Efficacy of two organophosphorus insecticides, Reldan & Dursban against the larvae of Culex quinquefasciatus

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1 Indian J Med Res 117, January 2003, pp Efficacy of two organophosphorus insecticides, Reldan & Dursban against the larvae of Culex quinquefasciatus M. Kalyanasundaram, P. Jambulingam, S.S. Sahu, P.S. Bhoopathi Doss D. Dominic Amalraj & P.K. Das Vector Control Research Centre (ICMR), Pondicherry, India Received April 3, 2002 Background & objectives : The resistance in vectors to conventional insecticides warrants development of newer insecticides for mosquito control besides use of other countermeasure s. The present study was undertaken to test the efficacy of a new and toxicologically safe organophosphorus compound, Reldan (chlorpyrifos-methyl) and compare its efficacy with Dursban (chlorphyrifos-ethyl) against larvae of different mosquito vectors unde r laboratory conditions and against Culex quinquefasciatus in three larval habitats. Methods: The susceptibility of larvae of mosquito vector species to Reldan and Dursban was determined by following the standard procedure and the LC 50 in mg/l values were calculated by probit analysis. The insecticides were evaluated in the larval habitats of C. quinquefasciatus in and around Pondicherry. The percentage reduction in larval density was calculated using Mulla s formula and duration showing more than 80 per cent reduction was calculated by annotating the graph obtained by plotting percentage reduction in larval density against days of post-treatment. Results: Dursban and Reldan were effective against Anopheles fluviatilis larvae at lower LC 50 of 5.90 x 10-7 and 1.07 x 10-9 mg/l respectively compared to other species of mosquitoes. Dursban and Reldan were equally effective against Aedes aegypti, An. stephensi and An. culicifacies. Reldan was about nine times more effective than Dursban against C. quinquefasciatus with respective LC 50 of 1.17 x 10-4 and 1.34 x 10-3 mg/1. Field evaluation of these insecticides against C. quinquefasciatus showed that Reldan and Dursban at the application rate of 500 g (ai) /ha reduced immature density by more than 80 per cent for a mean duration of 4.0 and 4.5 days and 4.6 and 3.7 days in cesspits and cement tanks respectively. In U-drains, at 500 g (ai)/ha, Reldan was effective for a period of 3.6 days whereas, Dursban was not effective. It was effective for 4.0 days only at 1000 g(ai)/ha. There was no significant increase in the residual activity of both Dursban and Reldan with increase in application rates. Interpretation & conclusion: In view of the lack of sustained control with the applications, it is inferred that these insecticides will be of limited use as larvicides against C. quinquefasciatus breeding in polluted habitats. Key words Culex quinquefasciatus - Dursban - organophosphorus insecticides - Reldan Mosquito control is becoming increasingly difficult because of the development of resistance in vectors to conventional insecticides 1. Many organophosphorus (OP) insecticides have been used for controlling Culex pipiens complex mainly C. quinquefasciatus, the vector of bancroftian filariasis in many countries and the corresponding resistance genes have also been identified 2-5. The resistance in vectors warrants development of newer insecticides for mosquito control besides use of other counter-measures. Dursban (chlorpyrifos-ethyl), an OP insecticide has been used in the operational programme for mosquito larval control. A methyl ether analogue of

2 chlorpyrifos (Dursban), Reldan [O, O-dimethyl-O-(3,5,6-trichloro-2-pyridinyl) phosphorothioate] is another OP insecticide reported to be suitable for mosquito larval control 6. Reldan has relatively higher safety margin with a low mammalian toxicity [LD 50 (oral to rat) mg/kg] compared to Dursban for which LD 50 (oral to rat) was 135 mg/kg 7. However, the efficacy of Reldan under Indian conditions has not yet been determined. The present study was therefore undertaken to test the efficacy of the new and toxicologically safe organophosphorus compound, Reldan and to compare its efficacy with Dursban against larvae of different mosquito vectors