An enzyme test for determining isomalathion impurities in water-dispersible powders of malathion

Bulletin of the World Health Organization, 64 (3): 397-401 (1986) © World Health Organization 1986 An enzyme test for determining isomalathion impurities in water-dispersible powders of malathion ELSA REINER' & ZORAN RADK92 An enzyme test for determining isomalathion (O,S-dimethyl-S-(1,2-dicarbethoxy- ethyl) phosphorodithioate) impurities in water-dispersible powders of malathion (WDP malathion) is described. The test is based on inhibition ofacetylcholinesterase (EC3.1.1. 7) by isomalathion extractedfrom WDP malathion. The lower limit ofdetection of the test is 0.01% (wlw) isomalathion. For 18 samples of WDP malathion there was good correlation between the levels ofisomalathionfound using the enzyme test and those obtained by thin- layer chromatography. Water-dispersible powders of malathion (WDP malathion) contain other organophosphorus com- pounds as impurities, the principal one of which is isomalathion. The impurities are toxic and potentiate malathion's own toxicity (1, 2). Before being used as an insecticide, WDP malathion has therefore to be analysed for isomalathion. The method described here for the determination of isomalathion in WDP malathion is an enzyme test based on inhibition of acetylcholinesterase (EC 3.1.1.7) by isomalathion. Malathion itself does not react with acetylcholinesterase and therefore does not interfere in the test. A field method for measuring cholinesterase activity is already in usea and can also be employed to determine organophosphorus im- purities in WDP malathion. MATERIALS AND METHODS Principle of the method In the enzyme test, the organophosphorus im- purities in WDP malathion are first extracted with methanol. Some malathion is also extracted, but addition of water to the extract leaves the organo- phosphorus impurities in solution, while precipi- ' Research Adviser and Head ofthe Laboratory for Biochemistry, Institute for Medical Research and Occupational Health, M. Pijade 158, 41000 Zagreb, Yugoslavia. Requests for reprints should be sent to this author. 2 Research Assistant, Laboratory for Biochemistry, Institute for Medical Research and Occupational Health, Zagreb, Yugoslavia. a Spectrophotometric kit for measuring cholinesterase activity. Unpublished WHO document WHO/VBC/84.888. tating most of the malathion. The diluted extract is then incubated with acetylcholinesterase. After 10 minutes the substrate acetylthiocholine iodide is added, and the residual enzyme activity is measured spectrophotometrically in the presence of the thiol reagent 5,5 '-dithiobis-2-nitrobenzoate (DTNB). The degree ofenzyme inhibition is proportional to the con- centration of organophosphorus impurities, which is read off a calibration curve determined by the use of standard solutions of isomalathion as inhibitor. Con- sequently, concentrations of the organophosphorus impurities determined by the test are expressed as equivalents of isomalathion. Reagents Purified, powdered electric eel acetylcholinesterase was used.b This retains its activity for several years if stored undissolved at -20 'C. Stock solutions of acetylcholinesterase (1.0 g/l) were prepared in 0.1 mol/l sodium chloride containing 1.00o (w/v) gelatine, and 0.1 ml aliquots stored frozen at this tem- perature. Before each determination an aliquot was thawed and diluted 100-fold by addition of 0.1 mol/l sodium chloride containing 1.0% (w/v) gelatine. Samples of malathion containing 50% (w/w) mala- thion were obtained from WHO. Pure malathion and 90/o pure isomalathion were kindly donated by Dr J. Miles and Mr D. Mount (Centers for Disease Control, Atlanta, GA, USA) and were dissolved in methanol to give solutions containing 0.1, 10-2, and 2.7 x 10-2 mol/l, respectively, of the pure substance. Prepared b From BDH, Poole, England. 4678 -397- 398 E. REINER & Z. RADIC in this way, the solutions are stable for up to 2 months at 4 'C. Acetylthiocholine iodideC was used as substrate and aqueous stock solutions (11 mmol/l) were prepared on a daily basis. A solution of the thiol reagent DTNBC (10 mmol/l) was prepared in 0.1 mol/l phosphate buffer (pH 7.4) and was stable for up to 15 days at 4 'C. Thin-layer chromatography (TLC) plates precoated with silica gel 60 F254 to a thickness of 0.25 mm were used.d Hexane, acetone, methanol, and brilliant green (all of analytical grade) were obtained commercially.e Solutions of brilliant green in acetone (0.1% (w/v)) were stable at room temperature for up to 2 weeks. Pure