Analyst, December, 1979, Vol. 104, pp. 1129-1134 1129 Modified Colorimetric Method for the Determination of Ma I at hion E. R. Clark and I. A. Qazi Department of Chemistry, University of Aston in Birmingham, Gosta Green, Birmingham, B 4 7ET A thorough investigation has been made of the recommended method for the determination of malathion, and the major cause of two of the most serious problems in this method has been resolved. The method that uses a copper(I1) complex as the basis of colorimetric measurements suffers from the disadvantage that the colour fades quickly and that an increase of a few seconds in the contact time of the copper(I1) solution and the hydrolysis product of malathion results in a reduction in the intensity of the yellow colour. Attempts to overcome these drawbacks have been reported, some of which are tedious and others only partially successful, but it is suggested that if copper is replaced with bismuth the problems may be more simply resolved.Isomalathion does not react in this method. Keywords : Malathion alkaline hydrolysis ; malathion determination ; copper chelate colorimetry ; colour instability ; bismuth chelate The first colorimetric method for the determination of malathion was devised by Norris et aZ.,1 who applied it to technical product analyses and malathion residue analyses. In this method, malathion is decomposed by alkali to dimethyl dithiophosphate, sodium fumarate and ethanol. The dithiophosphate is then converted into the copper( 11) complex, which is soluble in organic solvents such as carbon tetrachloride and hexane with the forma- tion of an intense yellow colour. The colour intensity is proportional to the concentration of dimethyl dithiophosphate and is measured colorimetrically a t 420 nm, the absorption peak. Iron(II1) reagent is added in order to oxidise materials that would reduce copper(I1) ions to copper(1) ions.With dithiophosphate, copper( I) ions form a colourless complex, which is reported to be more stable than the yellow copper(I1) complex. This method has been recommended by the Malathion Panel, set up jointly by the Scientific Sub-committee of the Inter-Departmental Advisory Committee on Poisonous Substances Used in Agri- culture and Food Storage, the Analytical Methods Committee of the Society for Analytical Chemistry and the Association of British Manufacturers of Agricultural Chemicals2 as suitable for malathion residue analysis in fruits, vegetables and agricultural crops.In addition, it is the recommended method of the US Association of Official Analytical Chemists3 and has been listed in the WHO’S “Specification for Pesticides.”* In spite of such widespread use, the method suffers from the disadvantages that the yellow colour produced, which forms the basis of the colorimetric measurements, fades very quickly, and a slight increase in the contact time of the copper(I1) solution and the hydroylsis product of malathion results in a large decrease in the absolute ab~orbance.~ Many attempts have been made to overcome these drawbacks. Hill6 presented evidence to show that the copper(I1) complex of dimethyldithiophosphate exists in reversible equi- librium with its two dissociation products, copper( I) dimethyldithiophosphate and bis- (dimethoxyphosphorothiono) disulphide and that this dissociation accounts in part for the instability of the yellow complex observed by many workers.It was shown by Hill6 that incorporation of the disulphide would limit the dissociation of the coloured complex. Roussow7 found that the colour is stable for 1545 min if the temperature of the reagents is maintained between 16 and 20 “C. Wayne et aL8 developed a non-aqueous copper colorimetric method based on the reaction of Norris et aZ.l for the analysis of technical products containing malathion, but it has not been favoured by some workers because it is tedious and requires a large amount of glass- ware.Also, the availability of anhydrous solvents can be a problem in some in~tances.