Pesticide Cycle and Quantification of Organochlorine Pesticides.(Food analysis)

In this slide contains introduction, Pesticide Cycle and Quantification of Organochlorine Pesticides.
Presented by: K.Sandhya Rani. ( Department of pharmaceutical analysis),
RIPER, anantapur.

1. 1 Presented by Ms. K. Sandhya Rani (Reg.No:20L81S0702 ) Under the guidance of Dr. P. Ramalingam, M. Pharm., PhD Director – R&D Division & Professor of Pharmaceutical Analysis and Medicinal Chemistry, President – IPA Local Branch – Anantapuramu . A Seminar as a part of curricular requirement for 1 st year M . Pharm 1 st semester Pesticide Cycle & Quantification of Organochlorine Pesticides

2. 2 Introduction Pesticide cycle Organochlorine Pesticides Quantification of organochlorine pesticides References Contents

3. 3 Pesticides: Introduction Pesticides   are the substances that are meant to control pests. The term pesticide includes all of the following:  Herbicide, insecticide, nematicide, molluscicide, piscicide, avicide, rodenticide,  bactericide, insect repellent, animal repellent, antimicrobial, and fungicide. The application of pest control agents is usually carried out by dispersing the chemical in an (often hydrocarbon-based) solvent-surfactant system to give a homogeneous preparation.

4. 4 Pesticide Cycle

5. 5 Adsorption is the binding of pesticides to soil particles. The amount a pesticide is adsorbed to the soil varies with the type of pesticide, soil, moisture, soil pH, and soil texture. Pesticides are strongly adsorbed to soils that are high in clay or organic matter. They are not as strongly adsorbed to sandy soils. Adsorption

6. 6 Volatilization is the process of solids or liquids converting into a gas, which can move away from the initial application site. This movement is called Vapour drift. Vapour drift from some herbicides can damage nearby crops. Pesticides volatize most readily from sandy and wet soils. Hot, dry, or windy weather and small spray drops increase volatilization. Where recommended, incorporating the pesticide into the soil can help reduce volatilization. Volatilization

7. 7 Spray Drift is the airborne movement of spray droplets away from a treatment site during application. Spray drift is affected by: spray droplet size - the smaller the droplets, the more likely they will drift wind speed - the stronger the wind, the more pesticide spray will drift distance between nozzle and target plant or ground - the greater the distance, the more the wind can affect the spray. Spray drift

8. 8 Runoff is the movement of pesticides in water over a sloping surface. The pesticides are either mixed in the water or bound to eroding soil. It also occur when water is added to a field faster than it can be absorbed into the soil. Pesticides may move with runoff as compounds dissolved in the water or attached to soil particles. The amount of pesticide runoff depends on: slope, texture of the soil ,moisture content, type of pesticide, amount and time of rain. Runoff

9. 9 Leaching is the movement of pesticides in water through the soil. Leaching occurs downward, upward, or sideways. The factors influencing whether pesticides will be leached into groundwater include characteristics of the soil and pesticide, and their interaction with water from a rain-event such as irrigation or rainfall. Leaching can be increased when: the pesticide is water soluble, the soil is sandy, pesticide is not strongly adsorbed, rain-event occurs shortly after spraying. Leaching

10. 10 Absorption is the uptake of pesticides and other chemicals into plants of microorganisms. Most pesticides break down once they are absorbed. Pesticide residues may be broken down or remain inside the plant or animal and be released back into the environment when the animal dies or as the plant decays. Some pesticides stay in the soil long enough to be absorbed by plants grown in afield years later. They may damage or leave residues in future crops . Absorption:

11. 11 Degradation is the process of pesticide breakdown after application. Pesticides are broken down by microbes, chemical reactions, and light or photodegradation. This process may take anywhere from hours or days to years, depending on environmental conditions and the chemical characteristics of the pesticide. Pesticides that break down quickly generally do not persist in the environment or on the crop. However pesticides that break down too rapidly may only provide short-term control. Degradation or Breakdown Processes

12. 12 Microbial breakdown is the breakdown of chemicals by microorganisms such as fungi and bacteria. Microbial breakdown tends to increase when: temperatures are warm soil pH is favourable soil moisture and oxygen are adequate soil fertility is good Microbial breakdown

