Exposure time versus concentration in the WHO standard test for mosquito resistance to chlorohydrocarbon insecticides*

Bull. Org. mond. Sant 19 Bull. Wid Hith Org. J 1969, 40, 561-567 Exposure Time versus Concentration in the WHO Standard Test for Mosquito Resistance to Chlorohydrocarbon Insecticides* V. ARIARATNAM 1 & A. W. A. BROWN2 In order that the WHO standard test for mosquitos may yield a median lethal dose value (LC5o) in the case of highly tolerant species and resistant strains, it is necessary to increase the exposure period. The experiments described in this paper, done with a DDT- tolerant strain of Culex pipiens fatigans, show that doubling the exposure period has the same effect as doubling the DDT concentration, since it doubles the uptake ofDDT into the mosquitos, and has the result ofhalving the LCso obtained. The WHO standard test for resistance in adult mosquitos as defined in 1963 (WHO Expert Com- mittee on Insecticides, 1963) did not provide the means of assessing resistance levels once they had passed the point where no more than 50% mortality was obtained by a 1-h exposure to papers impreg- nated with 4.0% DDT, the highest recommended concentration. Thus there was no way of obtaining resistance ratios either for anophelines which had developed full resistance, or for Culex pipiens fatigans even in its normal state. Only in 3 instances, namely, at Machemba and South Pare, Tanzania (Smith & Bransby-Williams, 1962) and in Suva, Fiji (Burnett & Ash, 1961), was it possible to obtain LC,0 values for C. p.fatigans based on a 1-h exposure. As a consequence, susceptibility levels in C. p. fatigans have been obtained either by deriving the LC50 for 24-h exposure (Bhatia, Deobhankar & Vittal, 1958) or the LT50 from continuous exposure to 4.0% DDT papers (Tadano & Brown, 1966). The question remained whether doubling the duration of the exposure was equivalent to doubling the con- centration. With Anopheles atroparvus, Garms & Rehm (1961) found that doubling the exposure time from 1 h to 2 h, or from 2 h to 4 h, had the effect of halving the LCQ0 found for DDT. When dieldrin * This investigation was performed in the Department of Zoology, University of Westem Ontario, London, Canada, and was supported by a grant-in-aid from the World Health Organization. 1 Present address: Department of Entomology, Uni- versity of California, Riverside, California 92502, USA. 'Present address: Vector Biology and Control, World Health Organization, Geneva, Switzerland. was the test insecticide, Busvine (1958) discovered with Aedes aegypti that the LC50 was halved for each doubling of the exposure time from 0.25 h up to 4.0 h. Similar results were obtained with this species when it was exposed to dieldrin for periods between 0.25 h and 8.0 h (Hamon, 1963). Moreover, it was found by Pennell, Miskus & Craig (1964) that the amount of uptake of dieldrin by the C. p. fatigans mosquitos exposed to the impregnated paper was directly proportional to the concentration on the one hand and to the duration of exposure on the other. Accordingly, in the present investigation individu- als of a slightly DDT-tolerant strain of C. p. fatigans were exposed to the full range ofDDT concentrations in the WHO standard test for periods of 1 h, 2 h, 4 h, 8 h, 16 h and 24 h, and from the percentage mortalities obtained the LC50 values were calculated for each exposure period, and the LT50 values for each concentration. The uptake of DDT at all these concentrations and exposure times was determined by means of gas chromatography. In addition, the determinations of mortality and uptake were repeated by employing 14C-DDT-papers followed by assessment by liquid scintillation counting. MATERIALS AND