Attraction of Mosquitoes to Diethyl Methylbenzamide

Snprnruspn 1990 A'rrnlcrlor,l on MosQurtoEs To REPELLENTS 469

ATTRACTION OF MOSQUITOESTO DIETHYL METHYLBENZAMIDE AND ETHYL HEXANEDIOL1

Z. A. MEHR,, L. C. RUTLEDGE, M. D. BUESCHER,3 RAJ K. GUPTAN IUO M. M. ZAKARIA

Lettennan Army Institute of Research,Presidin of san Francisco,cA 94129-6800

ABSTRACT' Studiesby prior workers have shown that insect repellen1gcan act as attractants when pr;;;t;l;;;oncentrations, depositsor r-esidues.In thc present study deet and etlvl hexanediolwere [".t.a i" 2-fold serial dosesfromi.g x 10-e to 1.6 x 10-2 mgf cm2on the forearms of volunteers against ,-Ji1;ia li"inaii albimanus,Aed.es aegypti andAe. taenioihynchus.Both_compounds were significantlv repellent at the high end of the dose ringe, as expected.Neither was significantly attractant to_42. ot6i^"i* in low d5ses.However, deet wasiignificantly attractant tn Ae. aegyptiin the doserapge 7.6 x i65;; ii t lb-,;;/;t;"J to'Ae. tocnioriynchus ii the doseranges 1.9 x-10-e to 3.1 x 10-8-mglcm2 and 2.0 i tg-u to 2.b"j<10-n mg/cm2. Ethyl heianediol was significantly attractantto Ae. taenio,rlry*Jr i" ifr" aor" runge 1.g x 10-t t;6.2 x 10-5-mg/cm2.Based on these results and prior work of V. G. Dethier and C. N. E. Rluscoe,a model sequenceof the effects of chemicalson insects with increasing dose-was J"*np"a.lt *"s concluded thai the labels of commercial repellents,should be amended to include instruitions to wash off or reapply the repellent when it is no longer effective.

INTRODUCTION al. (1977)found that rebemid on clothing and l% deet on the skin were attractant to Coquil- Several investigators have reported that re- lettidia richiardii (Ficalbi) and concluded that pellents can act as attractants when present as repellents should be washed off the skin when low concentrations,deposits or residues.In the their repellent action ceases. Iaboratory Hocking (1961) found that vapors of In an earlier study we observed attractancy at (butoxypropanediol butoxy polypropyleneglycol low dosesand repellency at high dosesin labo- polymer) were attractant to Aedes aegypti ratory trials of 2 cyclic analogs of lactic acid, (Linn.) in a T-tube olfactometer. Kost et al. methyl-6-pentyl-1 -cyclohexene- 1 -carboxylate (N,N-di- (1971) reported that vapors of deet and 4-butyl-2,3-morpholinedione, against Ae. (N- ethyl-3-methylbenzamide)and benzimine aegypti (Skinner et al. 1980).The present study benzoylhexamethylenimine)were attractant to was conducted to determine the attractancy or Ae. aegypti at low concentrations, and Potapov repellencyofdeet and ethyl hexanediol (2-ethyl- et al. (1977)reported similar effects for rebemid l,3-hexanedioll for Anophelesalbimanus Wied., (N,N-diethylbenzamide) and repellent P-633 Ae. aegypti, and Aedes taeniorhynchla (Wied.) (cyclopentanone-2-carboxylicacid) against Ae. over a wide range of doses.A preliminary report aegypti. was given by Mehr and Rutledge (1985). In the field Dubitskii (1966)found that vapors of dimethyl phthalate, repudin (composition not MATERIALS and METHODS grven), benzimine and deet were attractant to Anophclzs hyrcanus (Pallas), Aedes cinereus Test insects:Three laboratory coloniesof mos- Meigen, Aedes uexans (Meigen), Aedes caspius quitoes were used in the study: An. albimanus (Pallas) and Cul,exnodestu's Ficalbi. Potopov et and Ae. taeniorhynchus obtained from Carl E. Schreck, Insects Affecting Man and Animals 1The opinions and assertionscontained herein are ResearchLaboratory, U.S. Department of Ag- the private views of the authors and should not be riculture, Gainesville, Florida, and Ae. aegypti construed as official or as reflecting the views of the obtained from Abdul A. Khan, University of the Department of De- Department of the Army or California at San Francisco.Larvae were reared fense. Use of a trade name does not indicate official "Q catfish food (Con- endorsement or approval of the use of the product. at27 on a diet of floating Human subjectsparticipating in this study gave free tinental Grain, Chicago,IL). Adults were main- and informed voluntary consent,and the investigators tained under a L2:L2h photoperiod at 27"C and adhered to Army Regulation 70-25 and U.S. Army 807oRH on white rabbits and 10% sucrose so- Medical Researchand Development Command Reg- lution. Tests were conductedwith 5- to 15-day- ulation 70-25on the use of volunteers in research. old nulliparous females. 2 Current address:1Oth Medical Laboratory, United Test material,s:Test materials were technical States Army, Europe, APO New York, NY 09180- grade deet (Mclaughlin Gormley King Com- 3619. pany, Minneapolis, MN) and technical grade 3 address:686 CounselorsWay, Williams- Current (Eastman Chemicals, burg, VA 23185-4059. ethyl hexanediol Organic a Current address:U.S. Army Biomedical Research Rochester,NY). Materials were tested in 2-fold and Development Laboratory, Fort Detrick, MD serial dilutions in ethanol. Dilutions were cal- 21701-5010. culatedto provide dosesof 1.9 x 10-eto 1.6 x 470 Jounxer,oF THEAunnrceN Moseurro ColrRol AssocnuoN Vol.6, No.3

