Pesticides

1. Pesticides

2. The term ‘pesticide’ encompasses a wide variety of substances used to destroy unwanted life forms. Pesticides are applied in agriculture for crop protection and pest control, and in human and animal hygiene. They are classified as insecticides, herbicides, rodenticides, fungicides, nematocides, molluscicides and acaricides on the basis of their field of use. Commercial formulations can be mixtures of pesticides from different classes.

4. Most pesticides have common names agreed by the International Standardisation Organisation (ISO) through its Technical Committee. These common names are used throughout this chapter for convenience and brevity, but their equivalent systematic chemical names can be ascertained easily according to the rules of the International Union of Pure and Applied Chemistry (IUPAC) and the Chemical Abstracts Service Registry Number . Pesticides are also divided into different chemical subclasses. Often the type of chemical is also indicated by a stem in the common name (e.g. ‘uron’ for ureas, and ‘carb’ for carbamates).

6. Insecticides Insecticides may be classified into eight chemical groups, of which the following five are the most important: Organophosphorus (OP) compounds, which have the general structure 1. Carbamates (structure 2 , where R1=methyl, R2=H or methyl, and R3=aryl, heterocyclic or oxime groups) (e.g. aldicarb, structure 3)

7. Chlorinated hydrocarbons, which include dichlorodiphenyltrichlorethane (DDT) and its analogues (e.g. methoxychlor), hexachlorocyclohexane isomers (e.g. lindane) and bridged polycyclic chlorinated compounds (e.g. endosulfan, structure 4). Pyrethroids, both natural (e.g. pyrethrin II, structure 5) and synthetic (e.g. deltamethrin, structure 6 ) Substituted ureas (e.g. diflubenzuron, structure 7) . Other insecticide groups are organotin (e.g. cyhexatin, structure 8 ), and heterocyclic compounds (e.g. dazomet, structure 9 ).

8. Herbicides Herbicides may be classified into at least 12 groups, but the seven most important are: Chlorinated phenoxy acids (e.g. 2,4–dichlorophenoxyacetic acid (2,4-D), structure 10). Substituted ureas (e.g. metobromuron, structure 11). Triazines (e.g. atrazine, structure 12).

9. Uracils (e.g. lenacil, structure 13). • Quaternary ammonium compounds (e.g. paraquat, structure 14, and diquat). • Carbamates, which include not only the carbamates (e.g. propham), but also thiocarbamates (e.g. tri–allate, structure 15). • Carboxylic acids and esters (e.g. dicamba). • Other herbicides include amides chloroacetanilide, OP and organoarsenic compounds

10. Fungicides • Chemicals from many groups belong to this class: benzimidazoles (e.g. carbendazim), dithiocarbamates (e.g. thiram, structure 16, acylalanines (e.g. metalaxyl) and OP compounds (e.g. pyrazophos). The most important are: • Dithiocarbamate complexes with manganese, nickel and zinc. • Organic and inorganic compounds of copper and mercury

11. Rodenticides Three types of compounds are notable in this category Phosphines (derived by the reaction of moisture with magnesium, aluminium and zinc phosphides). Thallium salts, usually sulfates. Coumarin anticoagulants (e.g. brodifacoum, bromadiolone, coumatetralyl, difenacoum

12. Acaricides, molluscicides and nematicides These include organotin (acaricide), niclosamide (molluscicide) and phorate (nematicide). Some of these compounds that have more than one application can be found among those mentioned above

13. Toxicity • The large variety of chemical compounds that show pesticide properties means that there is a very wide range of toxicity in humans. It is believed that an oral dose of only several drops (100mg) of terbufos is fatal to most adults, whereas another pesticide (amitrole) is non–toxic in humans even when several hundred grams are ingested.

14. Within a particular class of pesticide the lethal dose may vary considerably. Moreover, the metabolites of many pesticides (e.g. oxygen analogues of phosphorothionates) are much more toxic than the parent compounds

15. The commercially available preparations usually contain an active substance mixed with filler (solids) or dissolved in an organic solvent (liquids). Although certain pesticides are unlikely to cause acute toxicity, the vehicle in which they are formulated (toluene, xylenes, butan-1-ol, cyclohexanone, and solvent naphthamay itself be toxic and, in some cases, can be the main causative agent for the symptoms observed.