METHODS Adult females of Culex pipiens fatigans from Rangoon, Burma, were used in these tests, 1-2 days after emergence. The strain employed was some- what DDT-tolerant (the larval LC50 was 0.13 ppm), 2322 561- 562 V. ARIARATNAM & A. W. A. BROWN being derived from about 100 females sent to this laboratory from Rangoon, Burma, in May 1963. The mosquitos were exposed to 0.25%, 0.5%, 1.0 %, 2.0% and 4.0% papers in lots of 25 according to theWHO standard method and using the standard test kit; the exposure periods investigated were 1 h, 2 h, 4 h, 8 h, 16 h, and 24 h. At the end of each exposure period the mosquitos were transferred to holding chambers and mortality counts were made after the usual 24-h observation period. In the case of 24-h exposure, the observation period was omitted and mortality counts were made at the end of the exposure period. Parallel exposures to control papers impregnated with Risella oil 1 provided a check on control mortality, which exceed- ed 5% in about one-quarter of the tests. In these cases the observed percentage mortalities were corrected by means of Abbott's formula.2 Tests in which the control mortality was in excess of 20% were discarded. For each exposure period the corrected percentage mortalities at each con- centration were plotted in probability units against the logarithms of the concentrations, and the regres- sion lines were fitted by eye for determination of the LC50 values. In the same manner, LT50 figures were derived by plotting the probit mortalities against the logarithms of the exposure times for each of the concentrations used. For the determination of uptake, papers were hand-impregnated with solutions of radioactive DDT to yield a volume impregnation rate of 3.6 mg solution per cm2, the same as in the standard WHO test papers. Sheets of Whatman No. 1 filter-paper, measuring 15 cmx 12 cm, were treated according to the method of Busvine & Nash (1953) with a 9: 1 mixture ofDDT (p,p'-isomer) and 14C-DDT dissolved in a 1: 2 mixture of Risella oil and diethyl ether. The "4C-DDT was ring-labelled on the para- carbon atoms and had a specific activity of 3.22 mCi/g. The series of tests for mortality on WHO test papers was repeated on the hand-impregnated papers. Each of these series was paralleled by a second series in which the exposed mosquitos were homogenized for 15 min in 25 ml acetone containing 1 g anhydrous sodium sulfate in a Sorvall Omni- mixer. The acetone extract was filtered and the filtrate was evaporated to dryness in a slow stream of air. Where the mosquitos had been exposed to IRisella 117, a refined mineral oil obtained from the Shell Petroleum Company. '(% test mortality - % control mortality) x 100 + (100-% control mortality). WHO test papers containing DDT, the dry residue was taken up in 10 ml hexane for assessment by gas chromatography; where they had been exposed to 14C-DDT in hand-impregnated papers, it was taken up in 15 ml toluene scintillation fluid for liquid scin- tillation counting. The gas chromatograph used was a Varian Aero- graph (Model 680) equipped with a tritium-foil, concentric-tube, electron-capture detector and a 1.6 m x 3 mm Pyrex glass column of chromosorb W (60/80 mesh) coated with 5% Dow 11 resin; the column was maintained at a temperature of 190°C and operated at a nitrogen-gas flow rate of 40 ml per min. A Beckman Liquid Scintillation System was employed to assess the scintillation fluid samples in 15-ml glass scintillation vials, 20 mm in diameter and of low 40K content, provided with foil-lined screw caps. The scintillation fluid consisted of 0.5% PPO (2,5-diphenyloxazole) and 0.03% POPOP (1,4-bis (2-(5-phenyl) oxazolyl) benzene) in fluorometric- grade toluene. The figure