April, 19501 NITROGEN IN CRAWFISH WASTE AND DERIVED PRODUCTS 189 The Analysis of p-Nitrophenyl Diethyl Thiophosphate, E605, Parathion BY J. C. GAGE SYNoPsrs-The high toxicity of the insecticidal compound generally known as parathion or E605 has necessitated a sensitive method for its determination in the atmosphere or in edible crops. A method is described in which the compound in toluene solution is reduced to the corresponding amino compound, which is extracted into acid, diazotised and coupled with N-sulphatoetliyl- m-toluidine. The coefficient of variation of a solution containing 5 pg. per ml. is of the order of 2 per cent.; the lower limit of sensitivity for the analysis of plant tissues depends upon the blank value for the material. THE historical development and chemical synthesis of the organic phosphorus insecticides have been discussed at a recent symposium of the Association of Applied Bio1ogists.l One of these compounds, 9-nitrophenyl diethyl thiophosphate (I), has been shown to be highly effective against a wide range of insect pests; it was synthesised in Germany and given the code reference E605, and in America the U.S.Department of Agriculture has used the official name “parathion.” I t is being manufactured in this country and both names are in current use; more recently the tendency, which will be followed in this communication, is to use the term parathion. / \ S OC,H, */ b C , H , (1) (11) (111) Analytical methods for determining parathion are not only necessary to control the strength of the crude commercial product, and of insecticidal dusts and solutions made therefrom, but also, on account of the high mammalian toxicity of the compound, to determine the concentration in the atmosphere during its manufacture and use.Moreover, if edible crops have been treated with such an insecticide, it may be desirable to know the residual a.mount of parathion present in the crop after harvesting. It is possible to base a colorimetric method on the hydrolysis of parathion to nitrophenol, which takes place readily in alkaline solution. Such a method is not, however, sufficiently sensitive for the determination of parathion in the atmosphere or in plant tissues. Averell and Norris2 have described a sensitive method which has been used to estimate the residual amount in a variety of plant materials; the tissues are extracted with benzene, which is then evaporated to dryness after treatment with an adsorbent to remove pigments.The residue is dissolved in aqueous alcohol and after reduction of the nitro group to an amino group with zinc dust and hydrochloric acid, an azo dye is developed by diazotisation and coupling with N-( 1-naphthylethylenediamine) after removal of excess of nitrite with ammonium190 GAGE: THE ANALYSIS OF p-NITROPHENYL DIETHYL [Vol. 75 sulphamate. In the method described in this paper, reduction is effected by heating a toluene solution, which may be a plant tissue extract or the absorbing liquid from a suitable air sampler, with zinc dust and acetic acid under refluxing conditions; the reduced parathion is then extracted from the toluene with dilute hydrochloric acid.The advantage of this procedure is that evaporation of the solvent is avoided and, as plant pigments are not extracted from the organic liquid into the aqueous phase, there may be no necessity for their removal. In the development of the azo dye it has been found advantageous to couple with.N-/3- sulphatoethyl-m-tol~idine,~ which is more readily available and more stable than the reagent used by Averell and Norris,2 and does not require the addition of ammonium sulphamate; it is, however, necessary to neutralise the hydrochloric acid and part of the extracted acetic acid to facilitate coupling, and the addition of alcohol gives greater stability to the colour. METHOD The following procedure was designed for th.e estimation of atmospheric contamination If or of residual parathion in plant material, in which only a few micrograms are present.larger amounts can be taken for analysis, suitable modifications may be made if desired. REAGENTS- ToZuene-Ten parts of commercial redistilled toluene shaken with 1 part of concentrated sulphuric acid for 2 hours and washed free from acid.* GZaciaZ acetic acid-Analytical reagent quality. Zinc dust4ommercial zinc metal dust. Hydrochloric acid, 0.5 N . Concentrated hydrochloric acid-Analytical reagent quality. Sodium hydroxide solution, 10 per cent.-Ten grams of sodium hydroxide of analytical N-~-suZphatoethyZ-m-toZuidine, 1 per cent .