16. The pesticides have been classified into five groups according to the World Health Organisation (WHO) toxicity classification for estimating the oral acute toxicity of pesticides. Toxicity was determined on the basis of LD50 for the rat and the estimated lethal doses related to a 70kg person. However, realistic human lethal doses of pesticides can be estimated only on the basis of well–documented cases of poisoning

17. WHO Pesticide Classification

18. WHO PESTICIDE LIST • Active ingredients believed to be obsolete or discontinued for use as pesticides • Extremely hazardous (Class 1a) technical grade active ingredients of pesticide • Highly hazardous (Class 1b) technical grade active ingredients of pesticides • Moderately hazardous (Class II) technical grade active ingredients of pesticides • Slightly hazardous (Class III) technical grade ingredients of pesticides

19. Colour tests Some colour tests can be very useful preliminary indicators of the class of compound and can confirm the constituents of a proprietary formulation. Simple quantification of compounds that belong to specific groups is also possible. To reduce false positives from artefactual sources, a blank solution should be subjected to the same procedure as the sample. It is also essential to check the viability of the reagents by analysing a reference compound

20. Sample preparation The major metabolites of many pesticides (e.g. carbamates and organophosphates) are sulfate and glucuronide conjugates. Cleavage of conjugates by enzymatic or acid hydrolysis is necessary before extraction. However, deconjugation of pesticides by acid hydrolysis drastically increases the formation of artefacts and can destroy analytes completely. Therefore, the gentle enzymatic method is recommended. The extraction of body fluids is further complicated because certain pesticides are decomposed readily by acids or alkalis

21. Enzyme hydrolysis (urine) To 5mL of urine specimen are added 1mL of 1M acetate buffer (pH 5) and 40μL of beta-glucuronidase plus arylsulfatase (30U/mL plus 60U/mL) and the mixture incubated overnight at 37° in a closed test tube. Incubation for 45min at 56° is less time–consuming, but the quantitative results are more variable – this shorter process can be used in emergency cases

22. Derivatisation procedures • methyl (ME, trifluoroacetyl (TFA) and acetyl derivatives (AC) derivatives of pesticides are usually obtained. Trifluoroacetylation and acetylation processes can be carried out for 5min under microwave irradiation at about 400W

23. Thin–layer chromatography • Four TLC systems are used; each consists of an independent mobile phase and a sequence of different spray reagents, widely used for pesticide visualisation. General systems TZ and TAA (are used to reveal any pesticide in an examined sample and to enable presumptive chemical classification. Two more systems, TX and TY, are used to identify the type of pesticide. The TX and TY systems give good reproducibility. TheRf values of the reference compounds chosen for the four screening systems are derived using 5 to 10μg of each substance. Each extract is spotted onto a TLC plate in an amount corresponding to 2g of the biological material being analysed. For additional information, two other solvent systems (TAB and TAC) can be applied

24. The chromatographic process uses silica–gel plates of 0.25mm layer thickness, without fluorescent indicator, and four mobile phases in saturated chambers in ascending mode. Seven spray reagents are suggested, which produce a variety of colours to facilitate differentiation. A large number of pesticides react with more than one reagent. The reagent sequences chosen allow the plates to be oversprayed

25. The LOD for most pesticides is 10μg after reagent overspraying and 2 to 5μg after single–reagent spray detection. After drying, all the chromatograms are first examined under ultraviolet (UV) light (366nm and 254nm) and then sprayed successively with the location reagents appropriate for each system. The plate is sprayed with a location reagent, dried and a note is taken of any colours. The plate is then oversprayed with another reagent and again any changes are noted.

26. Spray reagents Silver nitrate (AgNO3) • The plates are sprayed with a 0.1M aqueous solution of AgNO3. After spraying, the dry plates are exposed to UV radiation (254nm) for 10min. Many pesticides give white, grey and brown spots on a bright brown background Rhodamine B and sodium hydroxide • (RHB–NaOH) A 0.02% (w/v) solution of rhodamine B (RHB) in ethanol and a saturated solution of NaOH in ethanol are used as the spray. After both the RHB and NaOH solutions have been sprayed, compounds are located as navy–blue spots by examination under UV .

27. Diphenylamine and zinc chloride • (DPA–ZnCl2) The spray comprises 0.7% (w/v) diphenylamine (DPA) and 0.7% (w/v) ZnCl2 solution in acetone. After spraying, the plates are exposed to UV radiation for 10min and then heated at 100° until no further colour change is observed. Light blue, blue, green and pink spots are observed on a white background

28. 2,6-Dibromoquinone–4–chlorimide and sodium hydroxide • (DBQ–NaOH) A 0.2% (w/v) solution of 2,6–dibromoquinone–4–chlorimide (DBQ) in acetone and a saturated solution of NaOH in ethanol are used to spray the plates. After spraying, the plates are heated at 100° for 10min. Navy blue, pink, violet spots on a light blue background are observed

29. Palladium chloride • To make the spray, dissolve 0.5g PdCl2 in 2.5mL of 35% (v/v) HCl and carefully dilute with water to 100mL. After spraying, yellow and brown spots are observed