for uptake of the 9: 1 DDT mixture was obtained by multiplying the assay of 14C-DDT by 10. RESULTS With exposure periods of I h and 2 h no mortality was obtained at any concentration of DDT, and with 4 h only 11% mortality on the 4.0% DDT papers. With exposure periods of 8 h, 16 h and 24 h, a series of mortalities was obtained sufficient to yield dosage-mortality lines and LC50 values of 9.0%, 4.2% and 3.0% respectively (Fig. 1). When the percentage mortality figures were plotted as time- mortality lines at each concentration (Fig. 2), LT50 values could be derived for 2.0% and 4.0% DDT as 34 h and 15.5 h, respectively. It will be seen that the product of the LC50 and the exposure time is constant (see the table), amounting to a CT of 67-72 percent-hours, and that the product of the LT50 and the concentration is also constant, amount- ing to 62-68 percent-hours. When the entire series of exposures was repeated on hand-impregnated 14C-DDT papers, similar results were obtained (Fig. 3 and 4). The CT product obtained from the LC50 multiplied by the exposure time ranged between 40 h and 50 h, while that obtained from the LT50 multiplied by the con- centration showed a wider range. What is significant is the lower value of CT obtained with the hand- impregnated papers as compared with the machine- impregnated WHO papers. TIME VERSUS CONCENTRATION IN THE WHO TEST FOR MOSQUITO RESISTANCE TO DDT 563 FIG. 1 DOSAGE-MORTALITY RELATIONSHIPS OF CULEXPIPIENS FATIGANS EXPOSED TO WHO DDT PAPERS FOR DIFFERENT PERIODS 70 - * 8h 24h o 16 h x 60 - X 24 h /0 50 -6 h 40 _ 3 8 h 30 0 0 z 20- 00 a. 10 x o 5 K 21 _ 0.25 0.5 1.0 2.0 4.0 PERCENT DDT CONCENTRATION-TIME PRODUCTS DERIVED FROM EXPOSURE TO MULTIPLE CONCENTRATIONS FOR _ DIFFERENT TIMES AND FROM MULTIPLE-TIME EXPOSURES AT DIFFERENT CONCENTRATIONS WHO papers Hand-impregnatedpapers LCSO CT LC-o CT(% DDT) (%x h) (% DDT) (%x h) Multiple-concentration exposure 8-h exposure 9.0 72 5.0 40 18-h exposure 4.2 67 3.1 50 24-h exposure 3.0 72 1.9 46 Multiple-time exposure LT5s CT LTs CT(h) (%x h) (h) (%x h) 2.0% DDT 34 68 30 60 4.0% DDT 15.5 62 9.5 38 When the percentage mortalities obtained in all the tests in the entire series of experiments were plotted against the logarithm of the CT product of the concentration and exposure time involved, a linear relationship was revealed not only with the WHO papers (Fig. 5) but also with the hand- impregnated papers (Fig. 6). FIG. 2 EXPOSURE TIME-MORTALITY RELATIONSHIPS OF CULEX PIPIENS FATIGANS EXPOSED TO WHO DDT PAPERS 70 / 4.01 60_/ + 0.5% / 50 * 1.0% 0 2.0% /O% i40 - X 4.0% I-~~~~~~~~~~~~~~~~~~~~~c 1-30 0 0 t-20 -1.0% 1., L) a. / 0.5% 5 2 + ,1 2 4 8 16 24 HOURS OF EXPOSURE When the uptakes of DDT by the mosquitos, expressed in nanograms per female, are plotted graphically (Fig. 7), it is seen that they increase linearly with concentration at all the exposure times employed; moreover, the uptake lines show a regular stepped increase as the exposure time is increased., The same results are shown for the uptake of radioactive DDT (Fig. 8). It is clear that uptake increases linearly both with concentration and with exposure time. Since probit mortality has been found to increase with log concentration and with log exposure time, it would be expected to increase with log uptake, since uptake is directly proportional to concentration and exposure time. When the probit mortalities obtained with the WHO papers and the hand- impregnated papers are plotted against the deter- mined uptakes on a logarithmic scale (Fig. 9 and 10), the relationship is seen to be linear. There is, however, a tendency for the 24-h figures to show higher mortalities for a given uptake, which may I The DDT uptake includes the DDE