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Original article
Repellent efficacy of DEET, Icaridin, and EBAAP against Ixodes ricinus and Ixodes scapularis nymphs (Acari, Ixodidae)夽 Kerstin Büchel a , Juliane Bendin a , Amina Gharbi a , Sibylle Rahlenbeck b , Hans Dautel a,∗ a b
IS Insect Services GmbH, Haderslebener Str. 9, 12163 Berlin, Germany Umweltbundesamt, P.O. Box 33 00 22, 14191 Berlin, Germany
a r t i c l e
i n f o
Article history: Received 20 June 2014 Received in revised form 25 March 2015 Accepted 26 March 2015 Available online xxx Keywords: Repellent Ixodes ricinus Ixodes scapularis DEET EBAAP Icaridin
a b s t r a c t Repellent efficacy of 10% EBAAP (3-[N-butyl-N-acetyl]-aminopropionic acid, ethyl ester) and 10% Icaridin ((2-(2-hydroxyethyl)-1-piperidinecarboxylic acid 1-methylpropyl ester)) were evaluated against 20% DEET (N,N-diethyl-3-methylbenzamide) in human subject trials against ticks. Responses of host-seeking nymphs of the European castor bean tick (Ixodes ricinus L.; Acari: Ixodidae) and the North American blacklegged tick (I. scapularis Say; Acari: Ixodidae) were compared. Tests were carried out according to the US-EPA standard protocol with ethanolic solutions of the active ingredients of repellents being applied to the forearm of 10 volunteers. The upward movement of ticks was monitored until repellent failure taking up to 12.5 h. Application of 20% DEET resulted in median complete protection times (CPT; Kaplan–Meier median) between 4 and 4.5 h, while 10% EBAAP yielded CPTs of 3.5–4 h. No significant differences were found between the efficacies of two repellents nor between the two species tested. The median of the CPT of a 10% Icaridin solution was 5 h in nymphs of I. scapularis, but 8 h in those of I. ricinus (P < 0.01). Based on these studies, EBAAP and Icaridin are efficacious alternatives to DEET in their repellent activity against nymphs of the two Ixodes ticks with Icaridin demonstrating particularly promising results against I. ricinus. Future research should investigate whether similar results occur when adult Ixodes ticks or other tick species are tested. © 2015 Elsevier GmbH. All rights reserved.
Introduction Ticks are obligate blood-feeders known as vectors of a variety of infectious agents (Piesman and Eisen, 2008). The most frequently reported vector-borne diseases in the temperate zone of the Northern hemisphere are in fact related to ticks: in Europe and the United States, the number of tick-related zoonoses in humans not only exceeds those transmitted by mosquitoes, but the former ones often result in diseases that are more severe than those transmitted by the latter. In Europe, the sheep or castor bean tick, Ixodes ricinus is the most abundant species, infesting a wide range of hosts including mammals, birds, and even reptiles. The deer or blacklegged tick, I. scapularis is found in the eastern part and the Midwest of the United States currently expanding its occurrence further to the north into Canada. Humans are frequently bitten by both Ixodes
夽 This paper does not necessarily reflect the opinion or the policies of the German Federal Environment Agency. ∗ Corresponding author. Tel.: +49 3082096555; fax: +49 3082096555. E-mail address: [email protected] (H. Dautel).
species, whereby most tick bites stem from the nymphal stage (Wilhelmsson et al., 2010; Falco et al., 1996). Repellents are usually the primary method of personal protection against tick bites (Bissinger and Roe, 2010; Piesman and Eisen, 2008). While defining the ‘repelling’ action is straightforward insofar as flying insects are concerned, this cannot be defined as easily when it comes to crawling arthropods like ticks. In ambushing ticks, the process of questing, clinging to, and locating a suitable feeding site on a host is much more complex lasting for several hours to weeks. Each step in this interaction can be disrupted, but only certain steps would be defined as leading to a repellency ‘sensu stricto’, namely causing the tick to move away (Halos et al., 2012) leaving its host or avoiding it in the first place. Repellency is usually attributed to the vapor phase of a ‘repellent’ compound applied to the hosts’ skin. While DEET is the most widely used active ingredient of repellents worldwide, some researchers have speculated if other substances, notably EBAAP (IR3535) and Icaridin might be similarly efficacious (Schreck et al., 1995). However, only few studies are known comparing these active ingredients on human skin, which is more suitable to determine an effective concentration than an in vitro assay (Soares et al., 2010). While Pretorius et al. (2003) found ethanolic solutions of DEET
http://dx.doi.org/10.1016/j.ttbdis.2015.03.019 1877-959X/© 2015 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Büchel, K., et al., Repellent efficacy of DEET, Icaridin, and EBAAP against Ixodes ricinus and Ixodes scapularis nymphs (Acari, Ixodidae). Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.03.019
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(20%) to be slightly more effective than Icaridin (20%), no such differences between DEET, EBAAP and Icaridin dissolved in ethanol were detected by Kröber et al. (2013). In tests with commercial products, the results of Semmler et al. (2011) suggest, that products containing Icaridin and EBAAP are more effective than such ones containing DEET. Carroll et al. (2010) in an assay with human subjects tested different formulations of DEET, EBAAP, and Icaridin against nymphs of Ambylomma americanum at concentrations in the range of 20–33%, typical for repellent products (Pages et al., 2014). As all of those formulations repelled >80% of the ticks throughout the 12-h test period, no discrimination between products with different active ingredient was possible. The aim of the present study was to directly compare both, the active ingredients DEET, EBAAP, and Icaridin, as well as the responses of I. ricinus and I. scapularis against these. We, therefore, investigated ethanolic solutions of the three repellents at discriminating doses (i.e. lower concentrations than usual) in a well-defined simulated use test with human subjects against nymphs of both tick species. Materials and methods Test products EBAAP (3-[N-butyl-N-acetyl] aminopropionicacid ethylester), was obtained from Merck KGaA (Darmstadt, Germany) (IR 3535® , 100% purity) and Icaridin (2-(2-hydroxyethyl)-1piperidinecarboxylic acid 1-methylpropyl ester) from Saltiago GmbH (Langenfeld, Germany) (Saltidin® , 97% purity). DEET (N,Ndiethyl-3-methylbenzamide) was obtained from Sigma–Aldrich GmbH (Steinheim, Germany) (97% purity). All repellents were stored at 4 ◦ C in complete darkness. Ethanol (≥98.9%, Sigma–Aldrich GmbH) was used to prepare 10% solutions of (v/v) of Icaridin and EBAAP, while DEET was tested as a 20% ethanolic solution. The latter usually serves as a positive control and standard for comparison as recommended in the guideline of U.S. EPA (2010). Ticks Unfed nymphs of I. ricinus and I. scapularis from laboratory colonies were used throughout all trials. I. ricinus constituted the F2 generation (age: 20–24 d post ecdysis) of a lab colony originating from wild ticks collected near Berlin, Germany. Nymphs were kept at 20 ◦ C and 90% RH (relative humidity) at a photoperiod of 16:8 L:D. I. scapularis was obtained from the U.S. Center for Veterinary Parasitology at Oklahoma State University, Stillwater, OK. After transport, nymphs were acclimated at 23 ◦ C, 90% RH and a photoperiod of 16:8 (L:D) for 4 weeks. The age of nymphs when tested was between 8 and 12 weeks post ecdysis. Nymphs were randomized and placed in 10 ml glass vials (10–15 ticks per vial) covered by gauze. Only ostensibly healthy and ‘active’ nymphs were used for the tests. To ascertain activity, only nymphs moving up the glass vial or staying at the top of it were included. In addition, locomotive activity of those chosen was tested prior to each trial (see below). Each tick was used only once.
had been reviewed and were in accordance with the standards of the Local Health Authority. For each repellent, 10 volunteers (5 males and 5 females) between 18 and 54 years of age were tested in a controlled laboratory setting. The volunteers were instructed to avoid smoking, drinking of caffeinated or alcoholic beverages and/or applying fragrance products on the day of testing. Before testing, volunteers washed their arms for 30 s with water and 3 ml unscented soap, rinsing them once for 1 min with tap water, then wiping the skin with soft tissue soaked with ethanol (70%) twice, followed by rinsing with water for 1 min. Control arms were cleansed in the same fashion. Arms were then allowed to dry. Test formulations were applied at 1.0 l cm−2 to the entire test area. This constitutes 60% of the amount recommended by U.S. EPA (1.67 l cm−2 ). After pretesting with the full standard amount, the dose was reduced in order to yield protection times below 10 h. The calculated amount of the test solution was applied to the volunteers’ forearm with a syringe onto four to five stripes and was spread as evenly as possible by two gloved finger tips. Exposure began 20 min after application of the test formulations. During testing, temperature and RH as measured were 22.9 ± 0.8 ◦ C and 56.4 ± 5.6%, respectively. Repellent and control treatments were randomly assigned to subjects on the first day. The volunteers cycled through each repellent and both Ixodes species so that each subject tested each repellent and each Ixodes species once. The border of the treated zone was marked by a pen with a dashed line around the arm and a second line was marked at a distance of 1 cm below. The volunteer sat with their arm held vertically, the finger tips or the palm touching a small table. A single active nymph was placed about 1 cm below the marked border on untreated skin with an artist’s paintbrush (Rotmarder size 1, Fredericus Rex, Erwitte, Germany). While I. ricinus nymphs readily moved upwards, most I. scapularis nymphs did not. Therefore, they were gently ‘guided’ upwards using a paintbrush as described by Carroll (2008). The nymph was then observed for a maximum of 3 min. Ticks that crossed the ring mark within 3 min and stayed in the treated area for a minimum of 60 s were regarded as ‘not repelled’ and removed. Ticks that did not crawl onto the treated skin as well as those that walked down to the wrist or dropped off were regarded as ‘repelled’ and removed. A new tick was then taken for the next test period. In such a way, 5 ticks were tested every 30 min (one test period) until CPT was reached, which took up to 12.5 h after test formulation application. Thus, a total of 100 ticks was tested per hour (with 10 volunteers) and per repellent with each tick species. Exposure for a volunteer ended as soon as two nymphs within the 30-min test period were not repelled (=confirmed crossing). Technical personnel monitored the practice and the scoring of the volunteers. Brushes were replaced with new ones every hour. Controls The untreated arm served as control. It was prepared in the same fashion as the test arm and was also marked. Nymphs that crossed into the marked, but untreated area 1 cm above the mark and stayed there for 1 min were considered as sufficiently locomotive and used for testing on the other arm within the next 30 min. Data analysis
Trials Repellent testing was carried out following the guideline of U.S. EPA (2010) which currently is the only internationally specified guideline available for authorization of repellents. According to this guideline, all subjects gave informed consent prior to participation in the trial. Informed consent documents and study trial protocols
The complete protection time (CPT) was defined as the time interval between the application of repellent and the first confirmed crossing (U.S. EPA, 2010). It was calculated by means of a Kaplan–Meier analysis (WHO, 2009). The data are presented as median CPT and its 95% confidence intervals and interquartile range (25–75%). Differences between the CPTs of the three
Please cite this article in press as: Büchel, K., et al., Repellent efficacy of DEET, Icaridin, and EBAAP against Ixodes ricinus and Ixodes scapularis nymphs (Acari, Ixodidae). Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.03.019
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Table 1 Complete protection times (minutes) (CPTs) evaluated with DEET, EBAAP, and Icaridin against I. ricinus and I. scapularis nymphs. Repellent
Tick species
CPT (median)
95% Confidence interval
IQR (25–75%)
P
DEET (20%) DEET (20%)
I. ricinus I. scapularis
240 270
194–286 240–300
150–300 225–315
a ab
EBAAP (10%) EBAAP (10%)
I. ricinus I. scapularis
240 210
178–302 182–238
150–285 135–225
a a
Icaridin (10%) Icaridin (10%)
I. ricinus I. scapularis
480 300
418–542 253–347
405–525 210–360
c b
IQR: interquartile range. Significance: CPTs denoted by the same letter are not significantly different from each other (P > 0.05).
