.

Nerolidol is a natural compound isolated from piper species, having pesticidal activity against various mites. And another compound pipene isolated from volatile oil of peppercorn it is used as an odorant¹⁴.

Materials and Methods

Collection of Sample

Kodagu is one of the major pepper cultivated district in Karnataka state hence the pepper (Piper nigrum L) samples are collected from the different villages of ponnempet. The samples were collected in a zip polythene bags.And preserved in deep freezer.

Interview Questionnaires

Questionnaires was contained 4 sections to find out the details like Physical inspection (location, soil type and weather data) general information (type of crop, aware of pesticide effect and recommended dosage) cultivation steps (name of the verity, land preparation, spacing, month of plantation, intercropping, duration and common diseases) pesticide information (name of pesticide used, method of application etc.)

A total of 10 farmers from different villages of ponnempet, kodagu district were interviewed. They are majorly cultivated pepper plant in a distance of 10-15 feet of panniruverity with a support of forest trees or gliricidia plant and coffee is an intercropping in sand mixed and black soil and most of all the farmers not aware of pesticide effect on human health. Some of them were engaged in the application of pesticides for the high yielding purpose, and revealed via interview the generally used pesticides in this district were copper sulphate, bodomixture, trichoderma, copper oxichlorideetc through power/hand spraying for controlling black and yellow wilt diseases.

Chemicals / Materials Used

Acetone, petroleum ether, Dichloro methane. Diethyl ether, Sodiumsulphate, Florosil, sodium chloride, tween 20, Methanol, KBr, conc. Nitric acid, acetonitril, butter paper, flutted filter paper, micro fiber filter paper, IAC(immune affinity column) and  HPLC water.

Stock and Working Standard

Stock and working standard of organo phosphorous pesticides of appropriate concentration in acetone (standard from EPA of equivalent). Store the standards under refrigeration. (Shelf life for stock standard is one year and that for working standard is six months)

Analysis of Aflatoxin

Weigh the sample into a blender jar.

Add NaCl into blender jar.

Add 100 ml of 80:20 methanol & HPLC water mix.

Cap blender jar and seal with Parafilm.

Blend at high speed for 1 minute

Then filter it into a beaker through fluted filter paper.

Pipette 10 ml of filtrate into 50 ml graduated cylinder.

If the sample is nutmeg, oregano, or black pepper, add 40 mL of 20%Tween-20 solution to the graduated cylinder.  If the sample is not one of the products mentioned, add 40 ml of DI Distilled water to the cylinder.  Mix thoroughly.

Filter the contents of the graduated cylinder through a glass fiber filter into a 250 ml beaker. This filtrate will be used for the Aflatest column.

Immuno-Affinity Column Clean-Up

Attach an Aflatest-P column to the pump stand.

Pipette 10 ml of filtrate to the syringe and pass through the IAC column

Once the entire sample has passed through the column, rinse the column with 10 ml of HPLC water.  Repeat HPLC water rinse.

Place 20 ml stoppered test tube under the tip of the column and add 1 ml of methanol to the column.

Collect all the methanol eluent in the test tube.  And transfer the sample to a sample vial. The sample is now ready for injection to the HPLC.¹⁵

Analysis of Organo Phosphorous

Rinse thoroughly all the glass wares first with acetone and then with petroleum ether before starting the analysis

Always do a reagent blank along with sample

Sample preparation

whole spice:-The entire sample is reduced in six by mixing and quartering and a subsample of 100g taken for crushing/blending. A representative sample of 20g (in duplicate) is taken for analysis.

Crushed/ground spices

The entire sample is reduced in size by mixing and quartering and a subsample of 100g is taken. From this a representative sample of 20g is taken for analysis.

Extraction

Transfer the above weighed sample to a 500 ml stoppered conical flask

Add freshly prepared acetone-water mixture

Keep on the laboratory shaker for 1hour with occasional shaking

Filter through whatman No.1 filter paper in a bucher funnel, using vacuum pump

Shake well and transfer 100 ml of filtrate to the 250ml separating funnel

To this add 50ml petroleum ether first and then 50ml methylene dichloride

Shake vigorously for about 2 minutes and then allow to stand for the separation of layer

After clear separation layer, transfer the lower layer to another 250 ml separating funnel.

To this add 100 ml MDC. Shake and allow to stand.

Collect the upper layer of step-7 to a beaker after passing through a funnel containing anhydrous sodium sulphate which is supported by glass wool pre wetted with petroleum ether

After the separation of layer, collect the lower layer of the 2^nd separating funnel to the same beaker by passing through the same funnel containing sodium sulphate

Transfer this solution quantitatively to rotary evaporator flask/water bath and concentrate to minimum volume. This is ready for clean up.

Florosil Cleanup for Organo Phosphorous

Place activated florosil (about 4″) in the column; add about 0.5″ sodium sulphate pre wet column with 50 ml petroleum ether.

