EFFICACY OF A NOVEL AREA-REPELLENT AEROSOL FORMULATION AGAINST MOSQUITOES AND FLIES (DIPTERA: CULICIDAE, MUSCIDAE)

Proceedings of the Eighth Interntionl Conference on Urbn Pests Gbi Müller, Reiner Pospischil nd Willim H Robinson (editors) 2014 Printed by OOK-Press Kft., H-8200 Veszprém, Ppái ut 37/, Hungry EFFICACY OF A NOVEL AREA-REPELLENT AEROSOL FORMULATION AGAINST MOSQUITOES AND FLIES (DIPTERA: CULICIDAE, MUSCIDAE) 1 ANDREW M. SCRIVENER, 2 BRICE A. PETERS, AND 2 PETER F. MILLER 1 Pscoe s Pty Ltd, 40-46 Firfield Street, Firfield Est, NSW 2165, Austrli 2 Fculty of Science, University of Technology, Sydney, P.O. Box 123, Brodwy, NSW 2007, Austrli Abstrct Field studies of two re repellent erosol formultions with trnsfluthrin nd permethrin ctive ingredients were crried out ginst mixed freshwter mosquitoes ner Humpty Doo, Northern Territory nd ginst flies ( mixed popultion of house flies, Musc domestic nd bush flies Musc vetustissim) in Bell, South West Queenslnd, Austrli. The mosquito test site ws djcent to breeding re, with lrge popultion of biting mosquitoes. Fly repellency testing ws conducted in the milking shed of diry frm with lrge resident fly popultion. Non-bsorbent (glzed tile) nd bsorbent (plywood) substrtes were spryed with the formultions nd exposed to the mbient conditions. For the mosquito study, substrtes were locted djcent to seted humn test subjects. The test subjects counted ll mosquito lndings on the visible res of their body during 5 minute period every 2 hours for 6 hours. Fly evlution ws conducted in similr mnner, except tht humn ssessors counted ll fly lndings on surfces within 1 metre rdius of where they were seted. There were no significnt differences in efficcy between the high nd low ctive level formultions for both mosquitoes nd flies. The lower ctive level formultion (0.5% trnsfluthrin nd 0.05% permethrin) gve reduction of 96.3% over 6 hours for glzed tiles, compred to the untreted controls, nd reduction of 80.0% for plywood. For flies, the respective reductions for the sme rte were 81.4% nd 79.0%. These studies demonstrted tht both formultions provided effective re protection from mosquitoes nd flies for up to 6 hours fter ppliction, on both bsorbent nd non-bsorbent surfces. Key words Trnsfluthrin, re repellent, Musc domestic, Musc vetustissim, Anopheles bncroftii, Culex nnulirostris INTRODUCTION An re or sptil repellent hs been defined s: n inhibiting compound, dispensed into the tmosphere of three dimensionl spce which inhibits the bility of mosquitoes to locte nd trck trget such s humn or livestock (Nolen et l., 2002). This study ddresses the re repellency of novel erosol product ginst biting mosquitoes nd nuisnce flies. Outdoor re repellents re reltively new concept in consumer pest control. Formts include mosquito coils, lnterns, metered erosols, therml devices nd cndles (WHO, 1998). The mjority of these products re designed exclusively for mosquito repellency nd re generlly ineffective in repelling flies. In Austrli, the House fly, Musc domestic nd the Bush fly, Musc vetustissim re importnt nuisnce pests (Gerozisis et l., 2008). The bush fly is prticulrly nnoying pest over much of Austrli in the summer months becuse of the hbit of lnding on people (Hughes et l., 1972). Therefore, there is need for n re repellent tht is efficcious in repelling both mosquitoes nd Musc flies. Treting n inert surfce with vpour-ctive repellent is n ppeling concept. This ide is not new, however. The US Army tried sprying repellent on the ground in 1943 to repel mosquitoes, with Efficcy of Novel Are-Repellent Aerosol Formultion Aginst Mosquitoes nd Flies A.m. Scrivener, B.. Peters, And P.f. Miller

