Determining the Stability of Clove Oil (Eugenol) for Use as an Acaricide in Beeswax

December 15, 2014 — admin
AttachmentSize
stability_of_clove_oil.pdf294.32 KB
Embedded Scribd iPaper - Requires Javascript and Flash Player

Israel Journal of Veterinary Medicine  Vol. 69 (4)  December 2014 Girisgin, A.O. 192
INTRODUCTION
Acaricides are currently used globally for the control of the
honey bee parasite, Varroa destructor, a mite that endangers
the international beekeeping community. Colonies infest-
ed with V. destructor have signifcantly reduced worker bee
populations which may eventually die of if infestations are
uncontrolled (1). Tere are numerous drugs and modes of
applications to combat Varroosis. It has been widely docu-
mented for many years that some of these drugs may cause
contamination of bee products with acaricides (2, 3).
Te use of synthetic lipophilic acaricides leads to an ac-
cumulation of these substances in beeswax, but less so in
honey. Te accumulation in wax depends on the frequency,
lipophilicity and amount of active ingredient used (2). Te
persistence of pyrethroid and organophosphate acaricides in
beeswax has led to strains of V. destructor that are resistant
against fuvalinate, fumethrine and coumaphos (4, 5).
Tese problems have led to investigations to identify al-
ternative control methods. Some alternatives proposed have
included organic acids, thymol and essential oils or any of
their components (6). Essential oils are distilled from aro-
matic plants and possess an intense smell, exhibit low toxicity
Determining the Stability of Clove Oil (Eugenol)
For Use as an Acaricide in Beeswax
Girisgin, A.O.,
1,2
*, Barel, S,
3
Zilberman Barzilai, D.
4
and Girisgin, O.
5
1
Department of Parasitology, Faculty of Veterinary Medicine, Uludag University, Bursa, Turkey.
2
Beekeeping Development – Application and Research Center, Uludag University, Bursa, Turkey.
3
National Residue Control Laboratories, Veterinary Services, Israel Ministry of Agriculture and Rural Development,
Beit-Dagan, Israel.
4
Ichilov Sourasky Medical Center, Department of Epidemiology, Tel-Aviv, Israel.
5
Karacabey Vocational School, Uludag University, Bursa, Turkey.
*
Corresponding Author: Dr. Ahmet Onur Girisgin. Department of Parasitology, Faculty of Veterinary Medicine, Uludag University, Bursa, Turkey.
Tel: +902242941317, Fax: +902242940873, Email: onurgirisgin@gmail.com
ABSTRACT
Clove (Syzygium aromaticum) oil is primarily a mixture of monoterpenes, allylphenols and its major component is
eugenol. A study was conducted to determine the stability and sustainability of clove oil components in beeswax
samples under semi-feld conditions. Five wooden hives with ten comb foundations of frames were used; none
of the hives contained bees. For the assays in the four hives, 50 ml of 1% clove oil was tested using diferent
emulsifers and modes of application. Tween 80 and parafn oil emulsifers were used via both evaporation from
cups and the spraying method on each of the separate hives. One hive containing blank beeswax was kept as
control. Characterization of clove oil and contamination levels of its components in beeswax on days 1, 2, 5, 7,
14 and 28 were determined via gas chromatography/mass spectrophotometry analysis. Eugenol was detected
and identifed as the major component; its level in beeswax was determined to be stable for up to three weeks
with three of the dispensers, but it was not stable when used with parafn oil using the cup method application.
As a result of these studies, we found that if clove oil is used for honeybee Varroosis with diferent dispensers,
eugenol can be stable for a minimum of two weeks in beeswax. As a result, this period should be considered during
treatment of Varroosis and before honey harvesting. Absorption and accumulation of eugenol and other efective
volatile essential oils and monoterpenes into beeswax may make honeycombs as a secondary, sustain release source.
Keywords: Stability, Clove; Eugenol; Varroa destructor ; Beeswax.
DECEMBER Book.indb 192 04/12/2014 10:57:06
Israel Journal of Veterinary Medicine  Vol. 69 (4)  December 2014 193 Eugenol Stability in Beeswax
in mammals and bees and have less harmful environmental
efects than synthetic substances (7).
To date, over 150 essential oils and components of these
oils have been tested. However, only some of the compounds
have been found to be efective on V. destructor mites while
also remaining nontoxic to honeybees. Tey are applied topi-
cally either by pulverization or passive evaporation (6).