13. 13 Chemical breakdown is the breakdown of pesticides by chemical reactions in the soil. The rate and type of chemical reactions that occur are influenced by: the binding of pesticides to the soil soil temperatures pH levels - Many pesticides, especially the organophosphate insecticides, break down more rapidly in alkaline soils or in spray tank water with a high pH level moisture. Chemical breakdown

14. 14 Photodegradation is the breakdown of pesticides by sunlight. All pesticides are susceptible to photodegradation to some extent. The rate of breakdown is influenced by the intensity and spectrum of sunlight, length of exposure, and the properties of the pesticide. Pesticides applied to foliage are more exposed to sunlight than pesticides that are incorporated into the soil. Pesticides may break down faster inside plastic-covered greenhouses than inside glass greenhouses, since glass filters out much of the ultraviolet light that degrades pesticides. Photodegradation

15. 15 These are synthetic pesticides and are derivatives of chlorinated hydrocarbons. These are characterized by three kinds of chemicals: DDT (Dichlorodiphenyltrichloroethane)analogs BHC (Benzenehexachloride) isomers Cyclodiene compounds Examples: DDT, DDD, Dicofol, Eldrin, Dieldrin, Chlorobenziate, Lindane, BHC, Methoxychloro Aldrin, Chlordane, Heptaclor, Endosufan, Isodrin, Isobenzan, Toxaphene, Chloro propylate. Organochlorine Pesticides:

16. 16 Name of pesticide Structure Toxicity Dichlorodiphenyltrichloroethane (DDT) (C 14 H 9 Cl 5 ) Endocrine disruptor, risk factor for breast cancer. 1,1-dichloro-2,2bis (p- chlorophenyl)ethane (DDD) Seizures, respiratory failure, metabolic acidosis, cellulitis and coma Dichloro diphenyl dichloroethane (DDE) Excitability, tremors and seizures, increased chance of having premature baby. Dicofol (C 14 H 9 Cl 5 O) Conjuctivitis, CNS disorders, respiratory disorders. Endrin (C 12 H 8 Cl 6 O) Act as a neurotoxin

17. 17 Name of pesticide Structure Toxicity Dieldrin (C 12 H 8 Cl 6 O) Major motor convulsions, Endocrine disruptor, Dizziness, Headache. Methoxychlor (C 16 H 15 Cl 3 O 2 ) Neurotoxicity, development of leukemia, estrogenic effects Chlordane (C 10 H 6 Cl 8 ) Risk factor for type-2 diabetes, lymphoma, different types of cancers. Heptachlor (C 10 H 5 Cl 7 ) Neurological effects, irritability, salivation, dizziness Lindane (C 6 H 6 Cl 6 ) Uncontrolled seizures, psoriasis, dermatitis.

18. 18 Name of pesticide Structure Toxicity Endosulfan (C 9 H 6 Cl 6 O 3 S) Tonic- clonic convulsions, Lack of co-ordination, skin diseases etc. Toxaphene (Camphechlor) (C 10 H 10 Cl 8 ) Seizures, bronchial carcinoma, damage lungs, nervous system, kidneys. Isobenzan (C 9 H 4 Cl 8 O) Myocardial irritability and Cardiac arrhythmias disturbances of sensation, coordination, and mental status. Isodrin (C 12 H 8 Cl 6 ) Nervous System Disturbances

19. 19 List of foods containing Organochlorines: Spices and Seeds Fruits or Berries Poultry meat Eggs Milk Vegetables (tomatoes, brinjal, onion) Leafy vegetables Corn, Maize Soya bean, Moong beans

20. 20 Pesticide Maximum Residue Limit (MRL) (mg/kg) DDT 0.5 Dicofol 3 Endrin 0.01 Chlordane 0.02 Methoxychlor 0.01 Aldrin / Dieldrin 0.05 Lindane 1 Endosulfan 2 Alachlor 0.01 MRL: A Maximum Residue Level ( MRL ) is the highest level of a  pesticide  residue that is legally tolerated in or on food or feed when  pesticides  are applied correctly.