l0-2 mgf cm2of active ingredient when 0.02b ml range were either negative (attractancy) or pos- of solution was applied to a 6.6 cm2test area. itive (repellency); observed values of percent Test subjects;Five volunteers (4 male and 1 atftactancyfrepellency in the repellent range female) and 6 alternates (5 male and 1 female) were always positive (repellency). participated in the study. Three statistical tests were performed on the Test methads.'A method of the American So- values of percent attractancy/repellency ob- ciety for Testing and Materials (1g83) was served within the dilute range: 1) The t test adaptedfor use in the study.Five 2.g-cmdiam- (Steel and Torrie 1980)was performed to deter- (6.6 eter cm2)circular test areaswere imprinted mine if the mean percent attractancyf repellency on the flexor region of the forearm of a voiunteer within the dilute range was significantly less with a stamp and inking pad. The 5 test areas than zero (i.e., negative), indicating signifrcant were treated at random with 0.025ml of 4 serial attraction. 2) The numbers of attractant (nega- dilutions of the test repellent and a control tive) and repellent (positive) values observed (ethanol) using a micropipet and a glassspread- within the dilute range were compared with ing rod. After 5 min a 4 x b x 18 cm clear acrvlic tables of the binomial distribution (Beyer 1968) plastic test cage containing 15 mosquitoes was to determine if the occunence of attractant val- bound to the forearm with l-inch Velcro tape, ues was significantly more frequent than the and a slide was withdrawn to exposethe 5 test occurrenceof repellent values. 3) The runs test areasthrough matching holes in the floor ofthe (Beyer 1968)was performed to determine if the cage.The number of mosquitoesbiting in each sequenceof attractant and repellent values was of the 5 test areaswas recordedat the end of 90 random within the dilute range or if the attrac- sec. The mosquitoeswere then narcotized with tant (negative) and repellent (positive) values a jet of carbon dioxide, the slide was replaced were significantly clustered. The 5% error rate and the cagewas removed. was employedin all tests of significance. New mosquitoeswere used in each 90-sec trial. The initial range of dosesapplied (2.0 x RESULTS 10-3,4.0 x 10-3,8.0 x 10-3and 1.6 x l0-2 mg/ cmt) was progressively extended to lower and An. albimanus: A total of 2,8g5 bites were lower levels in successivetrials. A minimum of recordedin 278 single-cagetrials ofdeet against 8 replications on at least 2 volunteers was per- An. albimanus for an overall mean of 10.4 bites formed on each range of dosestested. per trial. The dilute rangewas 1.9 x 10-sto 2.0 Data analysis:The percent attractancy or re- x 10-3 mgfcm2(Fig. 1). Neither the f test, bi- pellency of the test material for a given species nomial probability nor runs test was statistically at a given dosewas calculated as: significant(Tables 1-3). A total of 1,745 bites were recorded in 144 Percent attr actancyf repellency single-cagetrials of ethyl hexanediol againstAn. total no. bites on treatment ahimanus for an overall mean of 72.1bites per :100- x 100 total no. bites on control trial. The dilute range was 1.9 x 10-e to 2.0 x 10-3mg/cm2 (Fig. 1).Neither the t test,binomial This value expressespercent attractancy/repel- probability, nor runs test was statistically sig- Iency in terms ofthe concurrent control to adjust nificant (Tables1-3). for variation due to differencesamong test sub- Ae. aeg5pti: A total of 4,307 bites were re- jects and the date and time of testing. It is corded in 297 single-cagetrials of deet against negativewhen more bites occur on the treatment Ae. oegypti for an overall mean of 14.5bites per than on the control (attractancy) and positive trial. The dilute range was 7.6 x 10-e b 2.5 x when more bites occur on the control than on l}-a mgfcm2(Fig.2). (The 2lowest dosesof the the treatment (repellency). standardrange, 1.9 x 10-eand 3.8 X 10-emg/ Since deet and ethyl hexanediol were known cm', werenot testedin this case.)Both the t test to be repellentto An. ahimanus, Ae. aegypti and, and the binomial probability were statistically Ae. taeniorhynchus at the highest doses used significant (Tables 1 and 2). The mean percent (Rutledge et al. 1983), the total range of doses attractancy/repellency within the dilute range tested (1.9 x 10-s to 1.6 x 10-2 mg/cm2) was was -5.6% (Table 1). The largest negative (at- divided into a dilute range and a repellent range tractant) value observedwas -33% at 3.1 x 10-5 for analysis.To be conservative,the dilute range mgf cm2(Fig. 2). Twelve of the 16 atttactancyf was defined to include all dosesup to and in- repellency values observed in the dilute range cluding the dose next higher than the highest were negative (attractant) (Table 2). The runs observed attractant dose (Figs. 1-3). The re- test was not statistically significant (Table 3). pellent range was defined to include all higher A total of 5,747 bites were recorded in 406 doses. By these rules, the observed values of single-cagetrials of ethyl hexanediolagainst Ae. percent atbactancy/repellency in the dilute aegypti for an overall mean of 14.2 bites per 477 SEPTEMBER1990 ArtRLctlolt or MosQUITonsro Rspnr,r,eNts

