SUBJECT FORENSIC SCIENCE Paper No. and Title PAPER No.10: Forensic Toxicology Module No. and Title MODULE No. 12: Pesticides-II Module Tag FSC_P10_M12 FORENSIC SCIENCE PAPER No.10: Forensic Toxicology MODULE No.12: Pesticides-II TABLE OF CONTENTS 1. Learning Outcomes 2. Introduction 3. Rodenticides 4. Fungicides 5. Nematicides 6. Acaricides 7. Molluscicides 8. Extraction of Pesticides 9. Summary FORENSIC SCIENCE PAPER No.10: Forensic Toxicology MODULE No.12: Pesticides-II 1. Learning Outcomes After studying this module, you shall be able to know about – Some unconventional Pesticides, which are used for specific categories of living organism. Their Mode of Actions and pharmacodynamics. The methods of extraction of Pesticides. 2. Introduction Pesticide is a substance or mixture of substances intended to prevent, destroy, repel, or mitigate a pest. In general, pesticides are classified according to their biological targets. Because of the physiological and biochemical similarities of target species and mammalian organisms, an inherent toxicity is associated with pesticides in mammalian organisms. In addition, within each classification, compounds are identified according to mechanism of action, chemical structure, or semi-synthetic source. Conventional Pesticides deals with common insects but there are other pests which are also a menace. To come across with those pests it is necessary to invent specific category pesticides. 3. Rodenticides These are the chemical compounds which exterminate rats, mice, moles, and other rodents. Thallium, Zinc and Aluminium Phosphide, vacor, phosphorus, alpha-naphthyl-thiourea, cholecalciferol, arsenic, barium carbonate, bromethalin, fluoroacetamide, sodium monofluoroacetamide, Red Squill, strychnine are some of the examples of common Rodenticides. Some of these are very commonly involved in human poisoning, e.g. phosphorus, Zinc and aluminium phosphide, long acting anticoagulants. 4. Fungicides These are compounds which kills fungus and moulds. The examples of fungicides are thiocarbamates, captan, captafol, bavistin, vitavax, hexachlorobenzene, and sodium azide. Thiocarbamates has an extensive range of specimens which includes benomyl, benthiocarb (or thiobenzcarb), cycloate, diallate, ferbam, molinate, thiram, thiophanate, triallate, zineb and ziram. Carbendazim (carbendazole), ETU, mancozeb, maneb, and vondozeb are benzimidazole (ethylene bis di-thio carbamate or ethylene thiourea) compounds that are also used as fungicides. These compounds have relatively low toxicity. They do not hinder acetylcholinesterase (unlike carbamates). Formulations of these compounds are broadly used for pest control in home gardens and in commercial agriculture. FORENSIC SCIENCE PAPER No.10: Forensic Toxicology MODULE No.12: Pesticides-II 5. Nematicides These are compounds which exterminates nematodes (i.e. worms). It exclusive example is Ethylene Dibromide. Basically, Ethylene Dibromide type fumigants are applied to control rodents, nematodes, insects, weed seeds, and fungi anywhere in the soil, structures, crop, grains, and commodities. Historically, diverse chemical classes have been used as fumigants, but only a few remained in use today. Most fumigants were abandoned because of their toxicity, many of which were halogenated solvents. The discontinued halogenated hydrocarbons includes Carbon Tetrachloride (CCl4), Chloroform, dibromochloropropane, 1, 2- dichloropropane, ethylene dibromide, and ethylene dichloride. Ethylene dibromide was previously approved for use as a fumigant to protect against insects, pests, and nematodes in citrus, vegetable, and grain crops, and as a fumigant for turf, particularly on golf courses. In 1984, the Environment Protection Agency (EPA) banned its usage as a soil and grain fumigant. Unfortunately, in India, it continues to be used widely, causing human poisoning not infrequently. 6. Acaricides Acaricide is a pesticide deliberated to control harmful species of mites. In crop protection practices, acaricides are used against phytophagous mites, pests affecting economic harms to agricultural crops and decorative plants. These are complexes which exterminates mites, ticks and spiders. Spider mites, generally polyphagous species, are common pests in modern agroecosystems worldwide, and some of them are among the most important crop pests. Phytophagous mites nourish on the liquid content of plant cells, thus disturbing the physiology of a host plant and causing various damages to plant tissues and organs, while some of the species can also act as vectors of plant viruses. In spite of comparatively small size, plant- feeding mites can cause significant damage in crop yield and quality losses, because they have short life span and under favourable conditions their populations quickly reach high abundance. Examples of Acaricides are Azobenzene, Chlorobenzilate, Tedion, and Kelthane. They are rarely encountered in human poisoning. 