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Research Article
Comparison of Repellency Effect of Mosquito Repellents for
DEET, Citronella, and Fennel Oil
Jong Kwang Yoon,1Kang-Chang Kim,1Yeondong Cho,1Yong-Dae Gwon,1
Han Sam Cho,1Yoonki Heo,1Kihoon Park,1Yang-Won Lee,2Mijeong Kim,3
Yu-Kyoung Oh,4and Young Bong Kim1
1Department of Bio-Industrial Technologies, Konkuk University, Seoul 143-701, Republic of Korea
2Department of Dermatology, Konkuk University Hospital, Seoul 143-729, Republic of Korea
3Cosmetics Research Team, Department of Pharmaceutical and Medical Device Research,
Ministry of Food and Drug Safety, Chungcheongbuk-do 363-700, Republic of Korea
4Department of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea
Correspondence should be addressed to Young Bong Kim; kimera@konkuk.ac.kr
Received April ; Revised September ; Accepted September
Academic Editor: Dave Chadee
Copyright © Jong Kwang Yoon et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
To conrm that Korean Food and Drug Administration (KFDA) guidelines are applicable to test the ecacy of mosquito repellents,
these guidelines were used to test the ecacy and complete protection times (CPTs) of three representative mosquito repellents:
N,N-diethyl--methylbenzamide (DEET), citronella, and fennel oil. e repellency of citronella oil decreased over time, from .%
at h to .% at h and .% at h, as did the repellency of fennel oil, from .% at h to .% at h and .% at h. In contrast,
the repellency of DEET remained over % for h. e CPT of DEET ( min) was much longer than the CPTs of citronella
(. min) and fennel oil (. min). ese results did not dier signicantly from previous ndings, and hence conrm that the
KFDA guidelines are applicable for testing the ecacy of mosquito repellents.
1. Introduction
Insect-borne diseases are a worldwide health problem, espe-
cially in tropical and subtropical climates. Mosquitoes trans-
mit many diseases, including yellow fever, dengue hem-
orrhagic fever, malaria, several forms of encephalitis, and
lariasis []. For example, malaria has been estimated to kill
million persons per year, including over million children.
Mosquito repellents may eectively protect humans from
vector-borne diseases as well as other problems caused by
mosquitoes.
N,N-Diethyl-m-toluamide (DEET) is a readily available
and frequently used mosquito repellent. However, adverse
eects of DEET have been reported, with some being severe
enough to cause sensory disturbances and aect motor
capacity, memory, and learning ability [–]. In addition,
DEET is not recommended for children, because high con-
centrations of DEET can cause encephalopathy and other side
eects [,].
Botanical mosquito repellents, which cause little risk to
the environment or human health, may be feasible alterna-
tives to synthetic chemical repellents such as DEET. us,
many people prefer to use natural repellents extracted from
plants, such as citronella oil from Cymbopogon nardus,p-
menthane-,-diol (PMD) from Eucalyptus maculata cit-
riodora,andfenneloilfromFoeniculum vulgare [–].
Little information is available, however, about the mosquito
repellent activities of these natural and herbal-based sub-
stances. is study evaluated the repellency of commercially
available natural mosquito repellents using the Korean FDA
guidelines and compared their activities with that of %
DEET.
Hindawi Publishing Corporation
Journal of Parasitology Research
Volume 2015, Article ID 361021, 6 pages
http://dx.doi.org/10.1155/2015/361021
Journal of Parasitology Research
2. Materials and Methods
2.1. Mosquitoes Used in Repellent Tests. Aedes albopictus
(Skuse) mosquitoes were used for repellent testing. Mosquito
larvae were obtained from the Division of Medical Entomol-
ogy of Korea Centers for Disease Control and Prevention
(KCDC).elarvaewererearedat
∘Cand%relative
humidity at a dedicated facility of Konkuk University. Adult
mosquitoes were fed and maintained on a % sucrose
solution, as described previously [].
2.2. Repellent Testing. ree kinds of mosquito repellents,
% citronella (California Baby Citronella spray, California
Baby, USA), % fennel oil (Moszero spray, Naturobiotech
Co., Korea), and % DEET (Insectan Spray, Green Cross,
Korea), were purchased. Aliquots of . mL were applied to
volunteers’ forearms to test repellent ecacy [].
2.3. Test Cage. Atestcage(× × cm) was constructed
with a metal frame to make decontamination easier. All sides
were covered with an observable white net to allow viewing.