~ It has also been pointed out that strict cleanliness of glassware must be maintained in order to obtain reproducible results and that the method is time consuming in comparison with the origmal method. Further, the non- Sometimes there is difficulty with colour stability.1°1130 CLARK AND QAZI MODIFIED COLORIMETRIC Analyst, Vol. 104 aqueous method suffers from the disadvantage that the absorbance has to be measured exactly 2 min after the addition of the copper reagent and there is also some doubt whether the method could be conveniently extended to residue analysis. Visweswriah and Jayaramll suggested that the reaction of palladium(I1) chloride with the acid hydrolysis product of malathion would give a yellow complex suitable for the deter- mination of malathion.Their observations are conflicting, however, because they state that the acid hydrolysis gives primarily dimethylthionophosphoric acid but that the yellow colour is due to the reaction of palladium(I1) with dimethyldithiophosphoric acid, a product not formed under their recommended acid hydrolysis conditions. In this paper, the most probable cause of the problems encountered in the copper method for the determination of malathion are discussed and the development of a modified colori- metric method that does not suffer from any of these drawbacks is described. Experimental Reagents material in 200 ml of distilled water. 3 ml of concentrated nitric acid and dilute to 100 ml with distilled water.Dimethyldithiophosphate (DMDTP) solution, 5 x Bismuth solution. M. Dissolve 0.175 2 g of purified Dissolve 0.1 g of bismuth oxide (Bi,O,) (BDH laboratory reagent) in 1.0 ml of solution = 10.45 mg of bismuth. Copper solution. Dissolve 2 g of copper(I1) sulphate (CuS0,.5H20) (AnalaR grade, Hopkin and Williams Limited) in 100 ml of distilled wa.ter. 1.0 ml of solution E- 5 mg of copper. Carbon tetrachloride. Ethanol. Medical grade (99.5%). Malathion anazytical standard. Malathion emulsijable concentrate. Re-distil commercial-grade material and store in a glass bottle. Supplied by the American Cyanamid Company. Supplied by Murphy Chemical Limited. Apparatus SP6-100) spectrophotometers with 1 .O-cm quartz cells were used.Spectrophotometers. Double-beam recording (Unicam SP8-100) and single-beam (Unicam Procedure Puri$cation of ammonium dimethyldithiophosphate A 10-g amount of the commercial product (Aldrich Chemical Co., 95% purity) was dissolved in 50 ml of ethanol. The solution was filtered and to the filtrate were added 30 ml of carbon tetrachloride. The solution was kept overnight at room temperature, after which time the purified compound had crystallised in well defined crystals. The crystals were washed several times with diethyl ether, dried and the purity of the compound was confirmed by determining the melting-point (143 "C). Absorption spectra of the copper and bismuth coznplexes of DMDTP (Fig. 1) To a 50-ml separating funnel containing 9 ml of water, 0.3 ml of the DMDTP solution were added, followed by 10 ml of carbon tetrachloride and 1 ml of the copper (or bismuth) solution. The funnel was stoppered and immediately shaken vigorously for exactly 1 min.The organic layer was allowed to separate and then transferred into the 1-cm quartz cell, via a cotton-wool plug placed in the stem of the funnel. The absorption spectra of the com- plexes were immediately recorded on the Unicam SP8-100 spectrophotometer using carbon tetrachloride as reference solution.December, 1979 METHOD FOR THE DETERMINATION OF MALATHION 1131 Wavelength/nm Fig. 1. Absorption spectra (1-cm quartz cell) of bismuth and copper complexes of dimethyl- A, 0.3 ml of 5 x 10-3 M DMDTP M DMDTP solution with excess of dithiophosphate extracted into 10 ml of carbon tetrachloride.solution with excess of bismuth; and B, 0.3 ml of 5 x copper. Persistence of colour (Fig. 2) The change in the absorbance of the copper and bismuth complexes (produced as described above) with time was studied on the single-beam spectrophotometer connected to a chart recorder. 0.6 I I I 1 I 0 10 20 30 40 0.3 Timelmin Fig. 2. Variation of absorbance (1-cm quartz cell) with time of bismuth and copper complexes of dimethyldithiophosphate extracted into 10 ml of carbon tetrachloride. A, 0.2 ml of 5 X 1 0 - 3 ~ DMDTP solution with excess of bismuth, X = 325nm; and B, 0.2ml of 5 x 10-3M DMDTP solution with excess of copper, X = 418 nm. Beer’s law studies (Fig. 3) The absorbances of the copper and the bismuth complexes of DMDTP, using different amounts of DMDTP solution, were measured as described above.For the copper complex the wavelength used was 418 nm, whereas for the bismuth complex both the 325- and 390-nm absorption peaks were used. The resulting absorbances were plotted against DMDTP concentration. E f e c t of reducing agents (Table I> In the original method of Norris