Side effects No side effects, i.e. cutaneous itching or flushing were observed or reported by any of the volunteers throughout the entire test period.
1.0 0.9
EBAAP DEET
0.8
Proportion of volunteers
repellents were tested by a multiple-sample test in Statistica® v.7.1, an extension of Gehan’s generalized Wilcoxon test, Peto and Peto’s generalized Wilcoxon test and/or the log-rank test. Two treatments were compared by Cox’s F-Test. The Wilcoxon matched pairs test was used to compare the number of ticks that entered the treated skin area, walked back to untreated skin, fell off from treated skin, or were not repelled. To do so, the proportion of ticks displaying a certain behavior were summarized over the first five (DEET, EBAAP) and six test hours (Icaridin). All statistics were carried out using the Statistika® v. 7.1 software (StatSoft (Europe) GmbH, Hamburg, Germany).
Icaridin
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0
50
100
150
200
Results
300
All three compounds performed well in repelling the nymphs of the two Ixodes species. As shown in Table 1, median Kaplan–Meier CPTs were between 4 and 8 h in I. ricinus nymphs and 3.5–5 h in I scapularis nymphs. While application of EBAAP (10%) yielded similar median protection times of about 4–5 h as DEET (20%) in both species, 10% Icaridin resulted in longer protection with a CPT of 5 h in I. scapularis and 8 h in I. ricinus. Figs. 1 and 2 show the Kaplan–Meier curves for I. ricinus and I. scapularis, respectively. In I. ricinus, a significant difference between repellents was observed (chi2 = 17.21; DF = 2; P ≤ 0.001, Fig. 1) with the CPT of Icaridin differing from DEET and EBAAP (P ≤ 0.01 for 1.0 0.9
EBAAP DEET
0.8
Icaridin
0.7 0.6
400
450
500
DEET/Icaridin, P ≤ 0.001 EBAAP/Icaridin, respectively). In I. scapularis, also a significant difference between repellents was observed (Fig. 2; chi2 = 6.84; DF = 2; P ≤ 0.05) with EBAAP showing a shorter protection time than Icaridin (P ≤ 0.05), but being not different from DEET (P = 0.11). There was no significant difference between DEET and Icaridin (P = 0.1).
Table 2 Percentages (mean ± SE) of I. ricinus and I. scapularis ticks displaying specific behaviors during the first five (DEET, EBAAP) and six (Icaridin) hours. Displayed are percentages of ticks walking back, falling off or remaining on treated skin for 60 s after moving onto treated skin. Behavior of ticks
I. ricinus
DEET Walking onto treated skin [%] Walking back from treated skin [%] Falling off from treated skin [%] Remaining on treated skin [%]
59 80 8 12
± ± ± ±
5*** 4* 2 3*
20 52 7 41
± ± ± ±
4 7 3 8
EBAAP Walking onto treated skin [%] Walking back from treated skin [%] Falling off from treated skin [%] Remaining on treated skin [%]
71 71 18 12
± ± ± ±
5** 4** 3 3**
51 47 25 28
± ± ± ±
4 5 6 5
Icaridin Walking onto treated skin [%] Walking back from treated skin [%] Falling off from treated skin [%] Remaining on treated skin [%]
60 78 20 3
± ± ± ±
4*** 3*** 3 1***
26 40 19 41
± ± ± ±
3 5 4 5
0.5 0.4 0.3 0.2 0.1 0.0 0
350
Fig. 2. Kaplan–Meier plots for DEET, EBAAP, and Icaridin tested against I. scapularis nymphs on 10 volunteers.
Repellency
Proportion of volunteers
250
Time [min]
100
200
300
400
500
600
700
800
Time [min] Fig. 1. Kaplan–Meier plots for DEET, EBAAP, and Icaridin tested against I. ricinus nymphs on 10 volunteers.
I. scapularis
Asterisks indicate significant differences between I. ricinus and I. scapularis. * P < 0.05. ** P < 0.01. *** P < 0.001.
Please cite this article in press as: Büchel, K., et al., Repellent efficacy of DEET, Icaridin, and EBAAP against Ixodes ricinus and Ixodes scapularis nymphs (Acari, Ixodidae). Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.03.019
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Specific tick behavior Looking at the more specific behavioral events, distinct differences were observed between both species. Table 2 shows the percentages of ticks that displayed certain behaviors within the first five (DEET, EBAAP) or six (Icaridin) hours of the test. Specifically, distinctly more I. ricinus than I. scapularis ticks crawled onto the treated skin. For example, with DEET a total of 59% of I. ricinus did so compared to only 20% of I. scapularis (Table 2). This trend was highly significant (P ≤ 0.01) in all three repellents tested (Wilcoxon paired rank tests: DEET: T = 24.5; Z = 4.67; P ≤ 0.001; EBAAP: T = 80.5; Z = 2.96; P ≤ 0.01; Icaridin: T = 33.5; Z = 5.62; P ≤ 0.001). Ticks that moved onto treated skin could remain there, fall off or crawl back onto untreated skin. Regardless of the repellent tested, always significantly higher percentages of I. ricinus than I. scapularis walked back to untreated skin (Table 2, Wilcoxon matched pairs test: DEET: T = 23.5; Z = 2.51; P ≤ 0.05; EBAAP: T = 79; Z = 3.61; P ≤ 0.01; Icaridin: T = 18; Z = 4.78; P ≤ 0.001). On the other hand, always higher percentages of I. scapularis than I. ricinus remained more or less motionless on treated skin for >60 s (Wilcoxon matched pairs test: DEET: T = 19; Z = 2.1; P ≤ 0.05; EBAAP: T = 50; Z = 3.03; P ≤ 0.01; Icaridin: T = 1; Z = 4.68; P ≤ 0.001). No such differences, however, were observed with regard to the percentages of ticks that fell off of treated skin (Table 2: Wilcoxon matched pairs test: DEET: T = 24; Z = 0.36; P = 0.7; EBAAP: T = 135; Z = 1.03; P = 0.3; Icaridin: T = 141; Z = 0.58; P = 0.6. Comparing the three repellents directly, it was observed that significantly more ticks (both I. ricinus and I. scapularis) walked onto skin treated with EBAAP than onto skin treated with Icaridin (I. ricinus: N = 42; T = 110.5; Z = 3.35; P ≤ 0.001; I. scapularis: N = 38; T = 30.5; Z = 4.75; P ≤ 0.001). In I. scapularis, also more ticks did so with EBAAP compared to DEET (N = 35; T = 42.5; Z = 4.36; P ≤ 0.001). Concerning the proportions of ticks returning to untreated skin, no significant differences were observed between the repellents in any of the two tick species. Finally, the percentages of non-repelled I. ricinus ticks were significantly lower when Icaridin was applied compared to EBAAP (N = 37; T = 5; Z = 2.98; P ≤ 0.01) or DEET (N = 37; T = 3; Z = 3.36; P ≤ 0.001), the difference between EBAAP and DEET being insignificant. No such differences were observed with I. scapularis.