range was 1'9 x 10-'to 2.0 x (Tables 1 and 3). The meanpercent attractancy/ trial. The dilute -12.5V9 10-3mg/cm2 (Fig. 2). Neither the t test,binomial repellency within the dilute range was probability nor runs test was statistically signif- (Table 1). The largest negative (attractant) val- icant (Tables1-3). Ae. taeniorhynchus:A total of 2,011bites were Table2. Datafor the binomialprobability test of recordedin 208 single-cagetrials of deet against frequencies of attractant and repellent observations Ae. tseniorhynchus for an overall mean of 9.7 (Figs. 1-3) in tests of deet and ethyl hexanediol in bites per trial. The dilute rangewas 1.9 x 10-e the dilute dose-range. P is the exact probability of to 5.0 x !0-a mgfcm2(Fig. 3). Both the t test observing an equal or Iarger number of attractant = and the runs test were statistically significant values if d 0.5 (Beyer 1968). Number of observations Attractant Repellent

An. aLbimanus Deet 11 10 0.50 Ethyl hexanediol I4 7 0.09 Ae. aegypti Deet 12 4 0.04 o Ethyl hexanediol 7 L4 0.96 ov- Bepellent o Ae.taeniorhynchus o Range Deet 13 6 0.08 o -100 Ethyl hexanediol 11 b U.I I

