340 MARTIN AND BATT: THE DETERMINATION OF [Vol. 83 The Determination of DDT in Plant Materials and Soil By J. T. MARTIN AND R. F. BATT (Research Statzon, Long A shtoii, Brzstol) Two procedures, based upon nitration and colour formation with ethanolic potassium hydroxide solution, are described for the determination of DDT in foliage, fruits, grain and soil. In the first, DDT is determined within the range 0 to 360 pg, and in the second, within the range 0 to 30 p g . Inter- ference by wax and pigments is overcome by treatment with alumina and oxidation with acidified permanganate, or by oxidation with acidified permanganate alone. IN work on the chemical control of plant pests and diseases, methods were required for the examination of spray deposits in the assessment of spraying efficiency.Progress reports have been made on the development of techniques for the determination of copper,l sulphur,2 captan (N-trichloromethylthio-4-cyclohexene-1 : 2-dicarboxyimide) ,3 DDT [ l : 1 : l-trichloro- 2 : 2-di($-~hlorophenyl)ethane],~?~ chlorobenzilate (ethyl 4 : 4'-di~hlorobenzilate)~ and of DDT and chlorbenside ($-chlorobenzyl p-chlorophenyl sulphide) occurring together.' The first, called for convenience the macro method, is designed €or the analysis of comparatively large samples of fresh foliage (10 g), fruits or grain (50 g) or soil (20 g), and DDT can be determined when present in amounts within the range 0 to 350pg. The second, a rapid micro method used in studies of the distribution of the insecticide over plants, is suitable for the analysis of single small fruits or samples of between 0.1 and 0-5 g of foliage or 5 g of grain or soil.Both procedures are based upon those of Schechter, Soloway, Hayes and Hailer* and Illing and Stephens~n,~ in which DDT, extracted from plant tissue with a suitable solvent, is nitrated and a colour is formed with sodium methylate or et hanolic alkali. Amsden and Wallbridgelo propose the use of isopropylamine for colour formation after nitration. MACRO METHOD OF EXTRACTING THE DDT- Foliage and fruits-An extraction procedure was used to assess the extent to which DDT could penetrate into plant tissue. To obtain the surface deposits, leaves and fruits were washed four times with carbon tetrachloride at room temperature. Most of the DDT was recovered in the first two washings; none was found in the fourth washing.The plant material was then dried in air, powdered and extracted by hot percolation with carbon tetrachloride, when additional amounts of DDT equivalent to 10 to 20 per cent. of the total deposits on the leaves were recovered." No absorbed DDT was detected in fruits such as blackcurrants and grapes. The ability of DDT to penetrate into foliage should be borne in mind in the analysis of leafy crops. Carbon tetrachloride was preferred as solvent since it removes the DDT with a minimum amount of pigment and gives no interference in the nitration procedure. Grain-The grain was percolated for 30 minutes with hot carbon tetrachloride. Soil-An examination was made of sandy loam soil samples (pH 6.2 to 6.4 and contain- ing 2.5 per cent. of organic matter) in which DDT had been incorporated.The soil was prepared for analysis by drying in air and passage through a &-inch square-hole sieve. Extraction was carried out on 20-g portions in three successive stages: (a) by hot percolation with carbon tetrachloride, (b) by setting the soil aside overnight in a (2 + 1) mixture of carbon tetrachloride and isopropyl alcohol, and (c) by hot percolation with isopropyl alcohol. Samples were also examined by direct hot percolation with acetone or isopropyl alcohol. The results of analysis showed that the treatment with carbon tetrachloride failed to extract all the DDT. Extraction with carbon tetrachloride and then isopropyl alcohol gave values that agreed closely with those obtained with acetone or isopropyl alcohol alone.6 MICRO METHOD OF EXTRACTING THE DDT- made to distinguish between surface and absorbed deposits.Two procedures have been used for the determination of DDT in spray deposits. In this method, the range of DDT covered is 0 to 30 pg. EXPERIMENTAL Foliage and fruits-Because small amounts of plant tissue were used, no attempt was Acetone was used as the solventJune, 19581 DDT IN PLANT MATERIALS AND SOIL 34 1 to obtain the total deposits, five leaf discs, each of 1-cm diameter, or single small fruitlets being extracted by hot percolation for 15 minutes. Grain-Some 5-g samples of grain were extracted by hot percolation with acetone for 15 minutes. Soil-Tests showed that the recoveries of DDT from a sandy loam soil after extraction with acetone of 5 or 20-g samples agreed well with those after extraction with isopropyl alcohol.6 MACRO METHOD OF REMOVING INTERFERENCE BY WAX AND PIGMENTS- The carbon