a paintbrush to move. In addition, more I. ricinus than I. scapularis nymphs walked onto the treated skin. This was evident in all tests. Finally, of the ticks that moved onto treated skin, many I. ricinus nymphs returned to the untreated area, while I. scapularis tended to remain more inactive. Obviously, nymphs of I. scapularis differ from I. ricinus in their locomotion pattern once being on host skin: while I. ricinus nymphs were quite active in their upward activity, the majority of I. scapularis nymphs were not. This has been described previously (Schreck et al., 1995; Carroll, 2008; Carroll et al., 2008; Dautel et al., 2013; Kröber et al., 2013). In the present study, we chose to test nymphs because in humans, bites of I. ricinus and I. scapularis are more frequently caused by nymphs than by adults (Falco et al., 1996; Wilhelmsson et al., 2010). Another point is that nymphs, due to their small size, often go unnoticed on inspection. Thus, the mean attachment period before removal tends to be longer in nymphs than in adult ticks (Falco et al., 1996). This increases the probability of transmission at least with Borrelia burgdorferi sensu stricto (Piesman and Dolan, 2002; Hojgaard et al., 2008). EBAAP is a synthetic insect repellent that is additionally classified as biopesticide by the U.S. EPA. During its consumer use over the past three decades, no negative effects have been reported of yet. In this study, it was inferior to Icaridin, but demonstrated protection times comparable to DEET in both tick species. A good performance of a formulation containing EBAAP against nymphs of I. scapularis had been previously reported (Carroll, 2008). It should be noted that DEET was tested at double the dose of EBAAP in the present study, hence EBAAP might actually be more effective than DEET if comparable doses are used. This is in accordance with the conclusion of Puccetti (2007). Staub et al. (2002) did a placebo-controlled double-blind field test in Switzerland and found a product containing DEET (15%) plus EBAAP (15%) to be 41% effective against I. ricinus compared to the control group treated with solvent only. It is known that product formulations could well be superior in their repellent properties and different results might be achieved at field conditions. It can be speculated, that a repellent product might be less efficient at field conditions than the results gained in the present study. Therefore, further testing is needed to ascertain the effectiveness of these repellents in the field, as well as against adults and other tick species.
Discussion Acknowledgements This is the first report comparing the efficacy of the three most commonly marketed repellent active ingredients against nymphs of I. scapularis and I. ricinus simultaneously. Ethanolic solutions of 10% Icaridin and EBAAP were compared to 20% DEET solutions according to standard procedures after skin application involving human volunteers. Ethanol was applied as it is a good solvent for all active ingredients tested. Although it is a commonly used solvent it might have an influence on the repellent properties. Of the repellents tested, Icaridin (10%) showed the highest repellent efficacy against both, I. ricinus and I. scapularis nymphs. Icaridin resulted in significantly longer complete protection times against I. ricinus than EBAAP (10%) and DEET (20%). However, in I. scapularis, DEET (20%) was similarly effective and only EBAAP (10%) showed a significantly shorter CPT. Distinct differences in certain behaviors between the tick species were observed. Quality and quantity of mobility, host seeking and attachment behavior is known to vary between tick species and with the age of ticks. In particular, blacklegged ticks are known for their comparably slow mobility compared to other ticks (Carroll et al., 2004). In this study, both species differed in their locomotor activity. While I. ricinus nymphs demonstrated good mobility when transferred onto the arm, I. scapularis had to be gently animated by
We thank all the volunteers for participating in the study. The study was supported by the German Environmental Agency, Dessau, through contract number 27962.