under laboratory conditions and against. quinquefasciatus in three larval habitats. Material & Methods Laboratory evaluation for larval susceptibility: Reldan (chlorpyrifos-methyl) 40 per cent EC (emulsifiable concentrate) and Dursban (chlorpyrifos -ethyl) 50 per cent EC (M/s DeNocil Crop Protection Chemicals Ltd., Mumbai, India) were used in the study. In the laboratory, the susceptibility of late third instar larvae offive mosquito vector species, namely, C. quinquefasciatus, An. stephensi and Ae. aegypti [maintained at the cyclic colony of Vector Control Research Centre (VCRC), Pondicherry] and An. fluviatilis and An. culicifacies (F l generation of field-caught mosquitoes from Koraput and Malkangiri Districts, Orissa state) to Reldan and Dursban was determined following the standard procedure 8. Larval susceptibility was expressed as LC 50 (lethal concentration for 50% mortality) in mg/l and the values were calculated by probit analysis 9. Field evaluation against C. quinquefasciatus: Field-testing of these insecticides was carried out in the larval habitats of C. quinquefasciatus viz., cesspits, cement tanks and U-drains in Pondicherry which is endemic for bancroftian filariasis. Three to four sites of each habitat type for each dosage were selected and monitored for immature density (number of larvae and pupae per dip) for 2 to 3 wk prior to application of the insecticide. A knapsack lever-operated sprayer with a discharge rate of 900 ml/min was used to treat the sites. Each insecticide formulation was suitably diluted with water to get 1000 ppm (0.1%) stock solution and the time of discharge was adjusted so as to conform to the required application rate. The sites were treated at 25, 50 and 100 g(ai)/ha [recommended dosage range being g(ai)/ha (gram active ingredient per hectare)] 10 and at higher dosages of 500 and 1000 g(ai)/ha. The sites selected for each dosage were treated at fortnightly intervals. Two sites of each type of habitat were left untreated to serve as control to monitor the natural fluctuation in the immature density. Following the treatment, the larval density was monitored bi-weekly in both treated and control sites. The larval density on different days of pre and post-treatment in both treated and check breeding sites was used to calculate the percentage larval reduction using Mulla s formula 11. The percentage reduction in larval density was plotted against days of observation - 1,4,7,10 and 13 during post-treatment and the graph obtained was annotated to 80 per cent reduction in larval density to arrive at the effective duration in days. A two-way ANOVA was used to compare the means of effective duration to test the level of significance between the two insecticides and the two application rates. Results & Discussion Laboratory evaluation for larval susceptibility: Dursban and Reldan were effective against An. fluviatilis larvae at lower LC 50 of 5.90 x 10-7 and mg/1 respectively compared to other species of mosquitoes. The two insecticides were equally effective against Ae. aegypti, An. stephensi and An. culicifacies. Reldan and Dursban were effective against C. quinquefasciatus

3 with the respective LC 50 of 1.17 x 10-4 and 1.34 x 10-3 mg/l (Table I). The ratio of the LC 50 indicated that Reldan was about nine times more effective than Dursban against C. quinquefasciatus. Contrary to this observation, Reldan was reported to be the least effective insecticide among the organophosphorus insecticides against C. quinquefasciatus (Dacca strain) with a LC 50 of mg/1 12. Table I. Laboratory evaluation of Reldan and Dursban against larvae of mosquito vectors Species LC 50 (mg/l) LC 50 (mg/l) (LCL-UCL) (LCL-UCL) Reldan Dursban 1. C. quinquefasciatus (lab. strain) ( ) ( ) 2. Ae. aegypti (lab. strain) ( ) ( ) 3. An. stephensi (lab. strain) ( ) ( ) 4. An. fluviatilis (field strain, Malkangiri) ( ) ( ) 5. An. culicifacies (field strain, Jeypore) ( ) ( ) LCL, lower confidential limits; UCL, upper confidential limits Field evaluation against larvae of C. quinquefasciatus: Application of Dursban or Reldan to the larval habitats at 25 and 50 g(ai)/ha had no significant effect on the larval density. Even at the application rate