154 A.m. Scrivener, B.. Peters, And P.f. Miller limited success (Moore nd Stge, 1943). For the concept to work well chemicl which vpourises t mbient tempertures is needed. The pyrethroid trnsfluthrin hs vpour pressure of 9 x10-4 P t 20 C (WHO,2014) nd is good cndidte for this use. Ogom et l. (2012) demonstrted the efficcy of this repellent ginst Anopheles rbiensis, when spryed onto hessin strips. Permethrin, the second pyrethroid included in our test product, hs lso been used s repellent spry on clothing nd ws shown to be effective (Debboun et l., 2007). Austrlin consumers embrce the erosol formt for domestic pest control. This drove the development of n erosol bsed re repellent tht cn be conveniently pplied to vriety of substrtes to provide re repellency ginst flies nd mosquitoes. MATERIALS AND METHODS Aerosol Formultions nd Appliction To Substrtes All erosol formultions in this study were provided by Pscoe s Pty Ltd,, 40-46 Firfield Street, Firfield Est, NSW 2165, Austrli. Aerosol formultion contined 1.0% trnsfluthrin nd 0.1% permethrin nd contined 0.5% trnsfluthrin nd 0.05% permethrin. For the mosquito study, 30.0g of erosol formultion ws spryed from distnce of 200mm onto 4 x 500mm x 500mm glzed floor tiles (totl re 1.0m 2 ) nd nother 30.0g ws pplied s bove to piece of unpinted plywood 1,200mm x 800mm (totl re 0.96m 2 ). The ppliction ws repeted to give 5 sets of pproximtely 1.0m 2 re of ech of the bove substrtes. All the bove ws repeted for erosol formultion. Once the substrtes hd been treted they were left for 30 minutes to dry outdoors, wy from the test site. For the fly study, 60.0g of erosol formultion ws spryed from distnce of 200mm onto 9 x 450mm x 450mm glzed floor tiles (totl re 1.82m 2 ) nd nother 60.0g ws pplied, s bove, to 2 pieces of unpinted plywood 1,200mm x 800mm (totl re 1.92m 2 ). The ppliction ws repeted to give 5 sets of pproximtely 1.0m 2 re of ech of the bove substrtes. All the bove ws repeted for erosol formultion. Once the substrtes hd been treted they were left for 30 minutes to dry outdoors, wy from the test site. Mosquito Study Mosquito species were identified primrily s Anopheles bncrofti, Culex nnulirostris nd Mnsoni uniformis. A lrge level clered section of ground in close proximity to mosquito breeding re t Thomsen s frm, Thomsen Rod, Humpty Doo, NT, Austrli, ws selected s the study site. Test subjects selected for the study were mles nd femles between the ges of 18 nd 70 yers. All test subjects were required to wer full body Elite Edition Originl Bug Shirt Ò mosquito suits (trousers nd jckets with hoods) disposble ltex gloves nd covered shoes for the durtion of the study. There were 5 test subjects, 4 of whom evluted the product nd the other cted s the untreted control. All test subjects st on folding cmping chirs during the pre-count nd evlution periods. To void experimentl bis, the control ws different subject on every evening of the study. Test subjects st minimum of 10m prt from one nother, with the control sited t one end of the test re to minimize eril contmintion from the treted subjects. The study commenced when biting mosquitoes were first noticed ech evening (pproximtely 7pm). All subjects simultneously conducted pre-count of 5 minutes durtion, counting ll mosquito lndings of greter thn one-second durtion on the visible prts of their body (primrily the bdomen, legs nd rms). Product evlution ws then strted if ech subject recorded minimum of 10 lndings during the 5-minute ssessment (i.e. 2 per minute).