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Schreck, C.E., Fish, D., McGovern, T.P., 1995. Activity of repellents applied to skin for protection against Amblyomma americanum and Ixodes scapularis ticks (Acari: Ixodidae). J. Am. Mosq. Control Assoc. 11, 136–140. Semmler, M., Abdel-Ghaffar, F., Al-Rasheid, K., Mehlhorn, H., 2011. Comparison of the tick repellent efficacy of chemical and biological products originating from Europe and the U.S.A. Parasitol. Res. 108, 899–904. Soares, S.F., Braga R de, S., Ferreira, L.L., Louly, C.C.B., Sousa, L.A.D., de Silva, A.C., da Borges, L.M.F., 2010. Repellent activity of DEET against Amblyomma cajennense (Acari: Ixodidae) nymphs submitted to different laboratory bioassays. Rev. Bras. Parasitol. Vet. 19, 12–16. Staub, D., Debrunner, M., Amsler, L., Steffen, R., 2002. Effectiveness of a repellent containing DEET and EBAAP for preventing tick bites. Wilderness Environ. Med. 13, 12–20. U.S. EPA, 2010, July. Product Performance Test Guidelines. OPPTS 810.3700: Insect Repellents to be applied to Human Skin, EPA 712-C-100-01. WHO, 2009. Annex 3. Estimation of median and confidence interval of complete protection time using Kaplan–Meier survivor function. In: Guidelines for Efficacy Testing of Mosquito Repellents for Human Skin (WHO/HTM/NTD/ WHOPES/2009.4). Wilhelmsson, P., Fryland, L., Börjesson, S., Nordgren, J., Bergström, S., Ernerudh, J., Forsberg, P., Lindgren, P.-E., 2010. Prevalence and diversity of Borrelia species in ticks that have bitten humans in Sweden. J. Clin. Microbiol. 48, 4169–4176.
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Original article
Repellent efficacy of DEET, Icaridin, and EBAAP against Ixodes ricinus and Ixodes scapularis nymphs (Acari, Ixodidae)夽 Kerstin Büchel a , Juliane Bendin a , Amina Gharbi a , Sibylle Rahlenbeck b , Hans Dautel a,∗ a b
IS Insect Services GmbH, Haderslebener Str. 9, 12163 Berlin, Germany Umweltbundesamt, P.O. Box 33 00 22, 14191 Berlin, Germany
a r t i c l e
i n f o
Article history: Received 20 June 2014 Received in revised form 25 March 2015 Accepted 26 March 2015 Available online xxx Keywords: Repellent Ixodes ricinus Ixodes scapularis DEET EBAAP Icaridin
a b s t r a c t Repellent efficacy of 10% EBAAP (3-[N-butyl-N-acetyl]-aminopropionic acid, ethyl ester) and 10% Icaridin ((2-(2-hydroxyethyl)-1-piperidinecarboxylic acid 1-methylpropyl ester)) were evaluated against 20% DEET (N,N-diethyl-3-methylbenzamide) in human subject trials against ticks. Responses of host-seeking nymphs of the European castor bean tick (Ixodes ricinus L.; Acari: Ixodidae) and the North American blacklegged tick (I. scapularis Say; Acari: Ixodidae) were compared. Tests were carried out according to the US-EPA standard protocol with ethanolic solutions of the active ingredients of repellents being applied to the forearm of 10 volunteers. The upward movement of ticks was monitored until repellent failure taking up to 12.5 h. Application of 20% DEET resulted in median complete protection times (CPT; Kaplan–Meier median) between 4 and 4.5 h, while 10% EBAAP yielded CPTs of 3.5–4 h. No significant differences were found between the efficacies of two repellents nor between the two species tested. The median of the CPT of a 10% Icaridin solution was 5 h in nymphs of I. scapularis, but 8 h in those of I. ricinus (P < 0.01). Based on these studies, EBAAP and Icaridin are efficacious alternatives to DEET in their repellent activity against nymphs of the two Ixodes ticks with Icaridin demonstrating particularly promising results against I. ricinus. Future research should investigate whether similar results occur when adult Ixodes ticks or other tick species are tested. © 2015 Elsevier GmbH. All rights reserved.
Introduction Ticks are obligate blood-feeders known as vectors of a variety of infectious agents (Piesman and Eisen, 2008). The most frequently reported vector-borne diseases in the temperate zone of the Northern hemisphere are in fact related to ticks: in Europe and the United States, the number of tick-related zoonoses in humans not only exceeds those transmitted by mosquitoes, but the former ones often result in diseases that are more severe than those transmitted by the latter. In Europe, the sheep or castor bean tick, Ixodes ricinus is the most abundant species, infesting a wide range of hosts including mammals, birds, and even reptiles. The deer or blacklegged tick, I. scapularis is found in the eastern part and the Midwest of the United States currently expanding its occurrence further to the north into Canada. Humans are frequently bitten by both Ixodes
夽 This paper does not necessarily reflect the opinion or the policies of the German Federal Environment Agency. ∗ Corresponding author. Tel.: +49 3082096555; fax: +49 3082096555. E-mail address: [email protected] (H. Dautel).