- tnn Table 3. Data for the runs tests of the sequence of o attractant and repellent values (Figs. 1-3) observed o for deet and ethyl hexanediol in the dilute dose- =(n range. P is the exact probability of occurrence of an (Beyer a equal or smaller number of runs 1968). o Number of runs Observed Expected P

An. albimaruts Deet 11 11.5 0.50 Ethyl hexanediol 11 10.3 0.72

'-' q q q q : : q q ol q c q e e - q q q q q q q q q Ae. degyptl F otsFOoFNl6NoFFO@FNOFN*oF -+ -+ Deet o 7.0 0.34 lO'9 tO-8 lO-7 10-6 to-s rO-4 10-S 1O-2 Ethyl hexanediol 11 10.3 0.72 Dose(mg/cm2) Ae taeniarhynchus Fig. 1. Percent repellency/attractancy of serial Deet A 9.2 0.01 doses of deet and ethyl hexanediol in tests against Ethyl hexanediol 10 8.8 0.82 Anophelesalbimanus.

Table 1. Data for the t test of observedvalues (Figs. 1-3) for attractancy and repellencyof deet and ethyl hexanediolin the dilute range.P is the probability of a smaller value of t (sign considered)if p = 0 (Fisher and Yates 1963).

Number of Standard observations Mean error t

An. aLbinanw Deet 2I -1.000 4.274 -0.234 0.44 Ethyl hexanediol 27 -5.476 3.569 -1.534 0.07 Ae. apgpti Deet 16 -5.562 3.036 -1.832 0.04 Ethyl hexanediol 2t +3.762 2.t31 +1.765 0.95 Ae. taeniorhynchus Deet 19 -12.474 5.445 -2.297 0.02 Ethyl hexanediol 1n -15.706 7.223 -2.t74 0.02 472 JounNal oF THE AunntcaNMoseurro Cournor,AssocrATroN Vor..6, No. 3

ues observedwere -45% at 1.5 x 10-omslcm. -67Vo and, at L.2 x 10-a mg/cm2 (Fig. B)l The longest runs of negative (atfractant) values observed were a run of 5 consecutive negative valuesfrom 1.9x 10-eto 8.1 x 10-8mg/cri2 and a run of 8 consecutivenegative values from 2.0 x 10-Gto 2.5 x t}-a mg/im2 (Fig. g). The bino- n llial probability was not statistically sigaificant (Table 2). A total of 2,344 bites were recordedin 281 o single-cagetrials of ethyl hexanediolagainst Ae. o o taeniorhynchusfor an overall mean of 10.1bites G per trial. The dilute rangewas 1.9 x 10-etu 1.2 o -100 x 10-nmgf cm2(Fig. 3). The t test was statisti- > 100 cally significant (Table 1). The percent o mean c atftactancy-frepellencywithin the o dilute range o was -I5.7%. The largest negative (attractant) 6 value observedwas -95% at 7.6 x 10-s mg/cm2 i50 (Fig. 3). Neither the binomial probability o nor o the runs test was statistically (Tables o significant Cn 2 and 3).

DISCUSSION The smallest dose of deet used in the studv was 1.9 x 10-e mgf cm2(Figs. 1-B). Convertin! 3 3ie:;;:i 3BR gf : ;S:RB:RgB: -<_-<_ .|o-4 r00 tO-9 tO-8 t0-7 tO-6 tO-S 1o-S tO-2 Dose(mg/cm2)

, Fig. 3. Percent repellency/attractancy of serial doses of deet and ethyl hexanediol in tests against Aedes taeniorhynchus.