of 100 g(ai)/ha, the percentage reduction in the larval density ranged from only per cent. Therefore the insecticides were evaluated at higher dosages of 500 and 1000 g(ai)/ha. At 500 and 1000 g(ai)/ha, Reldan caused 90 per cent reduction in larval density of C. quinquefasciatus on days 1 and 2 post-treatment in all the habitats. Subsequently, there was a steady increase in the density and the pre-control level was reached by day 7 post-treatment. Reldan was effective in causing more than 80 per cent reduction in larval density in cesspits for 4.0 ± 0.74 days at 500 g(ai)/ha and 4.3 ± 0.68 days at 1000 g(ai)/ha. In cement tanks the residual effect of Reldan at 500 g(ai)/ha lasted for 4.5 ± 0.84 days and at 1000 g(ai)/ha for 5.2 ± 0.82 days. In drains, Reldan was effective for 3.6 ± 1.34 days at 500 g(ai)/ha and for 4.6 ± 0.38 days at 1000 g(ai)/ha. There was no significant difference in the effective duration with increase in the application rate in all the habitats (P > 0.05) (Table II). Reldan in general was found to be effective for 4 to 5 days in the habitats tested. However, in a coastal village of North Sumatra, Indonesia, the residual effect of chlorpyrifos-methyl laste d for 7 days against An. sundaicus 13. The effectiveness of Reldan was comparatively less than that of fenthion, the OP larvicide used in the antifilarial campaign programme in Sri Lanka at 1 mg/l equivalent to 1000 g(ai)/ha at weekly intervals to control OP resistant C. quinquefasciatus breeding in polluted soakage pits 14. Application of Dursban at 500 and 1000 g(ai)/ha caused more than 90 per cent reduction in the larval density of C. quinquefasciatus on days 1 and 2 post-treatment in cesspits and cement tanks.

4 In drains, only at 1000 g(ai)/ha, the application of Dursban could bring down the immature density to > 90 per cent, 24 h after application. Subsequently, the density started steadily raising to that of the pre-control level by day 6 post-treatment. Table II. Field evaluation of Reldan and Dursban against C. quinquefasciatus S.No. Type of Application Effective days (>80% reduction in larval density) Mean habitat rate g(ai)/ha Rep.1* Rep.2* Rep.3* Rep.4* ±SE Reldan: 1. Cesspits ± ± Cement tanks ± ± U-drains ± ± 0.38 Dursban: 1. Cesspits ± ± Cement tanks ND 3.7 ± ND 3.8 ± U-drains ND 4.0 ± 0.82 *Rep. 1, 2, 3 & 4, Replicates 1, 2, 3 & 4; ND, not done Analysis of the results on percentage reduction in larval density during different days of posttreatment showed that in cesspits, at 500 g(ai)/ha, Dursban was effective for 4.6 ± 0.9 days and at 1000g(ai)/ha for 4.9 ± 0.90 days. In cement tanks, Dursban could effectively reduce the larval density of C. quinquefasciatus to more than 80 per cent for 3.7 ± 0.37 days at 500 g(ai)/ha and at 1000 g(ai)/ha for 3.8 ± 0.14 days. In U-drains, Dursban was not effective at the rate of 500 g(ai)/ha and at 1000 g(ai)/ha, it was effective for 4.0 ± 0.82 days (Table II). There was no significant difference (P > 0.05) in the residual effectiveness by enhancing the rate of application in cesspits and cement tanks. Overall, Dursban was found to be effective for 4 to 5 days in the habitats tested. Similar observation was made by Shim et al 15 in rice fields in Korea where application of Dursban at 1000 g(ai)/ha caused per cent mortality of An. sinensis larvae for 3 days. Ultra low volume (ULV) application of Dursba n was found to be effective against Ae. aegypti larvae for 5 days 16. In another study, the effect of monocrotophos at 10 mg/l and pirimiphosmethyl at 0.1 mg/l in clean rice fields against An. subpictus and An. nigerrimus was found to last for less than 10 days 17. In general, the lower residual effect might be due to the instability of these insecticides under field conditions. In the present study, treatment sites often contained mosquito larvae one day after treatment. This was due to continuous recruitment of mosquito immature in the breeding habitats and eggrafts were found in the treated sites on all days post-treatment. As the effect of Reldan and Dursban was found to last for 4 to 5 days in the habitats tested, these can play only a limited role as larvicides against C. quinquefasciatus breeding in such habitats.