Clove (Syzygium aromaticum) oil is one of the efective
essential oils against V. destructor mites that infest honey-
bee (Apis mellifera) colonies. Promising results have been
obtained in previous studies (8, 9, 10). Active components
of this oil have been demonstrated to have antimicrobial,
insecticidal, antioxidant, antitumor and anesthetic activity
(11). It is also recommended as an insect repellent during
pediatric nursing (12).
Beeswax is a highly lipophilic medium containing high
molecular weight acids. Synthetic acaricides can be stable and
persistent in beeswax, but essential oils show less persistence
and stability (13).
Tis study was undertaken to determine the stability of
clove essential oil with diferent excipients in beeswax under
semi-feld conditions, using gas chromatography/mass spectro-
photometry (GC/MS) for detection and analysis. Te evapora-
tion time of active ingredient(s) from beeswax and the possible
efect of diferent dispensers and excipients were investigated to
determine the time of the booster treatment of Varroosis and
the convenient time for honey harvest after treatment.
MATERIALS AND METHODS
Characterization of clove essential oil
In this study, Clove bud essential oil (EU pharmacopeia
grade) was cultivated in Germany and purchased from
Fagron GMBH & CO (Barsbüttel, Germany). It was dis-
tilled in Germany as follows: isolated from the buds using
steam distillation equipment for 4 hours, separated from wa-
ter by decantation, dried with anhydrous sodium sulphate,
and kept refrigerated to avoid deterioration (14).
Te characterization of the clove oil was conducted at the
National Residue Laboratory of Kimron Veterinary Institute
from the Ministry of Agriculture, Beit-Dagan, Israel, during
July 2010.
Te GC/MS analysis of the oil was conducted using a
GC (Agilent Technologies 6890N, California-U.S.A.) inter-
faced with a mass selective detector (MSD, Agilent 5973B)
and equipped with an auto sampler (ALS, Agilent7673). An
Agilent HP-5 MS (5%-phenyl methyl poly siloxane) capil-
lary column (30 m x 0.25 mm i.d. and 0.25 µm flm thick-
ness) was used for separation. Te carrier gas was helium with
a constant fow rate of 1 ml/min. Te oven temperature was
set at 50°C for 1 min, programmed at 150°C at a rate of 5°C/
min, was once more heated to 280°C at a rate of 30°C/min
and fnally held isothermally at this temperature for 25 min.
Injector and GC/MS interface temperatures were 250°C and
280°C, respectively. Te injection mode was splitless, and the
volume injected was 1 µl of the fnal extract volume. Te MS
operating parameters were as follows: ionization potential, 70
eV MS source and quad temperatures of 230°C and 150°C,
respectively. Te acquisition mass range was 40–400 m/z.
Identifcation and quantifcation of constituents
Te relative percentage of the volatile oil components was
evaluated from the total peak area (TIC) using apparatus
software. Identifcation of components in the volatile oil was
based on a comparison of their mass spectra and retention
time with those of the authentic compounds and by com-
puter matching with the NIST and WILEY libraries as well
as by comparison of the fragmentation pattern of the mass
spectral data with those reported in the literature.
Preparation of hives and application of clove oil
Location of the apiary was at Beit-Dagan, 31° 99’ N, 34° 82’
E, in Israel. Te study was performed in July 2010, in which
average temperature ranges were 27.5 – 31.3°C at daytime,
17.9 – 20.6°C at nights and average humidity ranges were
60 – 87% at daytime, 30 – 63% at nights. All procedures were
applied in the early morning to avoid the evaporation due to
high temperatures.
Four Langstroth hives with ten frames each were used.
All hives had a foundation of beeswax and contained no
bees in the hive at the feld site. Entrances of the hives were
open. Application of the clove oil was carried out using four
methods:
(1) Cup method with Tween 80 solution: 50 ml of 1% clove
oil was prepared in distilled water and emulsifed using a
Tween 80 solution (2 ml clove oil + 44 ml distilled water +
4 ml tween 80) and divided into two plastic cups (2×25 ml).
Te cups were placed at the bottom of the hive between the
2
nd
-3
rd
and 7
th
–8
th
frames. Two frames were removed to allow
enough space for the cups.
Research Articles
DECEMBER Book.indb 193 04/12/2014 10:57:06
Israel Journal of Veterinary Medicine  Vol. 69 (4)  December 2014 Girisgin, A.O. 194
(2) Spray method with Tween 80 solution: Te same mix-
ture used for the frst hive was applied with a spray bottle on
the 3
rd
–8
th
frames at an amount of 25 ml per frame, 12.5 ml
per each surface of combs and at a total of 50 ml in the hive.