21. 21 The basic steps of a quantitative analytical method for pesticide residues include the following: Sample preparation Extraction Clean Up Separation Detection and Quantification Quantification of Organochlorine Pesticides

22. 22 Sample Preparation: It can be done by Chopping, Grinding or Blending. The well equipped food analysis laboratory should have a variety of sample preparation equipment including mechanical choppers, mincers, grinders, blenders. The moisture content of a food also plays an important role in determining the food processing procedure or equipment to use. When a sample is processed by hand, it must be sufficiently finely divided to permit proper mixing and later sub-sampling of the mixture.

23. 23 Extraction process: This is a fundamental process in pesticide analysis. There are several approaches employed such as: Pressurized Liquid Extraction (PLE) Supercritical Fluid Extraction (SFE) Aqueous Extraction Microwave Assisted Extraction (MAE) Solid Phase Extraction (SPE) Matrix Solid Phase Dispersion (MSPD)

24. 24 1. Pressurized Liquid Extraction: Pressurized fluid extraction (PLE) is also known as pressurized solvent extraction (PSE), accelerated solvent extraction (ASE), pressurized accelerated solvent extraction (PASE) and enhanced solvent extraction (ESE). Samples are loaded into vessels, which require a drying agent to aid solvent flow. Extraction occurs at elevated temperature (40-200) and pressure 1000-3000 psi. The analyte is allowed to interact statically with the pressurized solvent for a predetermined time, after which the pressurized solvent is purged from the cell by a nitrogen stream and the extract is collected in a vial.

25. 25 2. Supercritical Fluid Extraction: SFE uses supercritical fluid as extraction solvent (CO 2 ) .With a low viscosity and high diffusivity, the component which is to be extracted can pass through the mobile phase easily. The higher diffusivity and lower viscosity of supercritical fluids, as compared to regular extraction liquids, help the components to be extracted faster than other techniques. Thus, an extraction process can take just 10-60 minutes with SFE.

26. 26 3. Microwave Assisted Extraction: In a MAE heating occurs in a targeted and selective manner. MAE process is as follows: Microwave radiation Moisture get heated up Generation of tremendous pressure on sample Sample swells and lysis occurs Leaching out of constituents

27. 27 Solid phase extraction uses the difference of affinity between an analyte and interferents, present in a liquid matrix, for a solid phase (sorbent). This affinity allows the separation of the target analyte from the interferants. A typical solid phase extraction involves four steps : First, the cartridge is equilibrated or conditioned with a solvent to wet the sorbent. Then the loading solution containing the analyte is percolated through the solid phase. The sorbent is then washed to remove impurities. The analyte is collected during this elution step. 4. Solid Phase Extraction:

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29. 29  MSPD is based on principle of involving forces applied to the sample by mechanical blending. It produce complete sample disruption and the interactions of the sample matrix with a solid support bonded-phase (SPE) or the surface chemistry of other solid support materials. 5. Matrix Solid Phase Dispersion:

30. 30 Take the concentrated petroleum ether extract into 250 ml separatory funnel. Add 100mL of petroleum ether saturated with acetonitrile, after vigorous shaking, discard the petroleum ether layer. collect the acetonitrile layer and reduce the volume to 10 ml. Add 20 ml of 10 % Sodium chloride solution; extract the residues with 75 ml of n-Hexane two times. Collect the n-hexane layers each time and reduce to small volume. Clean-Up:

31. 31 Acetonitrile free residue is dissolved in mixture of 10 parts dichloromethane and 90 parts petroleum ether Then it is placed on a prepared florisil column (diameter 2.5cm, length30-35 cm) The extract is evaporated to dryness in a rotary evaporator at 30℃ Extract is filtered through a filter paper over calcined sodium sulphate Extracted 3times with acetonitrile each time for 15 minutes 50gm of sample is taken General Procedure:

32. 32 The column is finally rinsed with 50ml pure dichloromethane The removal of interfering substances by means of concentrated sulphuric acid produces colorless elute. The elutes are concentrated to dryness separately in rotary evaporator at a maximum temperature of 40℃. Main fraction of chlorinated hydrocarbon is eluted with 100ml of mixture of 15 parts dichloromethane and 85 parts petroleum ether. The 2 nd elution with 100ml of mixture of 25 parts dichloromethane and 75 parts petroleum ether is collected in another receiving vessel.

