FUNGICIDE RESIDUES: ESTIMATION AND

MANAGEMENT

• FUNGICIDE:- A fungicide is a specific type of pesticide that controls fungal disease by specifically inhibiting or killing the fungus causing the disease.

• FUNGICIDE RESIDUE:- Pesticide residue refers to the pesticides that may remain on or in food or in soil after they are applied to crops.

• The amount of initially laid down pesticide after application on the surface or substrate is termed as deposit while the amount of pesticide left over after a lapse of time may be referred as residue.

• It is expressed as parts per million (ppm). (Gupta 1999)

Related Terms

• Persistence – Period for which the pesticide remain unaltered.

• Deposit –Amount of initially laid down chemical after application on the surface.

• Surface residue – Amount of insecticide that remain on treated surface after a lapse of time.

• Cuticular residue- Residue found in the cuticular region of plant.

• Harvest time residue- Residue found in the substrate at the time of harvest.

How pesticide residue occur in agriculture commodities

• Contamination of crop or animals exposed to chemical in the environment

• Intentional use of pesticide for protection of growing crops and stored products

• Unintentional exposure to pesticides such as would occur in crops, grown in soil treated previously or contaminated by foliar treatment of other crops grown earlier in the rotation

Why to study fungicide residue ?

• For safety of food and health• To reduce environmental pollution• To study effect of residues on non target

organisms

REGULATION OF FOOD AND SAFETY

At international level• Codex Alimentarius

Commission• Latin for “Food Law” or “Food

Code”• Founded by FAO in 1961• Responsible for the Joint

FAO/WHO Food Standards Programme since 1962

• The Secretariat of CAC is located at HQ of FAO (Rome)

• Collection of food standards, guidelines and codes of practice

At national level• "Codex India" the National

Codex Contact Point (NCCP) for India

• Located at Food Safety and Standards Authority of India (FSSAI) (Ministry of Health and Family Welfare), New Delhi.

• Coordinates and promotes Codex activities in India in association with the National Codex Committee

REGULATION

• Each country adopts their own agricultural policies and Maximum Residue Limits (MRL) and Acceptable Daily Intake (ADI).

• Some countries use the International Maximum Residue Limits -Codex Alimentarius to define the residue limits; this was established by Food and Agriculture Organization of the United Nations (FAO) and World Health Organization (WHO) in 1963 to develop international food standards, guidelines codes of practices, and recommendation for food safety.

• Currently the CODEX has 185 member countries.

• MRL:- maximum concentration of a residue that is legally permitted or recognized as acceptable in, or on, a food, agricultural commodity or animal feedstuff as set by codex or national regulatory authority (mg/kg).

• ADI:-estimate of the amount of pesticide in food and drinking water which can be ingested daily over a life time by humans without appreciable health risk(mg/kg body weight/day).

Maximum Residue Limits (MRL) in India

Central Codex committee of Food Standards(The Prevention of Food Adulteration Act, 1954)Pesticides Food MRL

(mg/kg)Dithiocarbamates Tomatoes 3.0Benomyl Mango

Other fruitsVegetables

2.01.00.50

Captafol Tomato 5.0Copper oxychloride Fruits and vegetables 20Carbendazim Mango

BananaOther fruitsDry FruitsVegetables

2.01.05.00.010.50

Hexaconazole Apple 0.1

Dodine Apple 5.0

Pesticides Food MRL (mg/kg)

Penconazole Grapes 0.2

Myclobutanil Grapes 1.0

Chlorothalonil Potato 0.1

Propiconazole Wheat 0.05

Captan Fruis and vegetables 15.0

Mancozeb chillies 1.0

Tricyclazole Rice 0.02

How to estimate of fungicide residue ?