bromine was obtained commercially. Procedure WDP malathion (50 mg) was added to 1.0 ml of methanol and stirred to obtain a homogeneous suspension (suspension A). Water (9.0 ml) was then added and the resultant mixture stirred to produce suspension B, which was centrifuged (800 g) until the supernatant liquid was clear (up to 30 min). After decantation, the supernatant liquid was diluted 10- fold with water to give solution C. All suspensions and solutions were prepared at room temperature, while enzyme inhibition experiments and measure- ment of enzyme activity were carried out at 25 'C. Enzyme inhibition. A mixture of buffer (2.5 ml), DTNB reagent (0.1 ml), and diluted stock solution of acetylcholinesterase (0.1 ml) was placed in a test tube. Solution C (0.3 ml) was then added (t = 0) and after 10 min the substrate (0.3 ml of the stock solution of acetylthiocholine iodide). The mixture was then placed in the cell of a spectrophotometerg and the absorbance followed at X = 412 nm for 3 min. If the residual enzyme activity was less than about 40% that of the control activity (see below), the experiment was repeated using solution C that had been further diluted (10-fold or 100-fold) until the residual enzyme activity was 40-80% that of the control. If, in contrast, the measured enzyme activity was more than 80/o that of the control activity, the supernatant liquid of suspension B (see above) was used instead of solution C to inhibit the enzyme. The procedure was also used to follow the inhib- ition of the enzyme with solutions of pure malathion or of malathion plus isomalathion, except that the inhibition times differed. ' From Fluka, Buchs, Switzerland. d From Merck, Darmstadt, Federal Republic of Germany. ' From Kemika, Zagreb, Yugoslavia. f From Schering-Kahlbaum AG, D-1000 Berlin. ' Unicam SP-500. Unicam Instruments Ltd., Cambridge, England. Control activity. A mixture of buffer (2.5 ml), DTNB (0.1 ml), acetylcholinesterase (0.1 ml of the diluted stock solution), and water (0.3 ml) was pipetted into a 10-ml test tube. A solution of the sub- strate was then added (0.3 ml of the stock solution), and the increase in absorbance at X=412 nm measured spectrophotometrically. Thin-layer chromatography. The procedure used for the thin-layer chromatography (TLC) has been described previously (3). WDP malathion (1.0 g) was added to methanol (10 ml), the mixture stirred, and centrifuged (800 g) until the supernatant liquid was clear. Aliquots of the decanted supernatant liquid (20 Al) were spotted on to TLC plates (three spots per plate) as was 20 Al of each of three standard solutions of isomalathion (containing 6.8, 3.4, and 1.7 mmol/l isomalathion, respectively), corresponding to con- centrations of 2.0, 1.0, and 0.5% (w/w) isomalathion in WDP malathion. The TLC plates were eluted with a mixture of hexane and acetone (3:2 (v/v)), air- dried, and then developed with 0.1% (w/v) brilliant green in acetone. Spots were visualized by immersing the plates in bromine vapour for 30 seconds. Quanti- tation was carried out by visual comparison with TLC spots obtained using standard solutions. For samples ofWDP malathion that contained less than 1.0% (w/w) isomalathion the suspension in methanol was made up to 0.5 g WDP malathion/ml. Four samples were spotted on each plate together with four isomalathion standards containing 17, 8.5, 5.1, and 2.5 mmol/l isomalathion, respectively (cor- responding to 1.0, 0.50, 0.30, and 0.15% (w/w) isomalathion in WDP malathion). 100 JL L ~~~~~~~0.09plmot/I & 50 A .9 ;AMO/II :~~~~~in9.0 pImol/I o 0 10 20 30 t inhibition(min) Fig. 1. Time plot of acetylcholinesterase inhibition by isomalathion (O, A ) and isomalathion + malathion (10% (v/v) saturated solution) (-, A). Concentrations of iso- malathion are shown in Mmol/l. DETERMINATION OF ISOMALATHION IN MALATHION POWDERS RESULTS AND DISCUSSION The time plot of the inhibition of acetylcholin- esterase by isomalathion is shown in Fig. 1. Initially, the rate of inhibition was fast, but, after about 10 minutes, approached a steady state. From the initial pseudo-linear part of the curve the second-order rate constant was calculated as 2.4 x 10 mol-1 -l min-'. The effect of malathion upon the rate of inhibition of acetylcholinesterase by isomalathion is also shown. The concentration during enzyme inhibition was 10% of the saturation value in methanol, which is equiv- alent to the maximum concentration of malathion when the supernatant liquid of suspension B was used (see above). These results show that the concentration