to which it is converted in vivo; the DDE amounted to 45% for uptakes of 60-100 nanograms, 75% for uptakes of 10-60 nanograms, and fully 90% for uptakes of less than 10 nanograms per female. The results for the 24-h exposure period, which had no following observation period, were not significantly different from those for the 16-h exposure period, which was followed by the usual 24-h observation period. V. ARIARATNAM & A. W. A. BROWN FIG. 3 DOSAGE-MORTALITY RELATIONSHIPS OF CULEX * PIPIENS FATIGANS EXPOSED TO 14C-DDT PAPERS FOR DIFFERENT PERIODS 90 * 8h s0 o16h x X 24 ht 70 0 60- 50- ~ 0 z 30- Q ~20K 0 FIG. 4 EXPOSURE TIME-MORTALITY RELATIONSHIPS OF CULEX PIPIENS FATIGANS EXPOSED TO '4C-DDT PAPERS :i 0 :L z a. 4 8 HOURS OF EXPOSURE FIG. 6 RELATIONSHIP BETWEEN MORTALITY AND CT PRODUCT ('4C-DDT PAPERS) FIG. 5 RELATIONSHIP BETWEEN MORTALITY AND CT PRODUCT (WHO PAPERS) x C:i W 0 E-- z hl 16 32 16 C xT 564 1.0 PERCENT DDT A 0.25% + 0.5% * 1% O 2% X 4%:i 21- z W. 0. x A 0.25% + 0.5% * I % 0 2 % x 4 % 0 0 +- C xT FIG. 7 RELATIONSHIP BETWEEN UPTAKE (ng per female) AND CONCENTRATION FOR DIFFERENT EXPOSURE PERIODS (WHO PAPERS) o I h A 2h + 4h * 8h o 16 h x 24 h 9/ U,- .- - - - -0.25 0.5 1.0 2.E PERCENT DDT 4.0 FIG. 8 RELATIONSHIP BETWEEN UPTAKE (ng per female) AND CONCENTRATION FOR DIFFERENT EXPOSURE PERIODS (14C-DDT PAPERS) o I h A 2h + 4h 0 8h o 16 h x 24 h * 0 x A 2.0 PERCENT DDT 120 r 100o m 80 0 co E,: CL 60 0 z 40 z 20 24 h 116 h 8h 4 h 140 r I h 1201- 1001- Cz: 0 0 cn 0 co a Ci) cn 0 z z. 801- 601- 401 20 F 0 4.00.25 0°5 1.0 V. ARIARATNAM & A. W. A. BROWN FIG. 9 RELATIONSHIP BETWEEN UPTAKE (ng per female) AND MORTALITY (WHO PAPERS) FIG. 10 RELATIONSHIP BETWEEN UPTAKE (ng per female) AND MORTALITY ('4C-DDT PAPERS) 90r80 r x +0 0 x I h 2 h 4 h 8 h 16 h 24 h x x 70 O 09 t 50 w 400 z 30 a. 20+ 0 0 5 nA+ i A n +A I I 10 20 50 UPTAKE OF DDT 100 be connected with starvation by the end of that period. DISCUSSION The determinations of DDT uptake show that mortality (as expressed in probits) is proportional to the logarithm of the uptake. They also demonstrate for DDT precisely what Pennell, Miskus & Craig (1964) have shown for dieldrin in Culex p. fatigans, that uptake is arithmetically proportional to con- centration and exposure time. Thus a rational basis has been provided for the observations of Busvine (1958), Garms & Rehm (1961) and Hamon (1963) that mortality in adult mosquitos exposed to chloro- hydrocarbon insecticides increases not only with the logarithm of the concentration, but also with the logarithm of the exposure time. Since exposure time has the same deciding value as concentration, uptake bears a direct relationship with the CT product of concentration and time, and probit mortality is proportional to the logarithm of the CT product, as was demonstrated in Fig. 5 and 6. The CT product necessary for 50% mortality proved to be lower with hand-impregnated papers than with the WHO papers, as shown in the table. This was because there was a slightly greater uptake of DDT from the hand-impregnated papers than from the WHO papers, as may be seen by comparing Fig. 8 with Fig. 7. On the other hand, the relation- ship between mortality and the amount of uptake was identical whichever paper was used, as may be seen by comparing Fig. 9 and 10, where in either case an average of 62% mortality was obtained for an uptake of 100 nanograms/female. The slightly lower uptake from machine-impreg- nated papers as compared to the hand-impregnated papers, at the same rate ofimpregnation (3.6 mg/cm2), observed in this investigation explains why the impregnation rate of 3.3 mg/cm2, employed in the original test of Busvine & Nash (1953), had to be increased to 3.6 mg/cm2 for the