• Residue of fungicide are present in micro quantities in the matrix. Hence involve a complicated procedure involving many step for their analysis. Analysis does not depend on high cost equipment like GC(gas liquid chromatograph) and HPLC (high performance liquid chromatograph). But based on series of cumulative operation like:-

1. Sampling 2. Extraction3. Clean-up4. Estimation

SAMPLING

• Sampling can be defined as the procedure or step adopted to obtain a representative quantity from the large consignment, so that selected representative quantity can be handled conveniently.

Sampling procedure for different food commodities:-

• Vegetables and fruits:- a sample of a fruit or a vegetable must be taken directly from the field or grower at the time of, or shortly after the harvest.

• In field sample should be collected randomly 10 areas from the field. Collect 0.5-1 kg portion each of these 10 areas and combine to form the sample.

• Grains :-grains are never sampled at the primary producer. Because residue on grains typically due to post harvest use of fumigants sample size of 5 kg should be collected

• Animal feed:- it include hay, silage, grains, bio product and commercial feed rations. Sampling procedure is typically selective not random. Collect 5 kg sample.

CONTINUEType of food Minimum amount of sample to collected

Medium sized Fruits and vegetable with weight between 25-250 gm

1 kg (at least 10 fruit )

Large sized Fruits and vegetable with weight more than 250 gm

2 kg (at least 5 fruit )

Dairy products like butter, cream, milk and cheese

0.5 kg

Spices (coriander,cumin) 0.25 kg

Cereals 1 kg

Animal feed (cotton seed cake) and fodder 1 kg, 5 kg

Water (running water) 15 kg

Soil 5 kg

Select the crop components of interest , reduce the size of the component parts, mix subdivide and systemically reduce the sample size 50 gm

EXTRACTION IN VEGETABLES• Once a valid, representative sub sample has been

selected for residue analysis, it is processed for isolation of pesticide or its matabolites having toxicological significance from the surrounding biological environment. Extraction must be adequate to yield quantitative removal of toxicant.

• Selection of extraction technique depends upon:-• Nature of matrix, whether soil, plant material or water• The analyte,s chemical group, its metabolites .• Safety (method to be adopted should not be hazardous).• One should be ensure that analyte/metabolite are fairly

soluble in the selected single solvent or mixture of solvent.

SOLVENTS• Some of commonly used solvent in order to increasing polarity are

given below:-• Non polar• Petroleum ether • N-hexane• Cyclohexane• Carbon tetrachloride• Benzene• Chloroform • Ethyl ether• Ethyl acetate• Actetone• Polar• water

EXTRACTION IN VEGETABLES• Take a sample of vegetable/fruit (1-2 kg).• Chop it into small pieces and mix properly.• After this take 20 gm representative sample.• Macerate it with 4-5 gm anhydrous sodium sulphate.• Add 100 ml acetone and extract by shaking on

mechanical shaker for one hour• Filter the extract through 2-3 cm layer of anhydrous

sodium sulphate.• Concentrate the extract to 40 ml on rotary flash

evaporator after adding a drop of mineral oil.

CONTINUE• Dilute the extract 4-5 times with 10% NaCL aqueous

solution.• Partition it thrice with ethyl acetate (50, 30, 30 ml) in a

separatory funnel by shaking vigorously for one minute.• Combine the organic (ethyl acetate) phases and filter

through anhydrous sodium sulphate.• Concentrate the organic phase up to 5 ml on rotary flash

evaporator.• Divide the concentrated extract into two equal parts

(one for organochlorines and synthetic pyrethroids and other for organophosphates and carbamates).

CLEAN-UP• In order to interference by co-extractives such as pigment,

waxes and fats present in the extract, isolation of the toxicant is achieved before Taking up estimation of pesticide residues. To achieve the necessary sensitivity, interfering substances have to be removed from the pesticides with one or more procedure that are commonly refered to as clean-up.

• For organochlorines and synthetic pyrethroids• Pack the glass column (60cm*22mm i.d) with adsorbent

mixture(5gm) florisil: activated charcoal (5:1w/w) in between two layers of anhydrous sodium sulphate.

• Tap the column gently to ensure uniform and compact packing.