33. 33 The bulb of evaporator is connected to 5ml measuring flask . The concentrated elute is made upto the mark with cyclohexane at 20℃. 1.5ml is injected into gas chromatography

34. 34 Column 1 30 m × 0.25 or 0.32 mm ID fused silica capillary column chemically bonded with SE-54 1µ film thickness. Carrier gas Helium Carrier gas pressure 16 psi Injector temperature 225°C Detector temperature 300°C Initial temperature 100°Chold 2 min Temperature program 100 °C to 160 °C at 15°C/min Final temperature 160°C to 270°C at 5°C/min Detection by Gas Chromatography:

35. 35 Column 2 30 m × 0.25 mm ID fused silica capillary column chemically bonded with 35 % phenyl methylpolysiloxane ,25 µm coating thickness. Carrier gas Nitrogen Carrier gas pressure 20 psi Injector temperature 225°C Detector temperature 300°C Initial temperature 160°C hold 2 min Temperature program 160°C to 290 °C at 5 °C/min Final temperature 290°C hold 1 min

36. 36 Measure the peak height of the pesticides obtained in the chromatograms and calculate the concentration of the residues in mg/kg. By using the following formula. Ht×10/W ×Wt/Ht Where, Ht- peak height obtained for the test solution in mm. W- Quantity of the sample in the purified extract(g). Wt- Quantity of pesticides in ng in reference solution injected. Ht- Peak height obtained for the reference solution in mm.

37. 37 Peak height of the sample Peak height of the standard Residue level(mg/kg) = vol of the standard ( μ l) vol of the sample injected ( μ l) X X Final vol of sample extract conc. (ml) Mass of sample solution (g) X Concentration in ppm of reference standard

38. 38 GC Chromatogram of Organochlorine pesticides

39. 39 Gas chromatogram of Chlordane

40. 40 It is a multiclass multiresidue method (MRM). QuEChERS can primarily be thought of as a two-step process:  The first step is Sample preparation and Extraction, where analytes of interest are extracted from your sample. The second step is Cleanup, where many potential interferences are removed from the extract.  A variety of QuEChERS methods exist to help ensure the efficient extraction of different types of compounds. QuEChERS: (Quick, Easy, Cheap, Effective, Rugged and Safe)

41. 41 Step1: All the materials are ground uniformly and extracted using required quantity of acetonitrile. The extract is centrifuged. Step2: Cleaned up using DSPE Bulk drying salts and DSPE sorbent packings - Remove excess water and unwanted contaminants. Agitation and centrifugation - Vortex mixing for 30 sec and centrifugation at 3000rpm for 5 minutes. Then the sample is make up to the volume an analyzed by GC-MS/LC-MS.

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43. 43 Conventional method QuEChERS Time consuming, complicated Simplified alternatives Sample processing/ homogenization No way around this Blending/shaking for long time Homogenization for 30 sec Filtration Centrifugation Multiple partitioning steps Single partitioning Separation/Transfer of entire extract Take aliquots Evaporation/Reconstitution of large volume Evaporation / Reconstitution of small volume SPE with columns Dispersive SPE Lot of glassware Few glassware Difference between conventional method and QuEChERS

44. 44 1. Ministry of Health and Family welfare, Government of India, fssai Manual of Methods of Analysis of foods pesticides residues,2015 lab manual 11;43-52. 2. I.C. Yadav, N.L. Devi; pesticides classification and its impact on Human and environment environ.sci &engg.vol 6: Toxicology 2017 p.140-158 3. Lal, R., Dhanraj, P.S., NaranjanRao, V.V.S., 1989. Residues of organochlorine insecticides Environ.Contam. Toxicol. 42, 45–49. 4. Camel V. Recent extraction techniques for solid matrices-supercritical fluid extraction, pressurized fluid extraction and microwave-assisted extraction: their potential and pitfalls. The Royal Soc. Chem. Analyst. 2001, 126:1182-1193. References:

45. 45 Thank You