Efficcy of Novel Are-Repellent Aerosol Formultion Aginst Mosquitoes nd Flies 155 Non-bsorbent (glzed tiles) nd bsorbent (unpinted plywood) substrtes were used to replicte typicl domestic outdoor surfces. These were spryed with one or other of the coded formultions detiled bove. Glzed tiles nd plywood for the 4 nd 6 hour ssessments were ged under mbient conditions (27-35ºC) prior to the study commencing, while substrtes for the 0 nd 2 hour ssessments were ged during the study. These were plced djcent to the test subjects, 10 minutes prior to the strt of ech ssessment. For ech evlution, 5-minute count of mosquitoes lnding for more thn 1 second ws conducted. Lndings were defined s the mosquito resting on the subject, not merely touching them. After mosquitoes hd settled, movement of the subject disturbed them. Musc Fly Study Fly species were the House fly, Musc domestic (pproximtely 80% of the popultion) nd the Bush fly, Musc vetustissim (pproximtely 20% of the popultion). A lrge milking shed locted on diry frm on Wlkers Creek Rod, Bell, Drling Downs District, Queenslnd, Austrli ws selected s the study site. The milking shed provided protection from strong winds nd rin, but ws open on one side nd therefore exposed to the elements (sun nd breeze), mking it suitble for testing outdoor pest control products. This shed hd very high resident fly popultion, which ws ttrcted to the cows (which were milked there twice dily) nd to the mny cowpts (cow feces) deposited round the shed. Humn ssessors were used to count the flies. There were 5 ssessors, 4 of whom evluted the product, while the other counted the untreted control. All ssessors st on folding cmping chirs during the pre-count nd evlution periods. To void experimentl bis, the control ws different subject on every dy of the study. Assessors st minimum of 5m prt from one nother, with the control ssessor sited t one end of the test re to minimize eril contmintion from the treted subjects. The study ws commenced when flies were first noticed ech morning (pproximtely 9 m). All ssessors simultneously conducted pre-count of 5 minutes durtion, counting ll fly lndings of greter thn one second within their ssessment re (n re with n outer perimeter 1m from the spryed surfces plced round the seted ssessor). Product evlution ws then strted if ech subject recorded minimum of 10 lndings during the 5 minute ssessment (i.e. 2 per minute). Non-bsorbent (glzed tiles) nd bsorbent substrtes (unpinted plywood) were used to replicte typicl domestic outdoor surfces. These were spryed with one or other of the coded formultions detiled bove. All glzed tiles nd plywood were ged in situ during the study under mbient conditions (14-26ºC). These test substrtes were exposed to sunlight nd wind during the study, to replicte domestic outdoor conditions. Substrtes were plced djcent to the test subjects, 10 minutes prior to the strt of ech ssessment. For ech evlution, 5 minute count of flies lnding for more thn 1 second ws conducted. After the flies hd settled, movement of the ssessor ws then used to disturbed them. Clcultion of Percentge Repellency The following formul ws used to clculte the percentge repellency for ech : (1 C 0 /C 1 x T 1 /T 0 ) x 100 = Percentge Repellency C 0 = Control subject count for pre- ssessment C 1 = Control subject count t given ssessment time T 0 = Test subject count for pre- ssessment T 1 = Test subject count t sme given ssessment time