CONTINUE

• Prewett the column with 50ml hexane and transfer the concentrated extract to the column.

• Elute the column with 125 ml solution of ethyl acetate: heaxne (3:7 v/v)

• Concentrate the elute to near dryness using rotary flash evaporator followed by gas manifold evaporator after adding one drop oil.

• Make the final volume to 2ml in ethyl acetate: n-hexane (3:7v/v).

ESTIMATION OF RESIDUE• Both Qualitative and quantitative measurement of

small amounts of pesticides in or on any treated surface is known as assay or estimation .

• Techniques of residue estimation -1. Micro bioassay• Direct exposure• Extraction method2. Enzyme inhibition method3. Spectrophotometric method

4. Chromatography• Paper chromatography• Thin Layer chromatography• Gas Chromatography• High pressure liquid chromatography• Micro bioassay – Bioassay has been defined as

measurement of potency of any stimulus, physical, chemical or biological, physiological by means of reactions which it produces in living matter.

Direct exposure - This method consists of macerating and blending the

plant tissue and exposing the test insect.• No extraction of clean up is required. Extraction method –a) Dry film method • Test organism is exposed to film of pesticide in

solution or deposited in crystals .b) Aqueous Suspension Method This method involves extraction of pesticides,

evaporating solvent, making suspension of residues in water and testing it.

2.Enzyme inhibition method –• Inhibition of cholinesterase enzyme system in animals

by pesticides particularly OP and carbamates is a basis of this method.

3.Spectrophotometeric method -• Amount of electromagnetic radiations absorbed is a

measure of quantity of sample present and provide information regarding chemical identity of sample.

4.Chromatography –• Flow of gas or liquid in a sorptive medium brings about

separation of substances by differential migration from narrow zone in a porous sorptive medium.

• Paper chromatography -• Stationary phase is a sheet of paper containing

water or some other polar phase.• Two opposing forces are in operation ,one is

driving which act to move substances in direction of solvent flow and other resistive action which impede movement of substances.

• Thin Layer chromatography -• Mixture of silica gel and water in the ratio 1:2

is prepared on carrier plates.• Spot of sample is poured on the plates.

• Gas Chromatography –• Commonly used .• Provide both qualitative and quantitative

analysis.• Mixture of pesticides can be analyzed.

Minimizing Exposure To Pesticide Residues

Organic vs. Non-organic• Even though some organic foods contain significantly

less amounts of pesticides than non-organically produced products, they still contain certain amounts of residue levels that are persistent in the environment.

• The methods of organic farming prohibit the use of many pesticides, however certain chemicals could be persistent in the soil, reach the organic produce via pesticide drift from nearby farms, or contaminate produce during transportation to the consumer.

CONTINUE• Certain measures that can be taken to minimize

exposure to harmful residues: • vegetables washes with water, NaCL, vinegar.• peeling fruits and vegetables.• Cooking.• Observe waiting periods may help remove chemical residues that have

known high levels of pesticide residues, and growing your own essential produce.

Eliminating The Residues• A pesticide can’t simply be classifies as ‘dangerous’ or ‘safe’. It

always depends on the amount or dose used.• Use pesticide as per recommended dose by (CIB-RC)• Establishment of MRLs for each pesticide on each food

commodity is required• Pesticides undergo vigorous research and trials before

introduction.• It includes manual removal, applying heat, covering weeds

with plastic, placing traps and lures, removing pest breeding sites, maintaining healthy soils that breed healthy, more resistant plants, cropping native species that are naturally more resistant to native pests and supporting bio control agents such as birds and other pest predators.

• Biological controls such as resistant plant varieties and the use of pheromones, have been successful and at times permanently resolve a pest problem

• Integrated Pest Management (IPM) /Integrated disease Management (IDM) employs chemical use only when other alternatives are ineffective.

• Biotechnology can also be an innovative way to control pests. Strains can be genetically modified (GM) to increase their resistance to pests.