(3) Cup method with parafn oil: 50 ml of 1% clove oil
was prepared in parafn oil emulsifer (2 ml clove oil + 48
ml parafn oil) and divided into two aliquots (2×25 ml) in
plastic cups. Te cups were placed at the bottom of the hive
between the 2
nd
–3
rd
and 6
th
–7
th
frames. Two frames were re-
moved for the cups.
(4) Spray method with parafn oil: Te same mixture as used
in hive 3 was applied. Tis mixture was sprayed on the 3
rd
-8
th
frames. It was applied at an amount of 25 ml per frame, 12.5
ml per each surface of combs and at a total of 50 ml in the hive.
(5) Control group: None of essential oil or any acaricide was
applied on beeswaxes.
Te essential oil concentrations and amounts were chosen
on average according to the results of previous published re-
search conducted on clove oil (6, 9, 10). Tese specifc meth-
ods were used to obtain similar conditions for commercial or
home-made drugs of essential oil based acaricides.
Wax Sampling
Using a scalpel blade, 10 cm
2
(approximately 4 g) of wax was
taken from each hive and placed in plastic test tubes. In hives
1 and 3, samples were taken from two diferent frames (2 ×
5 cm
2
of wax) close to the clove oil source; in hives 2 and 4,
samples were taken from the combs onto which the clove oil
was sprayed. In the control hive, samples were taken from
two diferent places. All wax samples were taken at days 1,
2, 5, 7, 14, 21 and 28 after treatment.
Sample preparation procedure
Wax samples for GC/MS analysis were stored in a freezer
(-20°C) for one night. For clove oil residue extraction, 15 ml
of acetonitrile was added to the beeswax samples in 50 ml
polypropylene test tubes. Te samples were placed in a 70°C
water bath for melting.
After 40 minutes, the samples were shaken and left to
separate in the 70°C bath for 5 min. Tubes with the ace-
tonitrile phase with extracted clove oil residues on top and
beeswax at the bottom were held at room temperature for at
least one hour. Te liquid on top was fltered using Millipore
flter paper into another test tube. Next, 1.5 ml of this liquid
was transferred to Eppendorf test tubes and centrifuged for
15 min at 45000 rpm to separate possible particulate materi-
als and wax particles. After centrifugation, all samples were
transferred to the auto-sampler vials and held in the freezer
(-20°C) until GC/MS analysis was performed.
RESULTS
Composition of clove essential oil (Syzygium aromaticum)
obtained by GC/MS analysis resulted under our test con-
ditions in the identifcation of thirty-six diferent compo-
nents. Eugenol and trans-Caryophyllene were the two major
components in the oil, together representing 84.45% of the
sample; other components were present in small amounts
(Table 1). None of clove oil components was found in the
beeswax of control group.
Table 1: Major components of Syzygiumaromaticum essential oil
determined by GC/MS analysis
Component Percentage*
Eugenol 75.35
trans-Caryophyllene 9.10
(-) Caryophyllene oxide 4.59
beta-Selinene 2.15
delta-Cadinene 0.98
* Relative percentage obtained from GC peak area
Table 1 illustrates the amounts of the major components
of clove essential oil in beeswax and Figure 1 illustrates the
levels of eugenol and it’s depletion compare with beeswax
diferent excipients and modes of applications in hives, over
time obtained using GC-MS. Levels (mg/g) were calculated
based on 10 cm
2
of beeswax foundation and corrected to 1g
of beeswax foundation in each test hive.
As can be observed from the data, the amount of eugenol
decreased until the 5
th
day. After this point, levels increased
up to the third week in all groups.
Te role of excipients can be seen in Figure 1 and Table
2. Te amount of eugenol in clove oil mixed with parafn oil
(hive three) was evaluated as very low, and it had evaporated
at the earliest time (two weeks) when compared to the oth-
ers. Te amount of eugenol in beeswax with the other usage
methods was exactly at the same level in the 4
th
week. Tis
indicates that 1% clove oil is trapped and accumulated in
the wax for at least three weeks if it is mixed with tween 80
and is applied by evaporation from cups or spraying. Tis is
also true if it is mixed with parafn oil applied by spraying.
Research Articles
DECEMBER Book.indb 194 04/12/2014 10:57:06
Israel Journal of Veterinary Medicine  Vol. 69 (4)  December 2014 195 Eugenol Stability in Beeswax
DISCUSSION
Knowledge regarding the stability of the active ingredients of
acaricides is very imperative as residue risks in bee products
and residue studies should always include an investigation
into the stability of the analyzed active ingredient.