1.9 x 10-e mgf cm" to gf cm2and dividing by the + molecularweight of deet(191.3) gives 9.g x 10-15 o moles/cm2.Multiplying this figure by Avogad- ro's number (6.23x gives o 10'3) 6.2 x 10emole- o o cules/cm2of skin. The analogouscalculation for ethyl gives o -100 hexanediol 8.1 x 10emolecules/cm2 of skin. These figures distinguish our study sharply from that of Davenas et al. (1988) in

G which anti-immunoglobulin E was reported to o 6 induce the releaseof histamine by human poly- morphonuclearbasophils at dilutions up to 1 X 10120.At such high dilutions there is virtually no o possibility that any anti-immunoglobulin E re- o 0-0 mained in the reagentfluid (Maddox et al. 1988). In the present study low dosesof deet were attractant to A e. aegypti and Ae. toenbrlrynchtts but not to An. ahimanus (Tables 1-3). Since cornpletionofthe study the sameeffect has been obsewed in laboratory trials of controlled-re- gR:;;:i?3R3:: Ieaseformulations of deet against both Ae. ae- 3 ;8:R3:R33: gypti e€+<+ and Ae. taeniorhynchus (Gupta and Rut- 10-9 to-8 1o-7 ro-€ ro-s ro-4 lo-s io-2 ledge 1989).These results confirm the report of Dose(mg/cm2) Kost et al. (1971) that low concentrationsof Fig. 2. Percent repellency/attractancy of serial deet are attractant to Ae. aegypti and call into doses of deet and ethyl hexanediol in tests against question the report ofPotapov et al. (1977)that Aedes aegypti. l% deet is repellent to Ae. aegypti for up to Z SEPTEMBER1990 ArrnectroN on MosQutronsto Rnpnr-lnNrs 473