tetrachloride extracts of foliage, fruits and grain were passed through a column of activated alumina to remove pigments and most of the wax.This treatment also effectively removed interfering wax and pigments from extracts of light soils; some soils rich in organic matter and some samples of grain gave highly pigmented solutions, and for these oxidation with acidified potassium permanganate was required after passage through the alumina column. MICRO METHOD OF REMOVING INTERFERENCE BY WAX AND PIGMENTS- The complete removal of interference by pigments and fatty material extracted by the acetone was necessary to obtain significant values for DDT over the range 0 to 30 pg.This was achieved without loss of DDT by oxidation with potassium permanganate in dilute acetic acid. NITRATION OF THE DDT- Tests showed that, after removal of most of the wax, nitration at 100" C was completed in 10 minutes and this period of heating was adopted for both methods. Under the conditions of the test, the product formed is the tetranitro derivative, 1 : 1 : 1-trichloro-2 : 2-di(4-chloro- 3 : 5-dinitropheny1)ethane. SEPARATION OF THE NITRATED DDT- Separation of the nitrated DDT can be effected by extraction with ether, benzene, carbon tetrachloride or any other suitable solvent, In the macro method, we have preferred carbon tetrachloride, which occludes less water than ether and ensures fewer manipulations than when benzene is used.The nitrated DDT has a low solubility in carbon tetrachloride, but with the procedure recommended, which involves extraction from an aqueous solution saturated with salts, the recovery of the nitro- DDT is complete. In the micro method, benzene has been used as solvent for the extraction of the nitro compound. COLOUR FORMATION- Schechter, Soloway, Hayes and Hallers recommended the use of sodium methylate solution, standardised at 10 0.1 per cent., to form a colour with the nitro-DDT in benzene solution. We used ethanolic potassium hydr~xide,~ which forms a blue colour with the nitrated DDT in carbon tetrachloride (macro method) or benzene (micro method). The maximum development of colour occurs after 5 minutes.The concentration of potassium hydroxide is not critical, colours of equal intensity being given over the range 3.5 to 6.5 per cent? METHOD REAGENTS- Carbon tetrachloride-Fractionated before use. Acetone-Fractionated before use. Benzelze-Fractionated before use. Acetic acid, glacial. Potassium permanganate solution-A 2.5 per cent. aqueous solution. Sodium metabisul9hite solution-A freshly prepared 5 per cent. aqueous solution. Aluminium oxide for chromatographic analysis. Nitration acid-Prepared by mixing equal volumes of fuming nitric acid and concentrated Potassium hydroxide solution, 4 N. Sodium sulphate, anhydrous. sulphuric acid.342 MARTIN AND BATT: THE DETERMINATION OF [Vol. 83 Ethanolic potassium hydroxide solution-Prepared by heating 5 g of potassium hydroxide with 100 ml of absolute ethanol under reflux until dissolution is complete.This solution must be freshly prepared and should be filtered immediately before use. MACRO PROCEDURE- Extract the plant material or soil as described under "Experimental," p. 340, using the minimum amount of carbon tetrachloride. If the initial volume is appreciable, reduce it to about 10 ml by gentle distillation over a low flame, or by drawing a current of dry air over the surface of the liquid at about 35" C. If acetone is used for the extraction of soil, evaporate to dryness by passing a current of dry air over the liquid at 35" C and dissolve the residue in 10 ml of carbon tetrachloride with warming, and then cool the solution. Pass the solution through 5 g of aluminium oxide (previously wetted with carbon tetra- chloride) supported on cotton-wool in a glass tube, 150 mm x 18 mm, having a constriction a t the lower end.Gently distil the solvent from the eluate until the volume is about 10 ml and then evaporate to dryness a t 35" C with a current of air. When experience has shown it to be necessary, add 4 ml of acetic acid, warm to dissolve the residue, add 5 ml of potassium permanganate solution and place on a boiling-water bath for 10 minutes. Cool and decolorise by the addition, drop by drop, of sodium metabisulphite solution and then add 10 ml of water. Extract, successively, with vigorous shaking for 30 seconds each time, with 15, 5 and 5-nd portions of carbon tetrachloride. Combine the carbon tetrachloride extracts, add 10 ml of water and then 4 N potassium hydroxide, drop by drop, with shaking, until the aqueous layer is just alkaline to litmus paper.Run off the carbon tetrachloride layer, wash the aqueous layer with a little carbon tetrachloride, combine the carbon tetrachloride layers and wash with 5 ml of water. Run the carbon tetrachloride extract through