5 Acknowledgment The authors are grateful to M/s DeNocil Crop Protection Chemicals Ltd., Mumbai for providing the funds for carrying out the evaluation. The authors acknowledge Shri S. Subramaniam, Technical Officer for statistical analysis of the results and the staff of Vector Biology and Control Division of the Centre in Pondicherry and at the VCRC field station, Malkangiri, Orissa for technical assistance. References 1. WHO. Vector resistance to pesticides - Fifteenth report of the WHO Expert Committee on vector biology and control, WHO Tech Rep Ser No. 818, pp 62. Geneva : World Health Organization; Bisset JA, Rodriguez MM, Diaz C, Ortiz E, Marquetti MC, Hemingway J. The mechanisms of organophosphate and carbamate resistance in Culex quinquefasciatus from Cuba. Bull Entomol Res 1990; 80 : Poirie M, Raymond M, Pasteur N. Identification of two distinct amplifications of the esterase B locus in Culex pipiens (L.) mosquit oes from Mediterranean countries. Biochem Genet 1992; 30 : Bourguet D, Capela R, Raymond M. An insensitive acetyl cholinesterase in Culex pipiens (Diptera : Culicidae) from Portugal. J Econ Entomol 1996; 89 : Ben Cheikh H, Ben Ali-Haouas Z, Marquine M, Pasteur N. Resistance to organophosphorus and pyrethroid insecticides in Culex pipiens (Diptera: Culicidae) from Tunisia. J Med Entomol 1998; 35 : The pesticide manual - a world compendium, Worthing CR, Walker SB, editors, The British Crop Protection Council, Thornton Heath, 8th ed p WHO. Data sheets on pesticides (No.33) Chlorpyrifos-methyl. Geneva : World Health Organization; 1978 (unpublished Document WHO/VBC/DS/78.33). 8. WHO. Instructions for determining the susceptibility or resistance of mosquito larvae to insecticides. Geneva: World Health Organization; 1975 (unpublished document WHO/VBC/75.583). 9. Finney DJ. Probit analysis, 3rd ed. Cambridge: Cambridge University Press; 1971 p Chavasse DC, Yap HH, editors. Chemical methods for the control of vectors and pests of public health importance. Geneva : World Health Organization Division of Control of Tropical Diseases, WHO Pesticide Evaluation Scheme (WHO PES), 1997 pp 136 (unpublished document WHO/CTD/WHOPES/97.2). 11. Mulla MS, Norland RL, Fanara DM, Darwazeh HA, Makean D. Control of chironomid midges in recreational lakes. J Econ Entomol 1971; 64 : Ali A, Chowdhury MA, Hossain MI, Mahmud-ul-Ameen, Habiba DB, Aslam AFM. Laboratory evaluation of selected larvicides and insect growth regulators against field-collected Culex quinquefasciatus larvae from urban Dhaka, Bangladesh. J Am Mosq Control Assoc 1999; 15 : Imai C, Yamugi H, Panjaitan W. Efficacy of several larvicides in laboratory and field tests against Anopheles sundaicus in a village, North Sumatra, Indonesia. Jpn J Sanit Zool 1987; 38 : Peiris HT, Hemingway J. Effect of fenthion treatment on larval densities of insecticide resistant Culex quinquefasciatus in an urban area of Sri Lanka. Med Vet Entomol 1996; 10 : Shim JC, Yoon YH, Kim CL, Lee WJ, Lee BL, Kim SC. Integrated control of vector mosquitoes in rice field. Korean J Entomol 1987; 17 : Kilpatrick JW, Eliason DA, Babbitt MF. Studies of the potential effectiveness of Ultra Low Volume aerial applications of insecticides against Aedes aegypti (L.) larvae. Mosq News 1970; 30 : Hemingway J, Bonning BC, Jayawardena KGI, Weerasinghe IS, Herath PRJ, Oouchi H. Possible selective advantage of Anopheles spp. (Diptera: Culicidae) with the oxidase- and acetylcholinesterase-based insecticide resistance genes after exposure to organophosphates or an insect growth regulator in Sri Lankan rice fields. Bull Entomol Res 1988; 78 : Reprint requests : Dr M. Kalyanasundaram, Deputy Director (S.G.), Vector Control Research Centre (ICMR) Indira Nagar, Pondicherry , India