Pesticide Residue Analysis

It describes about different steps and methods of pesticide residue analysis.

1. PESTICIDE RESIDUE ANALYSIS Presented By: Manish Joshi Department of Entomology, SDAU, SK Nagar (Gujarat) Submitted To: Dr. P. S. Patel (Assoc. Prof.) Department of Entomology, SDAU, SK Nagar (Gujarat)

2. Pesticide Pest- “Pest is any harmful, destructive, or troublesome animal, plant or microorganism.” Pesticide- “Pesticide is any substance or mixture of substances intended for preventing, destroying, repelling, or mitigating any pest.” US Environmental Protection Agency (EPA)

3. WHY DO WE NEED PESTICIDES ? Protect Crop losses / yield reduction Protection during Storage Vector Disease Control India produces 16% of the world’s food grain but using less than 2% pesticides used world-wide. Hence there is a good potential of growth.

4. Pesticide : Side effects on human body Agrochemicals and Security: Homeland Security and Pesticides Pest-09 Acute exposure Chronic exposure Inhalation Absorption through skin Through food or water How pesticides get in Exposure levels Central nervous system Skin irritation Eye irritation Health effects (depends on specific pesticide) Hormone imbalance Cancer Liver damage Reproductive effects

5. 5 Factors Responsible for Increase Pesticide Residues

6. Label Level of toxicity LD 50 Listed chemicals Extremely toxic 1-50 Monocrotophos , Zinc phosphide , Ethyl mercury acetate Highly toxic 51-500 Endosulfan , Carbaryl , Quinalphos Moderately toxic 501-5000 Malathion , Thiram , Glyphosate Slightly toxic More than 5000 Mancozeb , Oxyfluorfen , mosquito repellant oils and liquids, and most other household insecticides. Color code of different Pesticides

7. New Colour code submitted by CIB

8. Pesticide Residues means any specified substances in food, agricultural commodities, or animal feed resulting from the use of a pesticide. The term includes any derivatives of a pesticide, such as conversion products, metabolites, reaction products and impurities considered to be of toxicological significance. 9 What is Pesticide Residue?

9. Terminology Residue : When pesticides is applied on the commodity, its parent compound or metabolism having pharmacological property and/or toxicological nature, together termed as residue. Maximum residual limit (MRL) : The limit beyond which pesticide levels are not legally permitted. Acceptable daily intake (ADI) : It is the daily dose of chemical which during an entire life time, appears to be without appreciable risk on the basis of all facts known at that time. Waiting period (T MRL ) : The determination of the minimum interval required between application of pesticide and harvesting of crop. RL 50 or T ½ (Half-life value) : It is the time in days required to reduce the pesticide residues to half of its initial deposits. 10

10. Persistence and Degradation Persistence – The chemical that remain active for a long period of time after application. Degradation – The breakdown of a substance into simpler molecular or atomic components through chemical reaction(s) either in a plant or animal (metabolic degradation) or in the environment (environmental degradation) 11

11. Deposit: The amount of initially laid down pesticide on the surface is called deposit. Dissipation and persistence: In nature, disappearance of residues takes place in two steps. The first step is the initial phase in which the disappearance of the residue is fast. This phase is called " Dissipation ". The second phase, in which there is a slow decrease in the amount of residue, is known as " Persistence ". 12

12. Workflow for analysis of pesticide residues

13. STEPS IN PESTICIDE RESIDUE ANALYSIS SAMPLING SAMPLE PREPARATION EXTRACTION CLEANUP QUANTIFICATION CONFIRMATION 14

14. 15 SAMPLING PROCEDURES Sample collection is the process of taking portion (s) of a food or other substance, for laboratory analysis, so that resulting analytical data and conclusions can be applied to the original food or substance sampled .

15. 16 COLLECTION OF WATER SAMPLES

16. 17 Water Underground water Do not take a sample from tank Purge for 10 minutes Collect water in washed glass bottle Collect 2.5 L water to represent one sample Transport sample to the laboratory at 4 C

17. 18 SAMPLE SIZE Samples of 2.5 to 5 liter / sampling site These are reduced with through mixing and by filling samples in to 1 to 2 bottles

18. 19 SAMPLING PROTOCOL Label it immediately Also record : Geography depth & diameter Exact location Date and time of sampling Water volume Water and air temperature Weather parameter Sampling personal

19. 20 COLLECTION OF SOIL SAMPLES

20. 21 SAMPLING Sample taken with auger In grass land – 10 cm In forest land – 20 cm In orchard – 0 to 25 & 25 to 50 cm Sampling site spread evenly Sample should not taken from border strip or places where pesticides have been transferred from container to tank or where spray have been mixed In large field, the sampling site should be 5 m away from field periphery or above site

21. 22 SAMPLE SIZE In small field : 3 x 3 grid In medium size field : 4 x 4 grid In large size field : 5 x 5 grid Same in untreated plots