anhydrous sodium sulphate and wash the water layer and desiccant with a little more solvent. Reduce the volume by gentle distillation to about 5 ml and evaporate to dryness a t 35" C in a current of air. Continue with this residue, or with the residue obtained before addition of 4 ml of acetic icid if the permanganate oxidation procedure was unnecessary, as follows. Cool and slowly add 10 ml of water. Cool again and neutralise by the addition, with swirling and cooling, of 4 N potassium hydroxide until litmus paper just turns blue.Add 2 drops of 4 N potassium hydroxide in excess. Wash the cooled solution into a separating funnel with 5 m l of water. Rinse the flask with 15ml of carbon tetrachloride, add the washings to the funnel and extract with vigorous shaking for 30 seconds. Re-extract with two further 5-ml portions of carbon tetrachloride, rins,ing the flask. Combine the carbon tetrachloride extracts and wash them twice with 5-ml portions of water. Run the carbon tetrachloride solution through anhydrous sodium sulphate and wash the separating funnel and desiccant with a little more solvent. Reduce the volume by dis- tillation over a low flame to 5 ml and then evaporate to 0.5 ml a t 35" C in a current of air.Add 10 ml of 5 per cent, ethanolic potassium hydroxide solution, rotate for 15 seconds, and, after 5 minutes, measure the optical density spectrophotometrically in a 1-cm cell a t a wavelength of 600 m p when a Unicam SI'600 spectrophotometer is used, or with an Ilford No. 607 filter when a Spekker absorptiometer is used. MICRO PROCEDURE- Extract the plant material or soil by hot percolation with acetone as described under "Experimental," p. 340, using the minimum amount of solvent. Evaporate to dryness at 35°C in a current of air. Add 4 ml of acetic acid, warm to dissolve the residue, add 5 ml of potassium permanganate solution and heat on a boiling-water bath for 10 minutes. Cool and decolorise by the addition, drop by drop, of sodium metabisulphite solution.Extract, with vigorous shaking, with 10 and 5-ml portions of carbon tetrachloridc. Run the carbon tetrachloride extracts through anhydrous sodium sulphate and evaporate the solvent to dryness at 35" C in a stream of air. Cool, add 5 ml of water and make just alkaline to litnius by adding 4 A' potassium hydroxide solution. Wash through with five 5-ml portions of carbon tetrachloride. Add 2 ml of nitration acid and heat in a boiling-water bath for 10 minutes. Add 1 ml of nitration acid and heat in a boiling-water bath for 10 minutes.June, 19581 DDT I N PLANT MATERIALS AND SOIL 343 Extract with vigorous shaking for 30 seconds with 10 ml of benzene. Wash the benzene layer with two 3-ml portions of water, run the benzene solution through anhydrous sodium sulphate into a 25-ml graduated stoppered cylinder, wash the desiccant and adjust the volume to 10 ml with benzene and add 5 ml of 5 per cent. ethanolic potassium hydroxide solution.Mix and after 5 minutes measure the optical density spectrophotometrically in a 2-cm cell. RESULTS Calibration curves relating optical density to micrograms of $p'-DDT were prepared from the results of tests on @'-DDT alone, of tetranitro-DDT prepared by the method of Backeberg and Marais,ll of pp'-DDT taken through each method from the nitration stage and of p p -DDT added to the solvent before the extraction of foliage, grain and soil. The degree of concordance was good, indicating no loss in the method and satisfactory recovery of DDT from the extracts of the materials examined. In collaborative work-with the Infestation Control Division of the Ministry of Agriculture, Fisheries and Food at Tolworth, carbon tetrachloride extracts of untreated grain to which known amounts of pure p p -DDT and gamma-hexachlorocyclohexane had been added, were prepared at Tolworth and sent to Long Ashton for analysis by the macro method.The results are shown in Table I. TABLE I RECOVERY OF DDT FROM GRAIN EXTRACTS BY MACRO METHOD Sample DDT added, 7-Hexachlorocyclohexane added, p.p.m. p.p.m. c. 1 4.0 c.2 4.0 11.1 10.0 n.2 10.0 E 20.0 Xi1 Xi1 0.5 S i l 2.0 S i l 4.0 10.0 DDT recovered, p.p.m. 0.5, 0.5 1.6, 1.7 1.5, 1.4 4.1, 3.9 4.0, 3.8 10.0, 9.9 10.0, 10.1 19.7, 19.9 The recovery by the macro method of DDT addea to whole grain and from DDT-treated The recoveries by the micro method of DDT grain taken from storage is discussed later. added to acetone extracts of grain and soils are shown in Table 11.RECOVERY OF DDT Sample Grain . . . . .. Sandy soil . Organic soil . TABLE I1 FROX GRAIN ATD SOIL EXTRACTS BY MICRO METHOD IIDT added, p.p.m, DDT recovered, p.p.m .. 2.0 2.0 4.0 3.9 6.0 6.1 .. 