156 A.m. Scrivener, B.. Peters, And P.f. Miller Dt Anlysis The dt were nlysed by nlysis of vrince (ANOVA) generl liner model using SPSS for Windows TM Version 20 (SPSS Inc. 2011). The ssumption of norml distribution ws checked using P-P plot nd homogeneity of vrince using Levene s test of equlity of error vrinces. RESULTS AND DISCUSSION Mosquito Repellency Tble 1 gives the percentge repellency of ech t ech of the time points during the study, s well s the verge repellency over the six-hour ssessment period. Lnding inhibition using glzed tile substrte ws 93.6% for nd 96.3% for, verged over the 6 hour study. For plywood, the corresponding figures were 80.4% nd 80.0%, respectively. The men numbers of mosquito lndings t vrious times on glzed tile substrte re presented in Figure 1. Tble 2 shows sttisticl nlysis of the men number mosquito lndings on test subjects t vrious time point - on the sme substrte. These dt show no significnt differences between the two s t most time points -. The men number of mosquito lndings t vrious times on treted plywood substrte is presented in Figure 2. Tble 3 shows sttisticl nlysis of the men number mosquito lndings on test subjects t vrious time point - on the sme substrte. These dt show no significnt differences between the two s t most time points -. The wether conditions previling during the mosquito study were: temperture: 24.0 31.4 C; reltive humidity: 74 89%; wind speed: 0 9 km/h; rinfll: 0.2 mm. Tble 1. Percentge mosquito lnding inhibition of test formultions on two surfces over 6 hours Formultion Surfce Ageing Time (h) Lnding Inhibition (%) Number of Replictes 0 81.4 4 Glzed Tiles 2 96.8 4 4 97.3 4 6 99.1 4 Averge 93.6 4 0 76.1 4 Plywood 2 95.4 4 4 58.2 4 6 91.9 4 Averge 80.4 4 0 98.4 4 Glzed Tiles 2 98.5 4 4 90.4 4 6 98.0 4 Averge 96.3 4 0 81.5 4 Plywood 2 86.2 4 4 59.8 4 6 92.3 4 Averge 80.0 4

Efficcy of Novel Are-Repellent Aerosol Formultion Aginst Mosquitoes nd Flies 157 Musc Fly Repellency Tble 4 gives the percentge repellency of ech t ech of the time points during the study, s well s the verge repellency over the six-hour ssessment period. Lnding inhibition using glzed tile substrte ws 85.1% for nd 81.4% for, verged over the 6 hour study. For plywood, the corresponding figures were 80.9% nd 79.0%, respectively. The men numbers of fly lndings on glzed tile substrte t vrious times re presented in Figure 3. Tble 5 shows sttisticl nlysis of the men number fly lndings on test subjects t vrious time point - on the sme substrte. These dt show no significnt differences between the two s t most time points -. Tble 2. Summry of the sttisticl nlysis for the verge number of mosquito lndings t vrious times in the presence of treted glzed tile surfce Tretment Pre 1 0 hours 2 hours 4 hours 6 hours 42.25 * (12.39) 2.25 * (0.63) b** 0.25 * (0.25) 0.50 * (0.29) 0.50 * (0.50) 31.00 (5.99) 0.25 (0.25) 0.25 (0.25) 1.50 (0.65) 0.50 (0.50) * Vlues re men number of mosquito lndings (n=4). Stndrd errors of men re in prentheses. ** Tretments with sme letter do not differ significntly from ech other. 1 Dt were ln(x+1) trnsformed prior to nlysis. The men numbers of fly lndings on treted plywood substrte t vrious times re presented in Figure 4. Tble 6 shows sttisticl nlysis of the men number fly lndings on test subjects t vrious time point - on the sme substrte. These dt show no significnt differences between the two s t most time points -. The wether conditions previling during the fly study were: temperture: 13.7 26.1 C; reltive humidity: 21 62%; wind speed: 0 19 km/h; rinfll: 0 mm. Tble 3. Summry of sttisticl nlysis for verge number of mosquito lndings t vrious times in the presence of treted plywood surfce Tretment Pre 1 0 hours 1 2 hours 4 hours 6 hours 31.00 * (5.99) 12.25 * (4.11) b** 1.00 * (0.41) 5.75 * (0.85) 1.50 * (0.87) 31.50 (6.89) 2.25 (0.48) 2.00 (0.71) 7.00 (0.71) 1.50 (0.96) * Vlues re the men number of mosquito lndings (n=4). Stndrd errors of men re given in prentheses.** Tretments with the sme letter do not differ significntly from ech other. 1 Dt were ln(x+1) trnsformed prior to nlysis.