Eugenol, as well as thymol, eucalyptol, camphor and
menthol have a ‘Food and Agriculture Organization-
Generally Recognized as Safe (for human consumption)
(FAO-GRAS)’ status at a concentrations up to 50 mg/kg.
According to EU regulation No.2377/90, eugenol is within
group II of the nontoxic veterinary drugs, which do not re-
quire a maximum residue limit (MRL) (15). However, if clove
oil (eugenol) is used during honey fow, there is a signifcant
possibility that eugenol residues in honey might reach levels
above the taste threshold (16). As a result honey harvesting
time after essential oil treatments has gained importance.
Te primary component of clove oil was found to be eu-
genol, which, together with analogs and allylphenols, a mem-
ber of the phenylpropanoid class of chemical compounds.
Te acaricidal properties demonstrated by clove oil and
its major component eugenol have shown promising results
on Varroa spp. both during in vitro and in vivo conditions (6,
9, 10). Promising results have also been obtained from euge-
nol for use against scabies mites as a topical acaricide (17).
Clove oil is non-toxic to fsh and acts as an anesthetic for
some fsh species; it has a low toxicity to mammals and is tox-
ic to ants (11, 18). Toxicity tests have shown lethal efect of S.
aromaticum on the mite of honeybees V. destructor. Its toxicity
on honeybees can change depending on factors such as dose,
temperature and evaporation time, but it is agreed as being
safe if it is used at proper doses for Varroa mites at the concen-
tations of approximately 1% (10), as was done in this study.
Chemical composition of the clove essential oil may vary
widely due to the origin of plant and other environmental
variables (11). In our study, the major component of clove oil
was eugenol 75.35%, with trans-caryophyllene 9.1% and lesser
amounts of other components (Table 1). Nevertheless, some
researchers have found diferent proportions such as, eugenol
63.37% with β-caryophyllene 15.94% (19), and in another
study, eugenol 86.7% with β-caryophyllene 3.2% (10).
After the application of clove oil, there were consider-
able eugenol residues in the wax, especially during the frst
few days. Te increase of eugenol levels from the 7
th
day in
almost all groups is likely due to the trapping capacity of
wax and volatility of the oil. Similar results were observed
in a trial with thymol, whereby an increase was reported to
be present on the 50
th
day in brood combs placed in a hive
without bees (13).
We used only eugenol to indicate the stability of clove oil
in beeswax. Because eugenol is the major compound, com-
prising 75.35% in this research, it can be used as a represen-
tative and reference compound for clove oil and for the less
volatile compounds in clove oil.
Beeswax is complex to analyze because it is composed
of fatty esters and long chain hydrocarbons, which are both
readily extracted by organic solvents commonly used in resi-
due analysis. Tis causes important extraction interferences
because these lipids cannot be fully separated from pesticides
(20). Similarly, there were many components in blank bees-
wax samples used in our control group however none of them
belonged to clove oil.
Table 2: Levels (mg/g) of eugenolfrom clove oil in 1 g of sampled
beeswax foundation exposed with diferent excipients analyzed
by GC/MS in eachhive*
Day Hive 1
(mg/g)
Hive 2
(mg/g)
Hive 3
(mg/g)
Hive 4
(mg/g)
1 3.094 3.125 0.593 2.711
2 1.017 0.846 0.233 0.696
5 0.034 0.171 0.015 0.259
7 0.057 0.224 0.022 0.213
14 0.048 0.041 0** 0.081
21 0.005 0.007 0 0.021
28 0 0 0 0
* Levels were calculated basedon 10cm
2
of beeswax foundation and
corrected to 1 g of beeswax. Units are mg eugenol/g in beeswax
foundation
** Levels below limit of detection
Figure 1: Levels (mg/g) of eugenol in beeswax in hives analyzed and
measured in beeswax within the hives
Research Articles
DECEMBER Book.indb 195 04/12/2014 10:57:07
Israel Journal of Veterinary Medicine  Vol. 69 (4)  December 2014 Girisgin, A.O. 196
Te importance of carriers to be introduced to beehives
is stated for the use of clove oil in Varroosis management
program (10). Terefore the excipients used in this work and
quantities of oil in the wax may help in the assessment of its
bioactivity under semi-feld conditions. However, it is recom-
mended that similar studies with bees should be planned in
the next stage of this project.