days after application to the forearm. We have acid was an oviposition attractant for Culiseta found no prior report of the attraction of Ae. incidens (Thomson) at low concentrations and taeniorhynchusto deet. Attraction of Anopheles an oviposition repellent at high concentrations. farauti Laveran, Aedes hochi (Doenitz), Aedes Although carbon dioxide is a potent attractant carrnenti Edwards, and Culex annulirostris for mosquitoesin nature (Reeves1953), its ef- Skuseto residuesof deet and permethrin in the fects in laboratory experiments have been vari- field was reported earlier (Gupta et al. 1987). able (Gillies 1980).Willis (1947)and Willis and In the present study low dosesof ethyl hex- Roth (1952)demonstrated that it can be attrac- anediol were attractant to Ae. taeniorhynchus tant, inert or repellent, depending on the kind but not to An. albimanus or Ae. aegypti (Tables of olfactometer used. Their data for female Ae. 1-3). We have found no prior report of the aegypti in a small-cageolfactometer (Table 1 of attraction of mosquitoesto ethyl hexanediol. Willis and Roth 1952)indicate that the repellent Prior workers have reported that low concen- effect increaseswith increasing concentrations trations of deet and other repellents were at- (0.1-50%)of carbondioxide. tractant to other Diptera. Deet was attractant Nakagawa et al. (1971) reported that high to the sand fly Phlebotomus papatasi Scopoli concentrations of trimedlure were repellent to (Psychodidae)(Sabitov 1985). Deet, dimethyl the Mediteranean fruit fly Ceratitis capita phthalate, benzimine, and repudin were attrac- (Wied.) (Tephritidae).Barrows (1907)reported tant to the biting midges Culicoidespulicaris that 8% ethyl acetate was attractant to the Linn. and Culicoidespuncticollis Becker (Cera- vinegar fly Drosophila ampelophiloLoew (Dip- topogonidae) (Dubitskii 1966). Sulfobenzamide tera: Drosophilidae) at a distance,but repellent repellentswere attractant to the black flies Gnus at close range. In this case,it would seem that cholodkouskiy(Rubzov) and Simulium galera- the gradient in spaceproduced by diffusion and turn Edwards (Simuliidae) and the horse flies convectionthrough increasingdistance would Hybomitra bimaculataMacquart and Hybomitra be equivalent to the gradeddoses in the present distinguenda Verrall (Tabanidae) (Potapov et study. Reed (1938) reported that solutions of al. 7977). Diallyl phthalate repellents were at- acetic acid above57o and of ethanol above257o tractant to the olive fruit fly Dacus oleae were repellent to Drosophila melanogasterMei- Gmelin (Diptera: Tephritidae) (Wright 1982). gen. Triethylamine hydrochloride was repellent Butoxy polypropylene glycol was attractant to to Hippelatescollusor (Townsend) (Chloropidae) the house f"ly Musca dornesticaLinn. (Diptera: (Mulla et al. 1976). Aliphatic aldehydes were Muscidae)(Hocking 1961).MGK Repellent 11 repellent to the black blow fly Phormia regina (1,5a,6,9,9a,9b-hexahydro-4a(4H)-dibenzofur- Meigen (Calliphoridae) (Dethier 1954a). Sec- ancarboxyaldehyde)was attractant to the stable ondary amyl mercaptan was repellent to Lu.cilia fly Stomoxyscalcitrans (Linn.) (Muscidae) (Yeo- sericata (Meigen) (Calliphoridae) (Hoskins and man and Warren 1970). Craig 1934). Ammonia, ethanol and isovaleral- On the other hand many materials that are dehydewere repellent to the housefly (Wieting normally thought of as attractants have been and Hoskins 1939, Dethier et al. L952,Dethier reported to be repellent at high concentrations.s 1954a). Smith et al. (1970) reported that lactic acid was Dethier (1954a)published a figure showing attractant to Ae. aegypti at concentrations nor- the successionof subliminal. attractant and re- mally present on the skin and in the breath but pellent effects of increasing concentrations of repellent at a higher (3.6 mg/cm') concentra- isovaleraldehyde on the house fly.? This se- tion.6 Kramer et al. (1980)reported that butyric quencecan be representedby the series + Attractant + Neutral > Repellent 5 Neutral A related topic is the case in which a material is "Neutral" attractant to one species but repellent to another. in which the term includes both the Thus, geraniol (3,7-dimethyl-2,6-octadien-1-ol),a subliminal and the transitional effects. In the well-known commercialattractant for the Jananese-Scara- present study this sequenceof effects was ob- beetle.Popil/io japonica Newman (Coleoptera: served in tests of deet against Ae. aegypti (Fig. baeidae),was repellent to Ae. aegypti in an in uitro blood-feedingtest system:ED56 : 9.931mg/cm2 (95% CL : 0.000,0.060); slope : -1.614 (SE : 0.163) skin, and its consequentconcentration in the air above (unpublished data, Letterman Army Institute of Re- the skin, is not affected by atmosphericpressure. search). ?Dethier reprinted this samefigure in his review of 6 Kostin (1984)reported that lactic acid was repel- the physiologyof olfaction in insects (Dethier 1954b), Ient to Ae. aegypti at low atmospheric pressure but stating that "For everychemically pure attractant thus attractant at high atmosphericpressure. It is not clear far tested,there can be found a concentration at which how this observationrelates to the present discussion. it becomesrepellent." He did not mention the con- According to the Iaw of partial pressures (Dalton's verse, that repellents may become attractant at Iow law) the rate of evaporation of lactic acid from the concentrations. 474 JouRNu, oF THE Arr,tpRtclNMoseurro CoNrnor, AssocurroN Vor,.6, No. 3