1.5 2.5 3.5 4.5 5.5 1.5 2.5 3.5 4.5 5.5 1.4 2.3 3.1 4.5 5.5 1.6 2.5 3.2 4.3 5.9 For convenience, a standard disc for use with the macro method, which gives the colour intensities produced over the range 25 to 350 pg of DDT, has been prepared in collaboration with Tintometer Ltd. of Salisbury, Wilts., from whom it is available. The disc was designed only for analyses in which the permanganate treatment was not necessary.In the description of the method supplied by the firm, we now recommend the use of 5-g portions of alumina, with washing as described.344 MARTIN AND BATT [Vol. 83 DISCUSSION Certain precautions and some experience in the use of the methods are necessary for satisfactory results. Chemicals of analytical-reagent quality should be used whenever possible and all-glass apparatus should be used throughout. Adequate separations and drainage should be allowed in all liquid - liquid extractions, and the amount of stopcock grease used should be kept to an absolute minimum. In the macro method, an opalescence on dilution of the nitration mixture with water indicates that more than 50pg of DDT are present ; if an appreciable amount of precipitate results, the carbon tetrachloride solution of the nitro compound should be diluted to a specific volume and an aliquot taken for the colour development.The test should be completed with the minimum of delay, particularly after extraction of the nitro compound with carbon tetrachloride. If crystals separate from the small volume of solvent before the addition of ethanolic potassium hydroxide, the test should be rejected. The colour obtained in the examination of deposits on crops that had been subjected to weathering in the field was comparable with that obtained in the analysis of fresh deposits. This was probably due to the fact that any residual 4 : 4’-dichlorobenzophenone, which is formed from DDT under the influence of ultra-violet light and which gives a tangerine colour in the test, is removed by the alumina treatment.of-DDT, which may be present in small amounts in spray deposits, gives a colour of lower intensity than that given by $p‘-DDT and is unlikely to cause any significant error in the determination of the $$‘-isomer. The presence of gamma-hexachlorocyclohexane in grain does not cause interference in the deter- mination of DDT. Work has been carried out by B. S. J. Border and A. Taylor in the Infestation Control Division of the Ministry of Agriculture, ]Fisheries and Food on the recovery of DDT from grain by the macro method and we are indebted to Dr. E. E. Turtle for permission to refer to their results. Weighed amounts of DDT were added to 50-g portions of grain at concen- trations over the range of 4 to 16 p.p.m.and the samples were analysed immediately. Re- coveries of 88 to 110 per cent., with a mean value of 93 per cent., were found. These results are satisfactory in view of the difficulty of ensuring the even distribution of DDT crystals in the grain. Other samples, containing 8, 40 and 76 p.p.m. of added DDT, showed recoveries of 81 to 84 per cent. after storage for 5 weeks; on further extraction of the grain after grinding, the mean recovery of DDT was 88 per cent. The Infestation Control Division workers have also shown that extraction of the tetranitro-DDT with benzene followed by washing the extract with dilute alkali and the use of an aliquot without the removal of solvent in the colour formation gives results agreeing chsely with those by the macro method, but with considerable saving of time. It is desirable that a standard method based upon further collaborative work should be adopted for the determination of DDT in food commodities. Most of our work has been carried out on leaves and fruits from spraying trials, and for these materials the methods have proved to be satisfactory. Further work is required on the recovery of DDT from grain after storage and on the details of the method of analysis to be used. It may be that a combination of the principles of the macro and micro methods, as suggested by the Tolworth workers, would be most acceptable. Fatty materials, such as groundnuts and oil-cake, need special consideration. 1 . 2 . 3. 4 . 5 . 6 . 7 . 8 . 9 . 10. 1 1 . REFERENCES Martin, J. T., Ann. Rep. Agric. Hort. Ref. Sta., Bristol, 1956, 125. Krentos, V. D., Batt, R. F., and Martin, J. T., Ibid., 1956, 122. Martin, J. T., and Pickard, J. A., I b i d . , 1955, 103. Martin, J. T., and Batt, R. F., Ibid., 1953, 121. Batt, R. F., and Martin, J. T., Ibid., 19516, 127. Skerrett, E. J., and Baker, E. A., Ibid., 1956, 130. Martin, J. T., and Batt, R. F., Ibid., 1955, 106. Schechter, M. S., Soloway, S. B., Hayes, R. A., and Haller, H. L., I n d . Eng. Chem., Anal. Ed., Illing, E. T., and Stephenson, W. H., A d y s t , 1946, 71, 310. Amsden, R. C., and Wallbridge, D. J., J . Agric. Food Chem., 1954, 2, 1323. Backeberg, 0. G., and Marais, J. L. C., J Chem. Soc., 1945, 803. 1945, 17, 704. Received December 2nd, 1957