22. 23 Sample size Type Unit weight (g) Minimum amount to be collected Small or light units e.g. Berries, peas etc. 25 g or less 1 kg Medium sized units e.g. Apple, orange, carrot, potato 25 - 250 g 1 kg (At least 10 units) Large sized units e.g. Cabbage, cauliflower, melons >250 g 2 kg (At least 5 units) Dairy products e.g. Butter, Milk cream, Cheese 0.5 kg Poultry products e.g. egg 0.5 kg Animal tissue e.g. Meat, Chicken, Fish and their products 0.5 kg Oil and fats e.g. Cotton seed 0.25 kg Spices e.g. Cumin, Fennel 0.5 kg Cereals e.g. Wheat, Rice 1.0 kg

23. Sample preparation Reduce the sample – sub sampling Store reserve portion of the sample Homogenize the sub sample by :- Chopping Grinding Blending For this, knives, grinder, warning blender are used. 24

24. Extraction process Extraction is the process by which toxicant is transferred from the treated bulky biological material in to a solvent.

25. Pressurized liquid extraction(PLE)

26. Supercritical fluid extraction (SFE)

27. Solid-phase Extraction

28. Clean-up Isolation of toxicant from interfering substances or solvents.

29. Various method of estimation

30. Spectrophotometric Method Three kinds of spectrophotometric method used in pesticide residue analysis. Ultraviolet, visible and infra-red methods. Measure the conc. of pesticide in solution by measuring the amount of the light that is absorbed by the solution. Range 180nm to 400nm

31. Chromatography The chromatography consist in the unique distribution of the components of a mixture between stationary phase and mobile phase due to different affinity of a single compounds between forward both phase. It involve passing a mixture dissolved in a “mobile phase” through a stationary phase, which separates the analytes to be measured from other molecules in the mixture based on differential distribution coefficient and different net rate of migration between the mobile and stationary phase. Chromo means “colour” & Graphy means “ To write”

32. Various techniques of chromatography

33. Planar Chromatography Planar chromatography is a separation technique in which the stationary phase in or on a plane. The plane can be paper, serving as such or impregnated by a substance as the stationary bed ( Paper Chromatography ) OR A layer of solid particles spread on a support such asa glass plate ( Thin layer chromatography )

34. Column Chromatography Stationary phase is placed into glass or metal column. The mixture of analytes is then applied and the mobile phase, commonly referred to as the Eluent passed through column either gravity feed or by use of pumping system or applied gas pressure . The stationary phase either coated on to discrete small particles (Matrix) and packed in to column. As the Eluent flow through the column analyte separate on the basis of their distribution coefficient and emerge individually in the eluent as it leaves the column.

35. Gas Chromatography(GC) Volatility and thermal stability. Carrier gas- Helium , Argon and Nitrogen : Which are chemically inert. The stationary phase is adhered to the inside of a small-diameter glass tube ( a capillary column ) or a solid matrix inside a larger metal tube ( a packed column ). 80% of pesticide can be analysed. Organochlorines , Organophosphates, Carbamates

36. Pesticide detected using GC Organochlorines e.g. Dieldrine , DDT, Lindane Organophosphorus e.g. Diazinon , dichlorovos , malathion Pyrethroid e.g. cypermethrin , deltamethrin Aromatic fungicides e.g. Chlorothalonil , quintozene Carbamates, imidazole , benzoylureas , sulfonylureas

37. Liquid Chromatography (LC) It is a separation technique in which the mobile phase is a liquid. Present day liquid chromatography that generally utilizes very small packing particles and a relatively high pressure is referred to as high performance liquid chromatography (HPLC). In the HPLC techniques, the sample is forced through a column that is packed with irregularity or spherically shaped particles or a porous monolithic layer ( Stationary phase) by a liquid (Mobile phase) at high pressure.

38. Carbamates e.g. Carbendazim Conazoles e.g. Fenamidone Sulfonylureas e.g. triasulfuron Triazines e.g. Simazine Phenylureas e.g. linuron Strobilurins e.g. Azoxystrobin Neonicotinoids e.g. Acetamiprid All the above plus their metabolites can be analysed by liquid chromatography Pesticide detected using LC

39. QuEChERS- Multiresidue-Method ADVANTAGES Rapid (8 Samples in Less Than 30 min) Simple (No Laborious Steps, Minimal Sources of Errors) Cheap Low Solvent Consumption (10 ml Acetonitrile) Practically no Glassware Needed Wide Pesticide Range (Polar, pH-Dependent Compounds) Extract in Acetonitrile (GC- and LC-Amenable)

40. Avoid un-judicial use of pesticides Use only recommended dose of pesticides Use pesticides only need base Follow safe harvesting period i.e. waiting period Avoid to use of pesticide near to ripening stage of crop Follow washing and cooking before use of vegetables Use botanicals / microbial insecticides for the control of various crop pests Purchase pesticides only from authorized dealers Banned pesticides should not be used for pest control Get detailed information from the authorized people before mixing of various pesticides. Follow IPM & GAP Steps to overcome pesticide residue problems 41

41. “Technical development always follow the way from the primitive via the complicated to simple”