Afabricsleevewasaddedtothefrontsideofthetestcageto
allow access by a human forearm.
2.4. Patch Tests. A patch containing repellent agent was
applied to clean skin on the volunteer’s forearm and allowed
to remain on the skin for hours. Volunteers were not
permittedtoremoveorwetthepatchduringthistime[].
Aer hours, the patch was removed by medical personnel,
and initial results were determined. e patch region was
marked on the forearm and results were determined hours
aer initial patch placement.
2.5. Laboratory Tests of Mosquito Repellents. e repellent
tests followed KFDA guidelines modied from WHOPES []
and EPA methods []. Two hundred female mosquitoes (age
– days), which had never received a blood meal, were
placed into each test cage and starved of their sugar diet for
h before the test.
e arms of each volunteer were washed with unscented
soap, rinsed with water, and dried for min. A . mL aliquot
of each repellent solution was applied evenly on the right
forearm between the wrist and elbow using a pipette and
allowedtodryforapproximatelymin.euntreatedle
arm was placed into a test cage for min and the number of
mosquitoes landing on that arm was counted. If fewer than
mosquitoes landed on that arm, the volunteer was excluded
from further testing.
Repellent-treated right arms were placed into the test cage
for min at h intervals, DEET-treated arms for h, and arms
treated with fennel or citronella oil for h. e number of
mosquitoes that landed on or bit that arm was recorded every
hour.
Repellency () was calculated using the formula []
(%)=−
×100%,()
where is the number of mosquito bites on the control arm
and the number of bites on the treated arm.
e complete protection time (CPT) was dened as the
timetherstmosquitolandedonorbitatreatedarm.To
determine the CPT of mosquito repellents, the treated right
arm of each volunteer was inserted into the test cage for
min. If there were no bites, that arm was reinserted at min
intervals until the rst bite occurred.
2.6. Statistical Analysis. e repellency of the control and
treated arms was compared using -tests, with a value <
. considered statistically signicant. SPSS was used for
statistical analysis. e CPT of DEET repellent was replaced
with a Kaplan-Meier survival function, since there were no
bites over h.
2.7. Ethics. e study protocol was approved by the IRB
of Konkuk University Hospital (Approval number KUH
). Forty-three volunteers were enrolled, all of whom
provided written informed consent.
3. Results and Discussion
3.1. e Choice of Mosquito Species. To e v a l u a t e t h e e e c -
tiveness of repellent activity against mosquito, we per-
formed preparatory experiments with widespread kinds of
mosquitoes, Culex pipiens, Aedes togoi, and Aedes albopictus.
Culex pipiens, common house mosquito, however, is not
ideal for the repellency test in the laboratory setting because
itfedonhumanonlyatnighttimeduetoitsnocturnal
characteristic. On the other hand, Aedes togoi showed much
less biting activity compared to Aedes albopictus throughout
the experiment setting, which is not optimal to quantify
the biting rate to assess the eect of repellants. us, Aedes
albopictus waschosentoevaluatetheeectofrepellant
activities clearly in the experimental setting.
3.2. Patch Test for Mosquito Repellents. DEET, citronella, and
fenneloilweretestedon,,andvolunteers,respectively.
Initial skin tests on volunteers’ forearms were performed
to assess their allergic responses to the three repellents. As
determined by a dermatologist, none of the volunteers had
allergic reactions at h and h (data not shown).
3.3. Repellent Eect for DEET, Citronella, and Fennel Oil. As
hazards by mosquitoes have gradually increased, many kinds
of mosquito repellents have been manufactured to protect
humans against mosquito bites. Because mosquito repellents
have played an important role in protecting humans from
vector-borne diseases caused by mosquitoes, standardized
guidelines are needed to evaluate the ecacy of these repel-
lents.
In the United States, for example, repellents are tested
against mosquitoes and other pests according to the guide-
lines of the Environmental Protection Agency (US EPA;
[]) and the American Society for Testing and Materials
(ASTM; []). Although European guidelines have not been
developed, the ecacy of these repellents has been tested
Journal of Parasitology Research
T : Repellency and CPT of % DEET against Aedes albopictus in laboratory test.
Untreated Repel lency (%) (±SE) at hours aer treatment
CPT (min)
∼h h h h
(%) (%) (%) (%)
V Unknown
V
V
V
V Unknown
V .