Transfer the concentrate to sintered column using 8ml HPLC grade acetone, and rinse walls of chromatographic tube with additional small portion of acetone. Drain with 100 ml of petroleum ether and collect and collect it in a 500 ml beaker.

Elute column at about 5ml/minute with 200ml diethyl ether-petroleum ether (50:50). Collect the eluant in the beaker containing petroleum ether in step-2

This elute is ready for concentration step for organo phosphorous pesticides by roatary evaporator/water bath.

Concentrate the above extract to minimum volume.

Concentrate the extract using a water bath/ K. D Evaporator/ Rotary evaporator

Rinse the content with acetone and transfer to a graduated stopped test tube

Make up to 4ml with acetone. This is ready for GC analysis¹⁶.

Calculation

Aflatoxin

Organo Phosphorous

Table 1: Aflatoxin Standard

Peak	Name	Ret. Time	Area	Area%	Conc.
1	G2	7.067	264859	11.573	20.000
2	G1	8.114	660603	28.866	80.000
3	B2	8.916	445142	19.451	20.000
4	B1	10.402	917926	40.110	80.000
Total			2288527	100.000

Table 2: Organo phosphorous Standard

Peak	Ret. Time	Name	Area	Height	Conc.	Units
1	2.267	Dichlorovos	9179844	1568984	2000.00	pG
2	3.235	Acephate	1816787	389424	4000.00	pG
3	4.772	Omethoate	3891511	756206	4000.00	pG
4	5.519	Monocrotofos	141960	44365	4000.00	pG
5	5.670	Phorate	10316861	1888548	2000.00	pG
6	6.310	Dimethoate	2394844	611262	2000.00	pG
7	6.870	Diazinon	5129908	1414095	2000.00	pG
8	7.083	Disulfoton	3150731	900220	2000.00	pG
9	7.253	Etrimphos	2821356	850826	2000.00	pG
10	8.133	Parathion methyl	525406	1626396	2000.00	pG
11	8.720	Pirimiphos methyl	9816919	1781049	2000.00	pG
12	8.882	Malalthion	2870442	938102	2000.00	pG
13	10.128	Quinolphos	5806111	2071751	2000.00	pG
14	11.096	Profenophos	3184702	1170602	2000.00	pG
15	12.077	Ethion	749567	284785	1000.00	pG
16	12.397	Triazophos	4486193	1655339	2000.00	pG
17	13.804	Phosmet	1026677	389577	2000.00	pG
18	14.524	Phosalone	3009899	1017151	2000.00	pG
19	15.313	Azinphos ethyl	2254325	691752	2000.00	pG
Total			77302702	20050434

Table 3: Amount of Pesticides present in sample no. 3

Sl.no	Compound Name	Results in ppm	EU Limits in ppm
1.	Diazinon	0.01	0.1
2	Malathion	0.01	0.02
3	Quinophos	0.01	0.1
4	Profenophos	0.02	0.07
5	Ethion	0.02	5

 

Figure 1: Aflatoxin blank Click here to View figure

Figure 2:Aflatoxin standard   Click here to View figure

Figure 3: Aflatoxin sample               Click here to View figure

Figure 4: Organo phosphorous Standard Click here to View figure

Figure 5: Organo phosphorous Blank Click here to View figure

Figure 6: Organo phosphorous Sample-3 Click here to View figure

Figure 7: A and B are disease effected leaf, Fig C and D are chemicals used during the cultivation of pepper plant and Fig.E Gliricidia (Supporting Plant). Click here to View figure

 

Results and Discussion

Kodagu is one of the major pepper cultivated district of the state Karnataka so that ten samples were collected from different villages of ponnempet of the above district. For the high yield purpose some of the farmers are used pesticides such as copper sulphate,bodomixture, trichoderma, copper oxichlorideetc., through power/hand spraying for controlling black and yellow wilt diseases¹⁷.This study shows the quantitative estimation of fungicide, pesticide residual content in the collected pepper samples by ASTA method using HPLC and GC. All the analysis of ten samples the result shows the absence of aflotoxins (G2 G1 and B2, B1). In the pesticides (Organo phosphorous) totally analyzed 19 chemical compounds from each collected sample in that Diazinon, Malathion, Quinophos, Profenophos and ethion are present in sample number three that was collected from the mattur village. But it also in acceptable range (within EU limits) 1.e. 0.01, 0.01, 0.01, 0.02 and 0.02ppm respectively.

Conclusion

As per the survey and quantitative estimation of fungicide(Aflotoxin), pesticide(Organo phosphorous) analysis results shows the samples collected from the different villages of Ponnempet of Kodagu district Karnataka was within the EU limits hence it was  free from fungicide, pesticide content.

Acknowledgement

I thank the University Grants Commission (UGC) for providing financial support for this research work. I also wish to thank Department of Food Science and Nutrition, University of Agricultural Sciences,GKVK, Bangaluru for laboratory facility.

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