et al.,l the presence of any reducing agents with the hydro- lysis product of malathion is a major source of error. It was therefore decided to investigate the effect of a typical reducing agent (ascorbic acid) on the absorbance characteristics of the1132 CLARK AND QAZI : MODIFIED COLORIMETRIC Analyst, VoZ. 104 1.4 1.2 1 .o 8 0.8 m + 2 2 0.6 0.4 0 2 0 .o Volume of 5 x ~ O - ~ M DMDTP solution/ml Fig. 3.Beer's law gra.ph for DMDTP com- plexes of copper and bismuth: 5 x 1 0 - 3 ~ DMDTP solution extracted with excess of metal into 10ml of carbon tetrachloride. A, Copper complex, X = 418 nm; B, bismuth complex, X = 325nm; and C, bi!;muth complex, X = 390 nm. copper and bismuth complexes of DMDTP. For this purpose the absorbances of these complexes, for a fixed amount of DMDTP solu1:ion (0.3 ml), were measured after extraction from aqueous layers containing varying amounts of ascorbic acid. TABL~E I EFFECT OF ASCORBIC ACID ON THE ABSORBANCE OF COPPER AND BISMUTH COMPLEXES OF DIMETHYLDITHIOPHOSPHATE* Volume of M ascorbic acid/ml . . 0 3 4 6 8 10 Absorbance, Cu - DMDTP . . . . 0.82 0.62 0.51 0.44 0.38 0.37 Absorbance, Bi - DMDTP . . . . 0.74 0.'76 0.75 0.74 0.74 0.73 * 0.3 ml of DMDPT extracted with excess of copper and bismuth in the presence of various amounts of ascorbic acid.Lapse of time betweeiz the addition of metal reagent and subsequent extraction into organic solvent In the standard method for the determination of malathion it has been recommended that there should be a minimum lapse of time between the addition of the copper(I1) reagent and the subsequent shaking with the organic solvents. A delay of only a few seconds causes a decrease in the absolute absorbance and thus introduces errors. To confirm these findings and to investigate the effect of this lapse of time on the bismuth complex of DMDTP, a fixed amount of the DMDTP solution (0.7 ml) was used. It was found that if the complexes were extratcted immediately after addition of the metal ion, the absorbances obtained for the copper and bismuth complexes were 1.92 and 1.79, respectively.On the other hand, when the extraction was started 1 min after the addition of the metal reagents the absorbance for the bismuth complex remained virtually the same, whereas the average absorbance for the copper complex fell to 1.31 (average of ten readings).December, 1979 METHOD FOR THE DETERMINATION OF MALATHION 1133 Analysis of emulsiJiable concentrate For the analysis of 607' emulsifiable concentrate, the method in reference 5 was employed. When bismuth was used in place of copper, addition of iron(II1) solution was omitted. The results obtained for analyses of five samples by each method were 59.8 3 1.2% for the copper method and 60.2 & 0.8% for the bismuth method.Results and Discussion From the absorption spectra and Beer's law graphs (Figs. 1 and 3), it is clear that if bismuth is used for the determination of malathion in the visible range (390 nm) it is 4-5 times less sensitive than in the standard method. On the other hand, in the ultraviolet range (325 nm) the sensitivity is only slightly less than that of the standard method. From Fig. 2 it can be seen that the yellow colour due to the bismuth complex of DMDTP remains unchanged even after 40 min, whereas the absorbance of the copper complex falls appreciably during this time. In fact, it was found that the absorbance of the bismuth complex does not alter even 24 h after extraction. This makes the bismuth method for the determination of malathion much more attractive than the copper method because one of the major drawbacks in the standard method is removed without taking the extra trouble of developing the colour at a lower temperature,' using a precious metal such as palladiumll or incorporating the use of disulphide.6 Fig.3 shows that the bismuth complex obeys Beer's law at least within the concentration range for which the standard copper method is applicable. This is found to be true for absorption at both wavelengths of interest, i.e., 325 and 390 nm. Results of experiments with ascorbic acid show that whereas even a slight amount of reducing agent has a drastic effect on the results obtained by the copper method, no such detrimental effect is observed in the bismuth method.This is a distinct advantage in that the analyst has no longer to consider the impurities that have to be oxidised by the addition of iron(II1) reagent in the