2) and of ethyl hexanediol against Ae. taenior- Dethier by the series hynchus(Fig. 3). (Neutral -> However, no statistically significant attrac- + Attractant)N > Neutral tant dosewas observedin tests of deet and ethyl Repellent, Toxic hexanediol against An. albinanus (Fig. 1) or in in which the term "Repellent" includes both tests ofethyl hexanediolagainstAe. aegypti(Fig. repellent and antifeedant effects. 2). Moreover, the runs test (Table 3) identified Rani and Osmani (1984) have confirmed this 2 distinct attractant doseranges (1.9 x 10-eto model sequenceof effects in tests of methoxy- 3.1 x 10-8mgf cm2 and 2.0 x 10-6to 2.5 x 10-a chlor and cyphenothrin against the house fly. mg/cmz) in the caseof deet against Ae. taenior- Even so, the model requires further researchfor hynchus(Fig. 3). These variations of Dethier's full verification and should be regardedas ten- sequencecan be representedby the series ative. For example,Riha et al. (1986)reported that the toxic effectsof 0.05%permethrin ap- (Neutral + Attractant)y + Neutral + Repellent plied to horsesas a spraylasted Ionger than the repellent effects.This is contrary to expectation in which N can be 0, I or 2. from the model sequenceand contrary to the Potapov et al. (1977) reported that 40% deet observed sequencefor permethrin-treated cot- applied to the forearm was alternately repellent ton and cotton/nylon fabrics (Gupta et al. 1989). and attractant to Ae. comrnunisfor 3 days after application.s The results obtained in tests of deet against Ae. taeniorhynchusin the present CONCLUSIONS study (Table 3 and Fig. 3) could also be inter- Accordingto Sabitov (1985)instructions for preted in terms of alternating repellent and at- use of repellentsin the Soviet Union include tractant effects. However, we have interpreted information on the attractant effectsofrepellent the data in terms of alternating neutral and residueson the skin. From results of the present attractant effects becausethe repellent values study, we concludethat the U.S. Environmental were not statistically significant within the di- Protection Agency should require this informa- lute range. tion in the "Directions for IJse" section of re- A number of thiocyanate, organophosphate, pellent labels, along with instructions to wash chlorinated hydrocarbon and pyrethroid insec- off or reapply the repellent when it is no longer ticides, including permethrin, have been shown effective.In this connectionit may be noted that to be repellent to mosquitoesat sublethal doses, the Agencyhas recently issued a ConsumerBul- and a number of repellents, including deet and letin providing use precautions for deet based ethyl hexanediol, have been shown to be toxic on adverse reaction reports involving children to mosquitoes at high doses (Rutledge et al. (Anonymous 1989). The precautions recom- 1981).Potapov and Bogdanova(1974) reported mendedby the Agency include: "Do not reapply that 10-30%solutions of repellentsR-2 (benzoic or saturate. Wash treated skin with soap and acid diethylamide),R-31 (caproicacid diethyl- water after and betweenuses." amide),R-228 (m-toluic acid N-piperidylamide), R-320 (furan-2-carboxylic acid diethylamide) ACKNOWLEDGMENTS and R-386 (a-chloropropionic acid diethylamide) were more repellent than 40-50% solutions The volunteers and alternates received19,049 because of narcotic and toxic effects at the bites in the courseof the study. We thank J. L. higher concentrations. Inase,V. E. Meixsell,J. J. Newland,P. I. Plamp, Ruscoe(1977) published a figure showingthe H. G. Semey,E.A. Shipwashand R. A. Wirtz successionof toxic, repellent/antifeedant and for participation. subliminal effectsof progressivelyolder residues permethrin on the diamondback moth Plu- of REFERENCES CITED tella xylostello (Linn.) (Lepidoptera: Plutelli- dae).In this caseit would seemthat the gradient AmericanSociety for Testing and Materials.1983. in time produced by the decay and dissipation Standardtest methods for laboratorytesting of non- ofthe residueswould be equivalentto the graded commercialmosquito repellent formulations on the doses in the present study. Accordingly, Rus- skin. Americal Society for Testing and Materials coe'ssequence can be incorporatedinto that of StandardE95l-83. Anonymous. 1989. Deet use precautions issued by EPA: reactions, sensitivity noted. Pestic. Toxic I Potapovet al. (1977)interpreted this and related Chem.News 77(421:6-7. experiments (Potapov and Vladimirova 1970, Vladi- Barrows,W. M. 1907.The reactionsof the pomace mirova, 1969, 1970a, 1970b)primarily in terms of fly, Drosophila ampelophila Loew, to odorous sub- changesin ambient meterologicalconditions and the stances.J. Exp. Zool.4:515-537. physiologicalstate of the test insects during the test. Beyer,W. H. (ed.)1968. Handbook of tablesfor prob- SEPTEMBER1990 ATTRACTToNoF MoseulToEs ro REPELLENTS 475

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