species, whereby most tick bites stem from the nymphal stage (Wilhelmsson et al., 2010; Falco et al., 1996). Repellents are usually the primary method of personal protection against tick bites (Bissinger and Roe, 2010; Piesman and Eisen, 2008). While defining the ‘repelling’ action is straightforward insofar as flying insects are concerned, this cannot be defined as easily when it comes to crawling arthropods like ticks. In ambushing ticks, the process of questing, clinging to, and locating a suitable feeding site on a host is much more complex lasting for several hours to weeks. Each step in this interaction can be disrupted, but only certain steps would be defined as leading to a repellency ‘sensu stricto’, namely causing the tick to move away (Halos et al., 2012) leaving its host or avoiding it in the first place. Repellency is usually attributed to the vapor phase of a ‘repellent’ compound applied to the hosts’ skin. While DEET is the most widely used active ingredient of repellents worldwide, some researchers have speculated if other substances, notably EBAAP (IR3535) and Icaridin might be similarly efficacious (Schreck et al., 1995). However, only few studies are known comparing these active ingredients on human skin, which is more suitable to determine an effective concentration than an in vitro assay (Soares et al., 2010). While Pretorius et al. (2003) found ethanolic solutions of DEET
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Please cite this article in press as: Büchel, K., et al., Repellent efficacy of DEET, Icaridin, and EBAAP against Ixodes ricinus and Ixodes scapularis nymphs (Acari, Ixodidae). Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.03.019
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(20%) to be slightly more effective than Icaridin (20%), no such differences between DEET, EBAAP and Icaridin dissolved in ethanol were detected by Kröber et al. (2013). In tests with commercial products, the results of Semmler et al. (2011) suggest, that products containing Icaridin and EBAAP are more effective than such ones containing DEET. Carroll et al. (2010) in an assay with human subjects tested different formulations of DEET, EBAAP, and Icaridin against nymphs of Ambylomma americanum at concentrations in the range of 20–33%, typical for repellent products (Pages et al., 2014). As all of those formulations repelled >80% of the ticks throughout the 12-h test period, no discrimination between products with different active ingredient was possible. The aim of the present study was to directly compare both, the active ingredients DEET, EBAAP, and Icaridin, as well as the responses of I. ricinus and I. scapularis against these. We, therefore, investigated ethanolic solutions of the three repellents at discriminating doses (i.e. lower concentrations than usual) in a well-defined simulated use test with human subjects against nymphs of both tick species. Materials and methods Test products EBAAP (3-[N-butyl-N-acetyl] aminopropionicacid ethylester), was obtained from Merck KGaA (Darmstadt, Germany) (IR 3535® , 100% purity) and Icaridin (2-(2-hydroxyethyl)-1piperidinecarboxylic acid 1-methylpropyl ester) from Saltiago GmbH (Langenfeld, Germany) (Saltidin® , 97% purity). DEET (N,Ndiethyl-3-methylbenzamide) was obtained from Sigma–Aldrich GmbH (Steinheim, Germany) (97% purity). All repellents were stored at 4 ◦ C in complete darkness. Ethanol (≥98.9%, Sigma–Aldrich GmbH) was used to prepare 10% solutions of (v/v) of Icaridin and EBAAP, while DEET was tested as a 20% ethanolic solution. The latter usually serves as a positive control and standard for comparison as recommended in the guideline of U.S. EPA (2010). Ticks Unfed nymphs of I. ricinus and I. scapularis from laboratory colonies were used throughout all trials. I. ricinus constituted the F2 generation (age: 20–24 d post ecdysis) of a lab colony originating from wild ticks collected near Berlin, Germany. Nymphs were kept at 20 ◦ C and 90% RH (relative humidity) at a photoperiod of 16:8 L:D. I. scapularis was obtained from the U.S. Center for Veterinary Parasitology at Oklahoma State University, Stillwater, OK. After transport, nymphs were acclimated at 23 ◦ C, 90% RH and a photoperiod of 16:8 (L:D) for 4 weeks. The age of nymphs when tested was between 8 and 12 weeks post ecdysis. Nymphs were randomized and placed in 10 ml glass vials (10–15 ticks per vial) covered by gauze. Only ostensibly healthy and ‘active’ nymphs were used for the tests. To ascertain activity, only nymphs moving up the glass vial or staying at the top of it were included. In addition, locomotive activity of those chosen was tested prior to each trial (see below). Each tick was used only once.
had been reviewed and were in accordance with the standards of the Local Health Authority. For each repellent, 10 volunteers (5 males and 5 females) between 18 and 54 years of age were tested in a controlled laboratory setting. The volunteers were instructed to avoid smoking, drinking of caffeinated or alcoholic beverages and/or applying fragrance products on the day of testing. Before testing, volunteers washed their arms for 30 s with water and 3 ml unscented soap, rinsing them once for 1 min with tap water, then wiping the skin with soft tissue soaked with ethanol (70%) twice, followed by rinsing with water for 1 min. Control arms were cleansed in the same fashion. Arms were then allowed to dry. Test formulations were applied at 1.0 l cm−2 to the entire test area. This constitutes 60% of the amount recommended by U.S. EPA (1.67 l cm−2 ). After pretesting with the full standard amount, the dose was reduced in order to yield protection times below 10 h. The calculated amount of the test solution was applied to the volunteers’ forearm with a syringe onto four to five stripes and was spread as evenly as possible by two gloved finger tips. Exposure began 20 min after application of the test formulations. During testing, temperature and RH as measured were 22.9 ± 0.8 ◦ C and 56.4 ± 5.6%, respectively. Repellent and control treatments were randomly assigned to subjects on the first day. The volunteers cycled through each repellent and both Ixodes species so that each subject tested each repellent and each Ixodes species once. The border of the treated zone was marked by a pen with a dashed line around the arm and a second line was marked at a distance of 1 cm below. The volunteer sat with their arm held vertically, the finger tips or the palm touching a small table. A single active nymph was placed about 1 cm below the marked border on untreated skin with an artist’s paintbrush (Rotmarder size 1, Fredericus Rex, Erwitte, Germany). While I. ricinus nymphs readily moved upwards, most I. scapularis nymphs did not. Therefore, they were gently ‘guided’ upwards using a paintbrush as described by Carroll (2008). The nymph was then observed for a maximum of 3 min. Ticks that crossed the ring mark within 3 min and stayed in the treated area for a minimum of 60 s were regarded as ‘not repelled’ and removed. Ticks that did not crawl onto the treated skin as well as those that walked down to the wrist or dropped off were regarded as ‘repelled’ and removed. A new tick was then taken for the next test period. In such a way, 5 ticks were tested every 30 min (one test period) until CPT was reached, which took up to 12.5 h after test formulation application. Thus, a total of 100 ticks was tested per hour (with 10 volunteers) and per repellent with each tick species. Exposure for a volunteer ended as soon as two nymphs within the 30-min test period were not repelled (=confirmed crossing). Technical personnel monitored the practice and the scoring of the volunteers. Brushes were replaced with new ones every hour. Controls The untreated arm served as control. It was prepared in the same fashion as the test arm and was also marked. Nymphs that crossed into the marked, but untreated area 1 cm above the mark and stayed there for 1 min were considered as sufficiently locomotive and used for testing on the other arm within the next 30 min. Data analysis
Trials Repellent testing was carried out following the guideline of U.S. EPA (2010) which currently is the only internationally specified guideline available for authorization of repellents. According to this guideline, all subjects gave informed consent prior to participation in the trial. Informed consent documents and study trial protocols
The complete protection time (CPT) was defined as the time interval between the application of repellent and the first confirmed crossing (U.S. EPA, 2010). It was calculated by means of a Kaplan–Meier analysis (WHO, 2009). The data are presented as median CPT and its 95% confidence intervals and interquartile range (25–75%). Differences between the CPTs of the three
Please cite this article in press as: Büchel, K., et al., Repellent efficacy of DEET, Icaridin, and EBAAP against Ixodes ricinus and Ixodes scapularis nymphs (Acari, Ixodidae). Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.03.019
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Table 1 Complete protection times (minutes) (CPTs) evaluated with DEET, EBAAP, and Icaridin against I. ricinus and I. scapularis nymphs. Repellent
Tick species
CPT (median)
95% Confidence interval
IQR (25–75%)
P
DEET (20%) DEET (20%)
I. ricinus I. scapularis
240 270
194–286 240–300
150–300 225–315
a ab
EBAAP (10%) EBAAP (10%)
I. ricinus I. scapularis
240 210
178–302 182–238
150–285 135–225
a a
Icaridin (10%) Icaridin (10%)
I. ricinus I. scapularis
480 300
418–542 253–347
405–525 210–360
c b
IQR: interquartile range. Significance: CPTs denoted by the same letter are not significantly different from each other (P > 0.05).