of malathion in the extract of WDP malathion pre- pared as described does not interfere with inhibition of acetylcholinesterase by isomalathion. For the enzyme test 10 minutes was selected as the inhibition time since at longer intervals the rate gradually approaches a steady-state value. The calibration curve for isomalathion after 10 minutes' inhibition is shown in Fig. 2, where enzyme activity is plotted against isomalathion concentration. Enzyme activity decreases rapidly to about 407o for iso- malathion concentrations up to about 2 ,mol/l. Fur- ther increases in isomalathion concentrations cause a much slower decrease in activity. For this reason 100 > 50 0 0 1 2 3 4 5 6 7 8 9 Isomalathion conc. (pmol/l) Fig. 2. Inhibition of acetylcholinesterase activity by various concentrations of isomalathion after 10 minutes' inhibition time. % Inh % Act 2.0 1.0 0 0.1 0.3 0.5 0.7 0.9 Isomalathion conc. (,mol/1) Fig. 3. Calibration curve for inhibition of acetylcholin- esterase by isomalathion. Each point is the mean of 4-6 measurements and the bars indicate the range of values. The line was obtained by linear regression analysis; slope (k) = 1.791 ± 0.01 3; 1 Ik = 0. 56. enzyme activities less than about 40% of the control cannot be used to determine the concentration of isomalathion in WDP malathion. The initial part of the calibration curve in Fig. 2 (corresponding to isomalathion concentrations of <0.9 jLmol/l) can be linearized if the ratio of 07 inhibition: % activity is plotted against the con- centration of isomalathion (Fig. 3). If the curve is used in this form, the above ratio is multiplied by the reciprocal slope of the calibration line to obtain the isomalathion concentration in itmol/l during enzyme inhibition and, hence, its concentration in WDP malathion, as shown in Annex 1. To establish whether all the isomalathion in WDP malathion is extracted using the method described here, we added a known amount of isomalathion (0.5 ml, equivalent to 297 ug isomalathion) to a sample of WDP malathion ("suspension A") and measured the enzyme inhibition before and after the addition. The degree of inhibition corresponded to the amount of isomalathion already present as impurity in the malathion plus that from the additional amount. Eighteen samples of WDP malathion (50% w/w nominal malathion content) were analysed using the enzyme test. Acetylcholinesterase was first inhibited using solution C, and in 9 of the 18 samples the degree 399 E. REINER & Z. RADIe of inhibition was between 40%o and 80%o. For the remaining 9 samples solution C had to be diluted 10-fold to inhibit acetylcholinesterase activity by 40-800%o. The concentrations of isomalathion in the samples were calculated from the calibration curve (see Annex 1) and are shown in Table 1. The results are expressed as "%lo isomalathion equivalents" and not as "% isomalathion", since WDP malathion contains as impurities several phosphorothiolates that potentiate its toxicity (1); however, the concen- tration of isomalathion in commercial samples of malathion is about 10 times that of other impurities. Furthermore, the rate constants for inhibition of acetylcholinesterase by the other phosphorothiolate impurities are considerably lower than the rate con- stant for inhibition by isomalathion (4), and the enzyme is most probably inhibited by isomalathion alone. The lower limit of detection of the method is 0.01% (w/w) isomalathion, and this level cor- responds to 20% inhibition if the supernatant liquid from solution B is used to inhibit acetylcholin- esterase. This limit is more than adequate for deter- mining the concentration of isomalathion in samples of WDP malathion since the recommended maxi- mum level of this impurity is 1.87o (w/w) that of the nominal malathion content after storage for 6 days at 55 °C (5). The 18 samples of WDP malathion analysed using the enzyme test were also screened by thin-layer chromatography. Comparison of the results with those obtained using a range of standards (containing 0.5, 1.0, and 2.0% (w/w) isomalathion, respectively), indicated that in 10 samples the isomalathion content was less than 1.0% (w/w). The remaining 8 samples were rechromatographed and compared with stan- dards containing 0.15, 0.30, 0.50, and 1.0%0 (w/w) isomalathion, respectively, in WDP malathion. The concentrations of isomalathion were estimated by visual inspection and are shown in Table 1. The Table 1. Comparison of the results of the analysis of WDP malathion by the enzyme test and by thin-layer chromatography (TLC); the