standard method. In fact, the addition of iron(II1) is no longer required. A further advantage of the bismuth method over the copper method is shown by the lapse of time between the addition of metal and subsequent shaking studies. Whereas a delay of 1 min has a noticeable effect on the absorbance of the copper complex of DMDTP, no such problem is encounFered with bismuth. This makes the bismuth method much simpler and reduces the probability of errors. Whereas the original method of Norris et a1.l requires strict adherence to time factors, the proposed method does not, and it is suggested that it gives equally good results.The application of the bismuth method for the determination of malathion to a technical product (60% malathion emulsifiable concentrate) confirmed its usefulness. As observed, within experimental error both the copper and bismuth methods yield similar results, proving that the standard method could well be replaced with the modified bismuth method with distinct advantages. The results not only prove the superiority of using bismuth in place of copper in the deter- mination of malathion but also give a good indication of the cause of all of the problems inherent in the standard method. All of the evidence supports the idea that the basic cause of all such problems is the ability of copper(I1) to be reduced to copper(1) not only by the impurities present in the hydrolysis product of malathion, but also by the major hydrolysis product DMDTP itself.It is known that aqueous solutions of diethyldithiophosphate reduce copper(I1) and iron(II1) to copper(1) and iron(II), respectively,12 and it can be assumed that DMDTP would behave similarly. This would account for the reduced absorbance of the copper - DMDTP complex when there is a delay between the addition of the copper reagent and subsequent extraction, i.e., with longer time more and more copper(I1) is reduced to copper(I), forming a colourless complex with DMDTP and resulting in a lower absorbance. Also, as suggested by this could account for the instability of the yellow complex in the organic solvent. Mention may also be made that, owing to the possibility of the oxidation of DMDTP, the addition of large amounts of iron(II1) solution used in the recommended method5 may be a source of error in itself.The dangers are two-fold in residue analysis, where some workers recommend repeated shaking of the aqueous solution of DMDTP and iron(IT.1) with the1134 CLARK AND QAZI organic solvent and discarding the extract. In this instance it is likely that even AnalaR- grade iron(II1) chloride (FeCl,.GH,O) may contribute sufficient copper(I1) to make serious losses of DMDTP possible. In conclusion, the advantages of using bismuth in place of copper for the determination of malathion can be summarised as follows: (1) less reagents are needed [there is no need to use iron(II1) solution or carbon disulphide]; (2) there is no need to use cooled solutions; (3) there is no interference from reducing agents; (4) there is no need for strict adherence to time factors; (5) the yellow colour is stable for extended periods of time; and (6) isomalathion has been found not to interfere.The authors thank the American Cyanamid Company and Murphy Chemical Limited for supplying malathion standard samples. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. Norris, M. V., Vail, W. A., and Averell, P. R., J . Agric. Fd Chem., 1954, 2, 570. Malathion Panel, Analyst, 1960, 85, 915. “Official Methods of Analysis of the Association of Official Analytical Chemists,” Eleventh Edition, “Specification for Pesticides,” Fourth Edition, World Health Organization, Geneva, 1973. Zweig, G., Editor, “Analytical Methods for Pesticides, Plant Growth Regulators and Food Addi- Hill, A. C., J . Sci. Fd Agric., 1969, 20, 4. Roussow, S. D., S. Afr. J . Agric. Sci., 1961, 4, 435. Wayne, R. S., Groth, W. C., Miles, J . W., and Guerrant, G. O., J . Ass. Off. Analyt. Chem., 1972, 55,926. Wayne, R. S., J . Ass. Off. Analyt. Chem., 1973, 56, 579. Stiles, A. R., Miles, J . W., Wayne, R. S., and Newton, W. H., J . Ass. 08. Analyt. Chem., 1977, 60, Visweswriah, K., and Jayaram, M., Agric. Biol. Chem., 1974, 38, 2031. Handley, T. H., Analyt. Chem., 1962, 34, 1312. Association of Official Analytical Chemists, Washington, D.C., 1970. tives,” Volume 11, Academic Press, New York, 1973. 1148. Received March 29th. 1979 Accepted July 9th, 1979