Side effects No side effects, i.e. cutaneous itching or flushing were observed or reported by any of the volunteers throughout the entire test period.
1.0 0.9
EBAAP DEET
0.8
Proportion of volunteers
repellents were tested by a multiple-sample test in Statistica® v.7.1, an extension of Gehan’s generalized Wilcoxon test, Peto and Peto’s generalized Wilcoxon test and/or the log-rank test. Two treatments were compared by Cox’s F-Test. The Wilcoxon matched pairs test was used to compare the number of ticks that entered the treated skin area, walked back to untreated skin, fell off from treated skin, or were not repelled. To do so, the proportion of ticks displaying a certain behavior were summarized over the first five (DEET, EBAAP) and six test hours (Icaridin). All statistics were carried out using the Statistika® v. 7.1 software (StatSoft (Europe) GmbH, Hamburg, Germany).
Icaridin
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0
50
100
150
200
Results
300
All three compounds performed well in repelling the nymphs of the two Ixodes species. As shown in Table 1, median Kaplan–Meier CPTs were between 4 and 8 h in I. ricinus nymphs and 3.5–5 h in I scapularis nymphs. While application of EBAAP (10%) yielded similar median protection times of about 4–5 h as DEET (20%) in both species, 10% Icaridin resulted in longer protection with a CPT of 5 h in I. scapularis and 8 h in I. ricinus. Figs. 1 and 2 show the Kaplan–Meier curves for I. ricinus and I. scapularis, respectively. In I. ricinus, a significant difference between repellents was observed (chi2 = 17.21; DF = 2; P ≤ 0.001, Fig. 1) with the CPT of Icaridin differing from DEET and EBAAP (P ≤ 0.01 for 1.0 0.9
EBAAP DEET
0.8
Icaridin
0.7 0.6
400
450
500
DEET/Icaridin, P ≤ 0.001 EBAAP/Icaridin, respectively). In I. scapularis, also a significant difference between repellents was observed (Fig. 2; chi2 = 6.84; DF = 2; P ≤ 0.05) with EBAAP showing a shorter protection time than Icaridin (P ≤ 0.05), but being not different from DEET (P = 0.11). There was no significant difference between DEET and Icaridin (P = 0.1).
Table 2 Percentages (mean ± SE) of I. ricinus and I. scapularis ticks displaying specific behaviors during the first five (DEET, EBAAP) and six (Icaridin) hours. Displayed are percentages of ticks walking back, falling off or remaining on treated skin for 60 s after moving onto treated skin. Behavior of ticks
I. ricinus
DEET Walking onto treated skin [%] Walking back from treated skin [%] Falling off from treated skin [%] Remaining on treated skin [%]
59 80 8 12
± ± ± ±
5*** 4* 2 3*
20 52 7 41
± ± ± ±
4 7 3 8
EBAAP Walking onto treated skin [%] Walking back from treated skin [%] Falling off from treated skin [%] Remaining on treated skin [%]
71 71 18 12
± ± ± ±
5** 4** 3 3**
51 47 25 28
± ± ± ±
4 5 6 5
Icaridin Walking onto treated skin [%] Walking back from treated skin [%] Falling off from treated skin [%] Remaining on treated skin [%]
60 78 20 3
± ± ± ±
4*** 3*** 3 1***
26 40 19 41
± ± ± ±
3 5 4 5
0.5 0.4 0.3 0.2 0.1 0.0 0
350
Fig. 2. Kaplan–Meier plots for DEET, EBAAP, and Icaridin tested against I. scapularis nymphs on 10 volunteers.
Repellency
Proportion of volunteers
250
Time [min]
100
200
300
400
500
600
700
800
Time [min] Fig. 1. Kaplan–Meier plots for DEET, EBAAP, and Icaridin tested against I. ricinus nymphs on 10 volunteers.
I. scapularis
Asterisks indicate significant differences between I. ricinus and I. scapularis. * P < 0.05. ** P < 0.01. *** P < 0.001.