V Unknown
V Unknown
V . .
V . . .
AVG ±. . ±. ±. . ±. . ±. . ±. . ±. . ±. . ±. .∼.
e number (𝑁) of mosquitoes landing on arm of each voluntee r was counted per hour for h. Repell ency (𝑅) was calculated each hour andcomplete protection
time (CPT) was determined by calculating the number of minutes from the time of repellent application to the rst mosquito landing.
according to the guidelines of the World Health Organization
Pesticide Evaluation Scheme (WHOPES; []) and the US
EPA, which are considered the international standard testing
guidelines.
In Korea, the standardized guideline to test the ecacy
of mosquito repellents has been established by modifying the
existing EPA and WHOPES methods in . In this study,
we applied a laboratory test and the semield test (data not
shown)totheecacyofDEETaccordingtoYoonetal.
[] and botanical mosquito repellents such as citronella and
fennel oils according to the KFDA guideline.
Table shows the mean numbers of mosquitoes landing
on untreated (control) and treated forearms of volunteers
over min. e mean number landing on the untreated
forearms of volunteers over min was 16.00 ± 1.71.
Testing of the repellency of treated forearms every hour
for h showed perfect repellency for % DEET over the
rst hours. One (V), two (V and V), and six (V,
V,V,V,V,andV)volunteerswerebittenat,
, and h, respectively, making the repellency at these
times 99.54 ± 0.46%, 97.89 ± 1.49%, and 90.33 ± 4.16%,
respectively. ese results indicated that % DEET had
>%repellencyforhours,withacompleteprotectiontime
(CPT)ofovermin.eotherfourvolunteerstreatedwith
DEET (V, V, V, and V) were not bitten by mosquitoes
for h, so the average CPT for all volunteers could
not be calculated. us, CPT in this group was estimated
using the Kaplan-Meier survival function, resulting in a
CPT between . and . min at % condence
interval.
e use of botanical mosquito repellents has increased
duetotheirlackofadverseeectsonhumans.Commercially
availablerepellentproductsbasedonplantessentialoils
include extracts of basil, citronella, fennel, cedar, cinnamon,
garlic, geranium, lavender, rosemary, thyme, pennyroyal,
peppermint, pine, and verbena oils, which have shown
repellent activity against dierent mosquito species as well as
Aedes albopictus [,–]. is study tested the repellency
and CPT of % citronella and fennel oil-containing products
according to KFDA guidelines.
e repellency of % citronella oil was tested in
volunteers. When their untreated le forearms were exposed
to mosquitoes for min, a mean (±SE) of 35.25 ± 2.81
mosquitoes landed.
TocalculatetheCPT,thetreatedrightarmofeach
volunteer was placed into the test cage for min at min
intervals until the rst mosquito landed (Table ). Seven
volunteers (V, V, V, V, V, V, and V) were bitten
within the rst min, another volunteers (V, V, V, V,
V,V,V,V,V,V,andV)duringthesecond
min exposure period ( min), and the last two (V and
V) during the third min exposure ( min). ese results
indicated that the average CPT of citronella oil for these
volunteers was 10.50 ± 1.20min.
Aer completing the CPTs for each volunteer, repellency
tests were performed at application and at h and h aer
treatment (Table ). Repellency at h, h, and h was 97.92±
0.69%, 71.42±3.05%, and 57.73±4.03%, respectively.
Repellency tests of fennel oil were performed on
volunteers. A mean (±SE) of 21.15 ± 0.36 mosquitoes landed
on their untreated le forearms during exposure to
mosquitoes for min (Ta b l e ).
TestingoftheCPTofcitronellaoilshowedthatnine
volunteers (V, V, V, V, V, V, V, V, and V) were
bitten within the rst min, one (V) was bitten during the
second min exposure period, and three (V, V, and V)
were bitten during the third min exposure period. ese
results indicated that the average CPT of fennel oil for these
volunteers was 8.38±1.12min.
Repellency tests of fennel oil were performed at applica-
tion andhand hlater. Repellencyat h, h, andhwas
88.57±2.96%, 61.15±3.85%, and 47.36±5.78%, respectively.
Many plant essential oils contain volatile components,
including alkanes, alcohols, aldehydes, terpenoids, and
Journal of Parasitology Research
T : Repellency and CPT of % citronella oil against Aedes albopictus in laboratory test.