30. 4-(4-Nitrobenzyl)pyridine and tetraethylenepentamine (NBP–Tetren) To make the spray, dissolve 5g of 4-(4–nitrobenzyl)pyridine (NBP) in 100mL of acetone and dilute 1:5 (v/v) of Tetren with acetone . Spray the plate with the NBP solution and dry at 110° for 10min. After cooling, spray the plate with the dilute Tetren solution and observe the blue–to–violet spots on a white background. The colours are not stable. The stability of the colours can be enhanced by spraying the plate with a 20% (v/v) solution of acetic acid and drying at room temperature and at 110° before using Tetren . The reagents should be freshly prepared

31. Dragendorff spray, ferric chloride, iodine and hydrochloric acid • The reagents are Dragendorff spray, a 5% (w/v) solution of FeCl3, 1g of iodine and 4g of KI dissolved in 100mL of ethanol, and finally a 25% (v/v) solution of concentrated HCl made up in ethanol. Spray the reagents consecutively and examine any spots and colour changes

32. Chromatography systems System TX Mobile phase is hexane:acetone (4:1). Reference compounds are trichlorfon (Rf 7), carbofuran (Rf 17), methoxychlor (Rf 43), dieldrin (Rf 65) and quintozene (Rf 84). Location systems are DPA–ZnCl2, NBP–Tetren and DBQ–NaOH

33. System TY Mobile phase is toluene:acetone (95:5). Reference compounds are thiophanate (Rf 8), 2,4-D (Rf 10), desmedipham (Rf 22), captan (Rf 42), tetramethrin (Rf 52) and fenitrothion (Rf 76). Location systems are AgNO3 and PdCl2

34. System TZ Mobile phase is chloroform:acetone (9:1). Reference compounds are trichlorfon (Rf 15), dimethoate (Rf 37), propoxur (Rf 66) and DDT (Rf 90). Location systems are AgNO3, RHB–NaOH, DBQ–NaOH and PdCl2

35. System TAA Mobile phase is chloroform. Reference compounds are methomyl (Rf 9), dichlorvos (Rf 36), chlorfenvinphos (Rf 42), methoxychlor (Rf 65) and fenvalerate (Rf 75). Location system is Dragendorff–FeCl3–I3- in KI–HCl

36. System TAB Mobile phase is dichloromethane. Reference compounds are any compounds examined in the TAB system System TAC Mobile phase is ethyl acetate:isooctane (15:85). Reference compounds are any compounds examined in the TAC system.

37. Location reagents for TAB and TAC Compounds are located with the DPA–ZnCl2 reagent. A second spray system is as follows: Reagent A (fluorescein in dimethylformamide): dilute 1mL of a 0.25% (w/v) solution of fluorescein in dimethyl formamide to 50mL with ethanol. Reagent B (silver nitrate and phenyl cellosolve): dissolve 1.7g of silver nitrate in 5mL of water and mix with 10mL of phenyl cellosolve and 185mL of acetone. To develop the colours, expose the developed plates to an atmosphere of bromine vapour for 1min and then spray sequentially with reagents A and B. Yellow spots on a pink background appear which, after exposure to UV radiation, produce yellow spots on a black background

38. Other Spray Reagents Chlorine and o-toluidine Dissolve 1g ofo-toluidine in 10mL of anhydrous acetic acid and 4g of potassium iodide in 10mL of distilled water. Mix the two solutions and dilute with distilled water to 1L. To develop the colours, put the plate in a closed tank with chlorine gas (prepared by adding 2mL of concentrated HCl to 1g of potassium permanganate) for 1min. Remove excess chlorine from the plate under a stream of air in a fume cupboard. Dip the plate in the reagent for about 3s. Yellow–orange spots appear against a white–to–blue background.

39. Specific pesticides Organophosphorus compounds Organophosphorus compounds are by far the most important class of pesticides, both in terms of worldwide usage and their toxicity to humans. They act by the irreversible inhibition of cholinesterases, which are responsible for hydrolysing, and thereby deactivating, the neurotransmitter acetylcholine (AcCh). Build–up of AcCh at the neural junction leaves the muscles, glands and nerves in a constant state of stimulation, which produces a wide range of acute symptoms. These include dizziness, confusion and blurred vision, excessive salivation and sweating, nausea and vomiting, and muscular weakness.