158 A.m. Scrivener, B.. Peters, And P.f. Miller Tble 4. Percentge fly lnding inhibition of test formultions on two surfces over 6 hours Formultion Surfce Ageing Time (h) Lnding Inhibition (%) Number of Replictes 0 81.7 4 Glzed Tiles 2 88.0 4 4 87.7 4 6 80.9 4 Averge 85.1 4 0 79.7 4 Plywood 2 85.0 4 4 82.8 4 6 76.2 4 Averge 80.9 4 0 65.2 4 Glzed Tiles 2 80.1 4 4 90.0 4 6 90.4 4 Averge 81.4 4 0 66.9 4 Plywood 2 81.1 4 4 91.2 4 6 76.9 4 Averge 79.0 4 Tble 5. Summry of the sttisticl nlysis for the verge number of fly lndings t vrious times in the presence of treted glzed tile surfce Tretment Pre 0 hours 2 hours 4 hours 6 hours 23.50 * (2.26) 7.00 * (3.00) 2.50 * (0.65) 5.50 * (0.87) 4.25 * (1.70) 51.00 (12.80) 11.50 (4.09) 6.75 (1.32) b 3.25 (0.63) 3.25 (0.95) * Vlues re men number of fly lndings (n=4). Stndrd errors of men re given in prentheses. ** Tretments with the sme letter do not differ significntly from ech other.

Efficcy of Novel Are-Repellent Aerosol Formultion Aginst Mosquitoes nd Flies 159 Tble 6. Summry of the sttisticl nlysis for verge number of fly lndings t vrious times in the presence of treted pinted plywood surfce Tretment Pre 0 hours 1 2 hours 4 hours 6 hours 23.50 * (2.26) 4.50 * (1.04) 4.50 * (1.71) 7.00 * (2.52) 6.25 * (2.02) 51.00 (12.80) 17.50 (5.72) b 4.00 (1.23) 3.25 (1.38) 7.75 (3.61) * Vlues re the men number of fly lndings (n=4). Stndrd errors of men re given in prentheses. ** Tretments with sme letter do not differ significntly. 1 Dt were ln(x+1) trnsformed prior to nlysis. Figure 1. The verge number of mosquito lndings t vrious times in the presence of treted glzed tile surfce (brs) with their stndrd errors of men Figure 2. The verge number of mosquito lndings t vrious times in the presence of treted plywood surfce (brs) with their stndrd errors of men

160 A.m. Scrivener, B.. Peters, And P.f. Miller Figure 3. The verge number of fly lndings t vrious times in the presence of treted glzed tile (brs) with stndrd errors of men Figure 4. Averge number of fly lndings t times in the presence of treted plywood (brs) with stndrd errors of men. CONCLUSIONS The mosquito nd fly field studies showed tht there were no significnt differences in efficcy between the high ctive rte nd the low ctive rte. Both formultions provided effective protection from biting mosquitoes nd nuisnce flies on both bsorbent nd non-bsorbent substrtes. The low ctive rte cn be used, without compromising efficcy. A spryed surfce re of 1m 2 ws sufficient to provide continuous protection for t lest 6 hours ginst biting mosquitoes, 2m 2 ws required for dequte fly protection. This product functions by vpour ction from spryed substrte, nd is dependent on mbient conditions for emntion nd consequently for mosquito nd fly efficcy. The product remined effective over rnge temperture nd humidity nd both still nd windy conditions. The product is suitble for use in repelling flying nuisnce insects in domestic environments. REFERENCES CITED Debboun, M., S.P. Frnces, nd D. Strickmn. 2007. (eds.). Insect Repellents: Principles Methods nd Uses. London: CRC Press. 495pp. Gerozisis, J., P. Hdlington, nd I. Stunton. 2008. Urbn Pest Mngement in Austrli. Sydney: UNSW Press. 326pp.

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