number of individual deter- minations is shown in parentheses Enzyme test: TLC: % isomalathion equivalents % isomalathion Sample (w/w) (w/w) 2.2 (4) 2.0 (3) 11 1.7 (4) 2.0 (3) III 1.8 (4) 2.0 (3) IV 1.2 (4) 1.5 (5) V 1.8 (2) 1.5 (3) VI 1.3 (2) 1.5 (3) VIl 1.4 (4) 1.5 (2) Vil 1.0 (4) 1.5 (4) IX 0.72 (2) 0.75 (2) X 0.43 (6) 0.45 (2) Xi 0.42 (2) 0.45 (2) XII 0.37 (2) 0.40 (2) XiII 0.21 (4) 0.30 (2) XIV 0.24 (2) 0.25 (2) XV 0.21 (6) 0.15 (2) XVI 0.16 (4) 0.15 (2) XVII 0.15 (4) 0.15 (2) XVIII 0.14 (2) 0.15 (1) retention times of malathion, isomalathion, and other phosphorothiolate impurities differ sufficiently to give well-resolved TLC spots (3). Only spots from malathion and isomalathion were, however, identi- fied by TLC. The TLC method and the enzyme test have similar sensitivities and there was good cor- relation between the results obtained (r= 0.97). Both tests are suitable for determining isomalathion in WDP malathion, although the TLC method has the advantage of permitting detection not only of iso- malathion but also of other phosphorothiolate impurities in WDP malathion. A disadvantage of the TLC procedure, however, is that it is not yet available as a field kit and in its present form requires access to a fume hood sincc bromine is used to develop the plates. Annex I Calculation of the isomalathion content (w/w) in WDP malathion If acetylcholinesterase is incubated with solution C and the measured degree of inhibition is 30%, i.e., residual activity = 70%0, the isomalathion content (o w/w) in WDP malathion is given by: 100[(30/70) x0.56x 330x 10-2 x 103x 1/(5 x 105)J =0.16 where: 0.56 330 10-2 lo, 5 x 105 reciprocal slope of curve in Fig. 3. relative molecular mass of isomalathion. volume of suspension A in litres. dilution factor of suspension A. sample weight of WDP malathion in gg. 400 DETERMINATION OF ISOMALATHION IN MALATHION POWDERS 401 ACKNOWLEDGEMENT This work was supported, in part, by the Vector Biology Control unit of WHO (Project No. V2/181/169). RtSUMt EPREUVE ENZYMATIQUE DE DOSAGE DE L'ISOMALATHION DANS LES POUDRES MOUILLABLES DE MALATHION L'article d6crit une 6preuve de routine pour 1'evaluation sur le terrain de la teneur des poudres mouillables de mala- thion en isomalathion ((dim&thoxy-thiophosphorylthio)-2 succinate d'6thyle). Cette methode est bas6e sur l'inhibition de I'ac6tylcholinest6rase (EC 3.1.1.7) par l'isomalathion. L'isomalathion est d'abord extrait de la poudre mouillable par le m6thanol, puis 1'extrait dilu6 est incube avec I'ac6tylcholinest6rase. Au bout de dix minutes, on ajoute un substrat constitu6 d'iodure d'acetylthiocholine, et on mesure l'activit6 r6siduelle de 1'enzyme par spectro- photom6trie en pr6sence de dithio-5,5 'bis(nitro-2 benzoate). Le degr6 d'inhibition de l'enzyme est proportionnel a la teneur en isomalathion, que l'on lit sur une courbe d'6talon- nage. Le malathion ne gene pas 1'6preuve et l'extraction est totale. La limite de detection de cette epreuve est de 0,01% (p/p) d'isomalathion, ce qui suffit pour verifier la purete d'6chantillons commerciaux de poudre mouillable de mala- thion (la teneur en isomalathion ne doit pas d6passer 1,8% (p/p)). On a analyse au moyen de cette epreuve 18 echan- tillons de poudre mouillable de malathion (teneur nominale en malathion: 50%) contenant 0,14-2,2% (p/p) d'isomala- thion et on a compare les r6sultats avec ceux obtenus par chromatographie en couche mince. On a observe une bonne correlation entre les r6sultats des deux 6preuves (r=0,97) qui conviennent donc pour le dosage de l'isomalathion dans la poudre mouillable de malathion; toutefois, 1'epreuve enzymatique a I'avantage de pouvoir etre utilisee sur le terrain. REFERENCES 1. ALDRIDGE, W. N. ET AL. The toxicological properties of impurities in malathion. Archives of toxicology, 42: 95-106 (1979). 2. LIN, P. T. ET AL. Studies on organophosphorus im- purities in technical malathion: inhibition of carboxyl- esterases and stability of isomalathion. Pesticide bio- chemistry and physiology, 21: 223-231 (1984). 3. VERSCHOYLE, R. D. ET AL. Dimethyl phosphoro- thioates. Reaction with malathion and effect on malathion toxicity. Archives oftoxicology, 49:293-301 (1982). 4. CLOTHIER, B. ET AL. Interaction of some trialkyl phosphorothiolates with acetylcholinesterase. Charac- terisation of inhibition, aging, and reactivation. Bio- chimica et biophysica acta, 660: 306-316 (1981). 5. WHO Technical Report Series, No. 620, 1978. (Chemistry and specifications of pesticides: second report of the WHO Expert Committee on Vector Biology and Control).