----he gram of the pure commercial product It should be stored in an amber Sodiztm nitrite solution, 0.25 per cent.-Freshly prepared each week from a 6 per cent.Ethanol-95 per cent. reagent quality dissolved and made up to 100 ml. with distilled water. dissolved and made up to 100ml. with distilled water. bottle and discarded when the solution turns pink. stock solution of sodium nitrite (analytical reagent quality) kept in a refrigerator. REDUCTION OF THE NITRO GROUP- Approximately 15 ml. of the toluene solution, containing not more than 7 pg. per ml. of parathion, is introduced into a 6 x 1 inch test tube fitted with a 30-cm. air condenser by means of a ground glass joint. To this is added 0.25 ml. of glacial acetic acid and 0.25 g. of zinc dust. The tube is then placed in an oil-bath at 130" to 140" C. and the contents heated under reflux for 15 minutes.After cooling, the zinc is allowed to settle and 10 ml. of the clear solution are transferred to a stoppered 6 x Q inch test tube, and shaken for 3 minutes with 2.5 ml. of 0 . 5 N hydrochloric acid. After separation of the layers, 2 ml. of the aqueous layer are removed by a pipette to a. tube graduated at 5 ml. DEVELOPMENT OF THE AZO COLOUR- Add 0.2 ml. of 0.25 per cent. sodium nitrite solution, mix and allow to stand for 15 minutes. Add 0.4 ml. of 1 per cent. N-sulphatoethyl-m-tcduidine followed by 0.6 ml. of 10 per cent. sodium hydroxide solution; mix and allow to stand for 15 minutes. Add 3 drops of concen- trated hydrochloric acid and 1 ml. of 95 per cent. ethanol and make up to 5 ml. with distilled water. COLOUR MEASUREMENT- diameter tubes and the azo colour read at 510mp.Ilford filter No. 604 is suitable. volume and is stable for several hours. For this investigation the Unicam D.G. spectrophotometer has been used with O-5-iiich With the Spekker absorptiometer, The colour ma.y be read immediately after making up to * Some supplies of toluene were not amenable to this treatment. It has since been found that the B.D.H. sulphur-free grade is satisfactory without acid treatment.April, 19501 THIOPHOSPHATE, E605, PARATHION 191 A standard curve relating optical density to concentration is constructed from toluene solutions containing known amounts of parathion and submitted to the above procedure, the colours being read against a blank from toluene similarly treated. For all determinations a toluene blank is employed; when plant material is investigated the toluene is first shaken with similar material that has not been treated with parathion. DISCUSSION- The method is specific for compounds that can be reduced to an amino compound capable of forming an azo dye.Amines may be excluded by acidifying the initial aqueous solution or washing the toluene solution with acid. $-Nitrophenol, which occurs in small quantity in commercial parathion, does not interfere, but bis-$-nitrophenyl ethyl thiophosphate (11), which is present as up to 10 per cent. of the crude product, does give a colour. Some difficulty has been experienced in obtaining a pure sample of parathion as a reference; many of the so-called pure samples obtained by distillation are grossly contaminated by an isomer (111), which is produced if parathion is heated above 140" C.4 Methods of analysis for these impurities are being elaborated and will be the subject of a later communication.Twelve determinations on a toluene solution containing 5 pg. per ml. gave a mean optical density 0690 with a standard deviation of 0.010 and a coefficient of variation of less than 2 per cent. In the analysis of plant tissues the lower limit of sensitivity is largely controlled by the blank value, the apparent parathion content of the untreated material; the optical density obtained with this blank may be of the order of 0.05. The method has been used satisfactorily to analyse the atmosphere during the manufacture of parathion insecticides, a sample of air being passed through toluene in a gas bubbler. An investigation of the residual parathion in a variety of plant materials is a t present in progress; the results will be reported in detail later. REFERENCES 1. 2. 3. 4. INDUSTRIAL HYGIENE RESEARCH LABORATORY Ann. Appl. Biol., 1949, 36, 153. Averell, P. R., and Norris, M. V., Anal. Chem., 1948, 20, 753. Rose, F. L., and Bevan, H. G. L., Hiochem. J., 1944, 38, 116. British Intelligence Objectives Sub-committee Report No. 1808, p. 9. IMPERIAL CHEMICAL INDUSTRIES LIMITED WELWYN, HERTS. Septembsr, 1949