Please cite this article in press as: Büchel, K., et al., Repellent efficacy of DEET, Icaridin, and EBAAP against Ixodes ricinus and Ixodes scapularis nymphs (Acari, Ixodidae). Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.03.019
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Specific tick behavior Looking at the more specific behavioral events, distinct differences were observed between both species. Table 2 shows the percentages of ticks that displayed certain behaviors within the first five (DEET, EBAAP) or six (Icaridin) hours of the test. Specifically, distinctly more I. ricinus than I. scapularis ticks crawled onto the treated skin. For example, with DEET a total of 59% of I. ricinus did so compared to only 20% of I. scapularis (Table 2). This trend was highly significant (P ≤ 0.01) in all three repellents tested (Wilcoxon paired rank tests: DEET: T = 24.5; Z = 4.67; P ≤ 0.001; EBAAP: T = 80.5; Z = 2.96; P ≤ 0.01; Icaridin: T = 33.5; Z = 5.62; P ≤ 0.001). Ticks that moved onto treated skin could remain there, fall off or crawl back onto untreated skin. Regardless of the repellent tested, always significantly higher percentages of I. ricinus than I. scapularis walked back to untreated skin (Table 2, Wilcoxon matched pairs test: DEET: T = 23.5; Z = 2.51; P ≤ 0.05; EBAAP: T = 79; Z = 3.61; P ≤ 0.01; Icaridin: T = 18; Z = 4.78; P ≤ 0.001). On the other hand, always higher percentages of I. scapularis than I. ricinus remained more or less motionless on treated skin for >60 s (Wilcoxon matched pairs test: DEET: T = 19; Z = 2.1; P ≤ 0.05; EBAAP: T = 50; Z = 3.03; P ≤ 0.01; Icaridin: T = 1; Z = 4.68; P ≤ 0.001). No such differences, however, were observed with regard to the percentages of ticks that fell off of treated skin (Table 2: Wilcoxon matched pairs test: DEET: T = 24; Z = 0.36; P = 0.7; EBAAP: T = 135; Z = 1.03; P = 0.3; Icaridin: T = 141; Z = 0.58; P = 0.6. Comparing the three repellents directly, it was observed that significantly more ticks (both I. ricinus and I. scapularis) walked onto skin treated with EBAAP than onto skin treated with Icaridin (I. ricinus: N = 42; T = 110.5; Z = 3.35; P ≤ 0.001; I. scapularis: N = 38; T = 30.5; Z = 4.75; P ≤ 0.001). In I. scapularis, also more ticks did so with EBAAP compared to DEET (N = 35; T = 42.5; Z = 4.36; P ≤ 0.001). Concerning the proportions of ticks returning to untreated skin, no significant differences were observed between the repellents in any of the two tick species. Finally, the percentages of non-repelled I. ricinus ticks were significantly lower when Icaridin was applied compared to EBAAP (N = 37; T = 5; Z = 2.98; P ≤ 0.01) or DEET (N = 37; T = 3; Z = 3.36; P ≤ 0.001), the difference between EBAAP and DEET being insignificant. No such differences were observed with I. scapularis.
a paintbrush to move. In addition, more I. ricinus than I. scapularis nymphs walked onto the treated skin. This was evident in all tests. Finally, of the ticks that moved onto treated skin, many I. ricinus nymphs returned to the untreated area, while I. scapularis tended to remain more inactive. Obviously, nymphs of I. scapularis differ from I. ricinus in their locomotion pattern once being on host skin: while I. ricinus nymphs were quite active in their upward activity, the majority of I. scapularis nymphs were not. This has been described previously (Schreck et al., 1995; Carroll, 2008; Carroll et al., 2008; Dautel et al., 2013; Kröber et al., 2013). In the present study, we chose to test nymphs because in humans, bites of I. ricinus and I. scapularis are more frequently caused by nymphs than by adults (Falco et al., 1996; Wilhelmsson et al., 2010). Another point is that nymphs, due to their small size, often go unnoticed on inspection. Thus, the mean attachment period before removal tends to be longer in nymphs than in adult ticks (Falco et al., 1996). This increases the probability of transmission at least with Borrelia burgdorferi sensu stricto (Piesman and Dolan, 2002; Hojgaard et al., 2008). EBAAP is a synthetic insect repellent that is additionally classified as biopesticide by the U.S. EPA. During its consumer use over the past three decades, no negative effects have been reported of yet. In this study, it was inferior to Icaridin, but demonstrated protection times comparable to DEET in both tick species. A good performance of a formulation containing EBAAP against nymphs of I. scapularis had been previously reported (Carroll, 2008). It should be noted that DEET was tested at double the dose of EBAAP in the present study, hence EBAAP might actually be more effective than DEET if comparable doses are used. This is in accordance with the conclusion of Puccetti (2007). Staub et al. (2002) did a placebo-controlled double-blind field test in Switzerland and found a product containing DEET (15%) plus EBAAP (15%) to be 41% effective against I. ricinus compared to the control group treated with solvent only. It is known that product formulations could well be superior in their repellent properties and different results might be achieved at field conditions. It can be speculated, that a repellent product might be less efficient at field conditions than the results gained in the present study. Therefore, further testing is needed to ascertain the effectiveness of these repellents in the field, as well as against adults and other tick species.
Discussion Acknowledgements This is the first report comparing the efficacy of the three most commonly marketed repellent active ingredients against nymphs of I. scapularis and I. ricinus simultaneously. Ethanolic solutions of 10% Icaridin and EBAAP were compared to 20% DEET solutions according to standard procedures after skin application involving human volunteers. Ethanol was applied as it is a good solvent for all active ingredients tested. Although it is a commonly used solvent it might have an influence on the repellent properties. Of the repellents tested, Icaridin (10%) showed the highest repellent efficacy against both, I. ricinus and I. scapularis nymphs. Icaridin resulted in significantly longer complete protection times against I. ricinus than EBAAP (10%) and DEET (20%). However, in I. scapularis, DEET (20%) was similarly effective and only EBAAP (10%) showed a significantly shorter CPT. Distinct differences in certain behaviors between the tick species were observed. Quality and quantity of mobility, host seeking and attachment behavior is known to vary between tick species and with the age of ticks. In particular, blacklegged ticks are known for their comparably slow mobility compared to other ticks (Carroll et al., 2004). In this study, both species differed in their locomotor activity. While I. ricinus nymphs demonstrated good mobility when transferred onto the arm, I. scapularis had to be gently animated by
We thank all the volunteers for participating in the study. The study was supported by the German Environmental Agency, Dessau, through contract number 27962.
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