papers used in the WHO test kit in order to obtain equivalent mortality. The results have shown that in Culex pipiens fatigans with DDT, as in Anopheles atroparvus with DDT (Garms & Rehm, 1961) and in Aedes aegypti 70p 60- 501- 801- 40- E- E- 0 z c=: W 0L 60- 30 20 10I 5 2 O Ih & 2h + 4h * 8h 0 16 h X 24 h x o 0 0 10- 1() 20 50 UPTAKE OF DDT 100 200 z 566 x TIME VERSUS CONCENTRATION IN THE WHO TEST FOR MOSQUITO RESISTANCE TO DDT 567 with dieldrin (Busvine, 1958; Hamon, 1963), an increase in exposure has the same quantitative value as an increase in concentration. Thus resistance ratios between resistant and normal strains based on the LT50 are equally valid as those based on the LC50. Moreover, where tests with a series of con- centrations are performed at exposure times which are a multiple of the usual l-h period, the LC5, obtained may be converted to the l-h LC5o0 equivalent by multiplying it by the hours of exposure. RISUME tPREUVE STANDARD DE L'OMS POUR LA MESURE DE LA RESISTANCE DES MOUSTIQUES AUX INSECTICIDES A BASE D'HYDROCARBURES CHLORES: UTILISATION COMME PARAMETRE DE LA DUREE D'EXPOSITION AU LIEU DE LA CONCENTRATION D'INSECTICIDE Au cours d'une epreuve de determination de la sensi- bilite aux insecticides, des moustiques adultes d'une souche de Culex pipiens fatigans moderement resistante au DDT ont ete exposes i des papiers impregnes de DDT pendant des periodes de 1, 2, 4, 8, 16 et 24 heures. L'absorption du DDT par les insectes, mesuree par chromatographie gazeuse, a e proportionnelle a la dur6e de 1'exposition ainsi qu'a la concentration de l'insecticide. Ce rapport a ete retrouv6 lors de 1'emploi de papiers impregnes de DDT radioactif (14C-DDT) suivi d'une determination de l'absorption de l'insecticide par scintil- lographie. On a constate que les probits de la mortalit6 6taient proportionnels a la fois au logarithme de la duree d'expo- sition et au logarithme de la concentration d'insecticide. Les valeurs de la CL50 (concentration ltale moyenne) correspondant a chacun des temps d'exposition dimi- nuaient de moitie chaque fois que l'on multipliait par deux la duree de ce demier. De meme, le TL50 (temps Ietal moyen) mesure pour chaque concentration de DDT etait reduit de moitie chaque fois que la dose d'insecti- cide etait doublee. II en resulte qu'a une mortalit6 de 50% correspond un produit CT (concentration x duree d'ex- position) constant quelles que soient la dur6e d'expo- sition utilisee au cours de 1'epreuve ou la dose d'insecti- cide ayant servi it determiner le TL50. Les probits de mortalite sont proportionnels au logarithme de CT independamment de la concentration d'insecticide ou de la duree d'exposition. Ce rapport est valable que les papiers soient impregnes de DDT ou de 14C-DDT. Ces rapports mathematiques permettent de comparer directement et arithmetiquement les valeurs de la CL50 obtenues pour les souches resistantes apres des exposi- tions d'une duree de 2, 4, 8, ... heures avec les valeurs de la CL50 observees apres exposition des souches sen- sibles pendant une duree standard de 1 heure. REFERENCES Bhatia, S. C., Deobhankar, R. B. & Vittal, M. (1958) Indian J. Malar., 12, 371 Burnett, G. F. & Ash, L. H. (1961) Bull. Wid Hlth Org., 24, 547 Busvine, J. R. (1958) Indian J. Malar., 12, 279 Busvine, J. R. & Nash, R. (1953) Bull. ent. Res., 44, 371 Garms, R. & Rehm, W. F. (1961) Z. angew. Zool., 48, 385 Hamon, J. (1963) Bull. Soc. ent. Fr., 68, 225 Pennell, J. T., Miskus, R. & Craig, R. (1964) Bull. Wid Hlth Org., 30, 91 Smith, A. & Bransby-Williams, W. R. (1962) Bull. Wld Hlth Org., 27, 603 Tadano, T. & Brown, A. W. A. (1966) Bull. Wld Hlth Org., 35, 189 WHO Expert Committee on Insecticides (1963) Wld Hlth Org. techn. Rep. Ser., 265, 41