40. Severe poisoning leads to coma, flaccid paralysis, breathing difficulties, cyanosis (blueness of skin) and irregular heartbeat. Atropine and pralidoxime are effective antidotes in severe cases. In acute clinical poisoning, diagnostic tests for depressed cholinesterase activity are the most crucial. Detecting, identifying and quantifying the particular agent responsible has less bearing on immediate treatment, although some of the lipophilic diethyl phosphothiolates can be sequestered in the tissues for several days and patients who appear to have recovered may suffer a recurrence of toxic effects. Identification of the agent involved can alert clinicians to this possibility

41. Determination of plasma or serum cholinesterase activity • Adjust the temperature of 3mL 0.02% (w/v) dithiobisnitrobenzoic acid in 0.1M sodium dihydrogen phosphate buffer solution (pH 7.4) to 25°, add 20μL of sample serum and 0.1mL of 5% (w/v) acetylthiocholine iodide solution, mix well, and record the absorbance of a 1cm layer at 405nm at 0.5min intervals for 2min. If the change in absorbance exceeds 0.2 to 30 seconds, dilute the sample (one in ten) with normal saline and repeat the measurements (the readings must then be multiplied by 10). The cholinesterase activity is calculated

42. Cholinesterase (mUnits/ml, at 25°)=change in absorbance in 30 seconds×23400. • Normal values of ChE activity in serum range from 1900 to 4000mU/ml. Commercial kits for the determination of ChE activity in plasma and serum are available (Sigma Chemical Co., St. Louis, MO; Biotron Diagnostics, Inc., Hemet, CA; Lovibond, Tintometer GmbH, Dortmund).

43. Determination of whole–blood acetylcholinesterase activity The reagents for this are: • Phosphate buffer (0.134M, pH 7.2). • AcCh (0.04M), prepared by dissolving 0.7266g of acetylcholine chloride in 100mL of 0.001M acetate buffer (pH 4.5); stable indefinitely in the cold. • AcCh (0.004M), prepared by diluting Solution 2 with nine volumes of phosphate buffer (Solution 1); made daily in the quantity required for the analyses.

44. Hydroxylamine hydrochloride (2M), made by dissolving 27.8g in water to 200mL. • NaOH (3.5M), made by dissolving 28g in water to 200mL. • Alkaline hydroxylamine prepared from equal volumes of Solutions 4 and 5, mixed shortly before use in a quantity required for the samples being analysed, and made up freshly for each set of samples analysed. • HCl (concentrated acid, specific gravity 1.18), diluted with two volumes of water. • Ferric chloride (FeCl3, 0.37M), made with 10g of FeCl3.6H2O dissolved in 100mL of 0.1M HCl

45. Prepare three test tubes. To the first tube (E), add 0.95mL of 0.01% saponin solution, 50μL of heparinised blood sample and then 1mL of 0.004M AcCh, mix well and incubate at 25° for 10min. For the control, 1mL of 0.004M AcCh solution is incubated in a second tube (C) alongside the experimental sample. After exactly 10min the reaction is stopped by the addition of 4mL of alkaline hydroxylamine reagent (with vigorous shaking) to both experimental and control samples. After a wait of at least 1min, 0.95mL of saponin and 50μL of blood sample are added to the control solution of AcCh. Then 2mL of HCl reagent is added to each sample, followed by 2mL of FeCl3 reagent, with mixing after each addition

46. The solutions are filtered through Whatman filter paper, and the absorbance of a 1cm layer at 520nm is recorded 10min after the addition of the FeCl3. The absorbance (AE, in mmol/L) is measured against a reagent mixture that consists of 4mL of alkaline hydroxylamine, 2mL of HCl and 2mL of FeCl3 reagent. The measured value of absorbance of the control sample (AC) should be in the range of 0.3 to 0.4. The activity of AChE in blood is calculated as follows:

47. 4 – (4AE/AC)× 2000=IU/ml (International Units per millilitre) The precision of the method is 210IU/ml. Normal values for AChE activity in whole blood range from 3500 to 8000IU/ml. Commercial kits for the determination of AChE activity in red blood cells, whole blood and plasma are available

48. Determination of organophosphates in urine Most compounds of this chemical class are hydrolysed rapidly by plasma and tissue enzymes with the production of many metabolites. Metabolites and their conjugates are excreted in urine and are known to be unstable in stored specimens. To derive data that accurately represent the true degree of exposure, as indicated by the concentration of OP compounds, it is essential to obtain and analyse samples as soon as possible after an incident. Urine samples should be analysed within a week of obtaining the sample and kept at –20° prior to analysis

49. Colorimetric procedure In the colorimetric procedure , to 1mL of urine (pH 5 to 8) add 0.1mL of 45% (w/v) of 4-(4-nitrobenzyl)pyridine (NBP) solution in acetone, vortex mix for 30s and heat at 100° in a heating block for 20min. After cooling to room temperature, add 0.1mL of tetraethylenepentamine (Tetren) and 1mL of diethyl ether, then close the tube and vortex mix for 3min. Measure the absorbance of the ethereal layer at 520nm against a reagent mixture. Construct a calibration graph for the analysis of the standard OP compound solutions and calculate the concentration in the sample. The limits of detection range from 0.1 to 3.0mg/L for 24 OP compounds .