7. Molluscicides These are the compounds which are capable of killing molluscs such as snails and slugs. e.g., Metaldehyde which is a tetramer compound with an eight member ring containing aldehyde molecules and also a cyclic polymer of acetaldehyde. Metaldehyde is a popular molluscicide being effective against snails and slugs. It is a tasteless substance with a mild characteristic odour. Instances of poisoning is however rare. It is a local irritant on skin and mucous membrane and a systemic convulsant. Metaldehyde overdosage results in lethargy, severe abdominal pain, nausea, vomiting, diarrhoea, hyperthermia, seizures, coma, and death. Profound hyperthermia may occur in association with seizures. Inhalation of metaldehyde fumes may cause CNS depression. The probable lethal dose LD50 is in the range of 100 mg/kg for adults. FORENSIC SCIENCE PAPER No.10: Forensic Toxicology MODULE No.12: Pesticides-II 8. Extraction of Pesticides The extraction of pesticides in biological materials like viscera, stomach contents, gastric lavage and blood is much meticulous due to the interferences of fat, degraded protein and coloring matter in the matrices. The extracts involve appropriate clean-up except for Micellar method. The extractions in case of non-biological matrices are less cumbersome and needs either slight clean-up or even no clean-up. The methods described hereunder are based on solvent extraction cum shedding under diverse conditions viz. nature and condition of matrices, use of organic solvent etc. A. Method- I: Sodium Sulphate and n- Hexane Method Biological materials (viscera, stomach content or gastric lavage etc.) are macerated into fine slurry by mixing with equal amount of anhydrous Sodium Sulphate (Na2SO4) and relocated into a conical flask with an air condenser. 50 ml of n-hexane is added to the flask and heated on a hot water bath for one hour. The contents are cooled and filtered. The remaining slurry is extracted twice with 25 ml portion of n-hexane. The filtered n-hexane fractions are combined and taken into a separating funnel. This hexane layer is vigorously shaken with 15 ml, 10 ml and again 10 ml portion of acetonitrile, which are previously saturated with n-hexane. The acetonitrile layers are mixed and taken into another clean separating funnel and diluted 10 times with distilled water. 25 ml of saturated Sodium Sulphate (Na2SO4) solution is added to it and extracted thrice with 25 ml portion of n-hexane. The n-hexane layers are combined, concentrated to 5 ml by evaporating on water bath and 5 gms of anhydrous Sodium Sulphate (Na2SO4) is added. The extract is evaporated as and when required for analysis. B. Method-II: Sodium Sulphate and Acetone Method 50 gm of macerated tissues or biological materials are mixed with equal amount of anhydrous Sodium Sulphate (Na2SO4) and 100 ml of Acetone in a conical flask and then refluxed on hot water bath for one hour. After cooling, the acetone extract is filtered. The residue is extracted twice with further 50 ml portion of acetone. The acetone fractions are combined and concentrated by evaporation up to 50 ml for further processing (cleanup). The above acetone extract (50 ml) is taken into a separating funnel and diluted with 150 ml of water. 20 ml of saturated solution of Sodium Sulphate (Na2SO4) is added to the same. The contents are extracted thrice with 25 ml portions of chloroform with gentle shaking. The chloroform extracts are combined, washed with water-acetone mixture (1:1) and finally with 50 ml of water. The washed chloroform layer is passed through anhydrous Sodium Sulphate (Na2SO4) and then evaporated to dryness by passing air. FORENSIC SCIENCE PAPER No.10: Forensic Toxicology MODULE No.12: Pesticides-II C. Method- III: Extraction of Pesticides in stomach-wash, urine and vomit The sample (20 ml of stomach wash or urine or 10- 20 gm of vomit) is taken in a conical flask. 50 ml of n-hexane is added. It is refluxed on a water bath for half-an-hour. After cooling, the liquid is filtered, mixed with 20 ml of n-hexane and taken in a separating funnel. The n-hexane layer is separated; passed through anhydrous Sodium Sulphate (Na2SO4) and evaporated to dryness by passing a current of dry air through it. D. Method-IV: Extraction of Pesticides in Blood 20 ml of blood is mixed with 10 ml of 10% Sodium Tungstate solution and 15 ml of 1N Sulphuric Acid, shaken for two minutes and then filtered. The filtrate is kept reserved. The residue is washed with two 15 ml portions of 0.1N Sulphuric Acid. The washings are collected, mixed with filtrate (kept reserved), transferred into a separating funnel