Untreated Rep ellency (%) (±SE) at hours aer treatment
CPT (min)
h h h
(%) (%) (%)
V
V . .
V . . .
V . .
V . .
V . .
V . .
V . . .
V .
V . .
V . . .
V . . .
V . . .
V ..
V .
V . .
V . . .
V .
V . .
V . .
AVG . ±. . ±. . ±. . ±. . ±. . ±. . ±. . ±.
e number (𝑁) of mosquitoes landing on arm of each voluntee r was counted per hour for h. Repell ency (𝑅)wascalculatedeachhourandcompleteprotection
time (CPT) was determined by calculating the number of minutes from the time of repellent application to the rst mosquito landing.
T : Repellency and CPT of % fennel oil against Aedes albopictus in laboratory test.
Untreated Repellency (%) (±SE) at hours aer treatment
CPT (min)
h h h
(%) (%) (%)
V . . .
V . . .
V . .
V
V . .
V
V
V
V .
V . . .
V
V . . .
V . .
AVG . ±. . ±. . ±. . ±. . ±. . ±. . ±. . ±.
e number (𝑁) of mosquitoes landing on arm of each voluntee r was counted per hour for h. Repell ency (𝑅)wascalculatedeachhourandcompleteprotection
time (CPT) was determined by calculating the number of minutes from the time of repellent application to the rst mosquito landing.
Journal of Parasitology Research
T : Comparative CPT of DEET and citronella oil against mosquito bites.
Product name Active ingredient Percentage (%) CPT (min) Reference (year)
Insectan Spray DEET .∼. Yoon et al. () []
( ±.)
Aero Bug O DEET EPA () []
AquaPel % DEET
Insect Repellent Pump DEET EPA () []
Spray
/ DEET avara et al. () []
OFF! Deep Woods DEET . . (±.) Fradin and Day () []
California Baby Citronella spray Citronella . (±.) in this study
Buzz Away Citronella . (±.) Fradin and Day () []
Complete protection time (CPT) was determined by calculating the number of minutes from the time of repellent application to the rst mosquito landing.
monoterpenoids, with some of these components showing
arepellencyeectinthevaporphase[]. Due to their
volatility, however, these components have a much shorter
protection time against mosquitoes than DEET [,].
erefore, several controlled-release formulations have been
developed to increase the duration of repellency [,–
]. erefore, Ecacy Data Sheets used to register repellent
products with the EPA specify CPTs.
Fradin and Day [] conducted the laboratory test with
the method modied from EPA and WHOPES method as
follows. mosquitoes were placed in a test cage measuring
cm × cm × cm and volunteers’ arms were inserted
for min every hour for a total of h to test repellency.
CPT was determined by inserting volunteers’ arms for min
every min for a total of min until the rst mosquito bite
occurred. Using this method, the mean CPTs of .% DEET
and % citronella were 301.5 ± 37.6 min and 13.5 ± 7.5min,
respectively (Table ).
In comparison, this study used a lower density of
mosquitoes, with mosquitoes in a cage measuring cm
× cm × cm, because the lower-density environment
more accurately mimics the biting pressures during outdoor
activities. e repellency and CPT of DEET were assessed
for min every h for a total of hours. In contrast, the
repellency of citronella and fennel oils was tested for h,
because their repellency was approximately % at h. e
mean CPTs of DEET and citronella repellent were min
and . min, respectively, similar to previous ndings [,
]. However, the CPT of % DEET repellents registered
with the EPA was reported to be min, which diered
from our results (Table ). Since four of our volunteers
(V, V, V, and V) were not bitten by any mosquito
hours aer DEET treatment, the average CPT would
likely have been longer had the experiment been continued
until each volunteer was bitten. us, the CPT measured
in this study was consistent with that specied by the
EPA.
e repellency and CPTs of DEET, citronella, and fennel
oil, measured according to KFDA guidelines, were consistent
with previous ndings. KFDA guidelines will therefore be
utilized to evaluate the ecacy of mosquito repellents.
Conflict of Interests
e authors declare that there is no conict of interests
regarding the publication of this paper.
Acknowledgments
is research was supported by a grant of the Ministry
of Food and Drug Safety (MFDS; KFDA), Korea
Institute of Planning and Evaluation for Technology in Food,
Agriculture, Forestry and Fisheries (IPET; ---
SB), and the Ministry of Trade, Industry & Energy (MI;
).
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