Clove oil or any aromatic oil residues in beeswax may
pose a problem for the taste of the honey (16). However,
this residue can become an advantage for the treatment
of Varroosis due to the trapping and releasing capacity of
beeswax for the efective compounds inside. Te eugenol
and other oil substances participate as sustained release dos-
age forms when they enter into beeswax or in situ retarding
release formulation, a process that can potentially prolong
the efect of clove oil against Varroa infestation (6). Both
situations should be considered before harvest and during
treatment period and therefore we aimed to determine the
duration of this period in our study.
Te results obtained show that if clove oil is used to
combat Varroosis of honeybee colonies, its most active in-
gredient, eugenol, can be stable for two to three weeks per
treatment period. Tis means that clove oil treatment should
be repeated once or twice after two weeks. Tus, the honey
harvest must be begun at least three weeks after the clove
oil treatment to prevent the presence of the taste of clove
in the honey. Additionally, the treatment of honeybees with
clove oil to prevent/combat Varroa destructor can be efective
for approximately two weeks; therefore, a booster treatment
should be applied after two weeks. Additional work to de-
termine the stability of clove or any diferent essential oils
used as acaricide with more hives and with bees, needs to be
carried out in order to broaden this research in future.
ACKNOWLEDGEMENTS
Tis work was supported by the Commission of Scientifc Research
Projects at Uludag University, project number: YDP(V)-2010/2.
Te authors thank Dr. Victoria Soroker for her contributions and
thank Prof. Levent Aydın for his eforts to promote collaboration
between Turkish and Israeli beekeepers and scientists. Tis paper
has been edited by the American Journal Experts (AJE).
REFERENCES
1. Rosenkranz, P., Aumeier, P. and Ziegelmann, B.: Biology and
control of Varroa destructor. J. Invert. Pathol. 103: Supplement 1,
S96-S119, 2010.
2. Bogdanov, S., Imdorf, A. and Kilchenmann, V.: Residues in wax and
honey after Apilife VAR treatment. Apidologie. 29: 513-524, 1998 a.
3. Chauzat, M.P. and Faucon, J.P.: Pesticide residues in beeswax
samples collected from honey bee colonies (Apis mellifera L.) in
France. Pest Manag. Sci. 63: 1100-1106, 2007.
4. Milani, N.: Te resistance of Varroa jacobsoni Oud. to pyrethroids:
a laboratory assay. Apidologie. 26: 415-429, 1995.
5. Pettis, J.: A scientifc note on Varroa destructor resistance to cou-
maphos in the United States. Apidologie. 35: 91-92, 2004.
6. Imdorf, A., Bogdanov, S., Ochoa, R.I. and Calderone, N.W.: Use
of essential oils for the control of Varroa jacobsoni Oud. in honey
bee colonies. Apidologie. 30: 209-228, 1999.
7. Isman, M.: Plant essential oils for pest and disease management.
Crop Prot. 19: 603-608, 2000.
8. Hoppe, H.: Vergleichende Untersuchungen zur biotechnischen
Bekämpfung der Varroatose, Dissertation, Justus-Liebig-Univer-
sität Giessen, 1990. Cited in Imdorf et al., 1999.
9. Al-Abbadi, A. and Nazer, I.K.: Control of Varroa mite (Varroa
destructor) on honeybees by aromatic oils and plant materials. J.
Agric. & Marine Sci. 8: 15-20, 2003.
10. Maggi, M.D., Rufnengo, S.R., Gende, L.B., Sarlo, E.G.,
Eguaras, M.J., Bailac, P.N. and Ponzi, M.I.: Laboratory evalu-
ations of Syzygium aromaticum (L.) merr. et perry essential oil
against Varroa destructor. J. Ess. Oil Res. 22: 119-122, 2010.
11. Chaieb, K., Hajlaoui, H., Zmantar, T., Kahla-Nakbi, A.B.,
Rouabhia, M., Mahdouani, K. and Bakhrouf, A.: Te chemical
composition and biological activity of clove essential oil, Eugenia
caryophyllata (Syzigium aromaticum L. Myrtaceae): a short review.
Phytother. Res. 21: 501–506, 2007.
12. Shapiro, R.: Prevention of vector transmitted diseases with clove
oil insect repellent. J. Pediatr. Nurs. 27: 346-349, 2012.
13. Bogdanov, S., Kilchenmann, V. and Imdorf, A.: Acaricide resi-
dues in some bee products. J. Apic. Res. 37: 57-67, 1998.
14. Aldicara, J.: Essential Oil. In: Encyclopedia of Chemical Process-
ing and Design. p. 262, Marcel Dekker, New York, 1976.
15. EU Council Regulation (EEC) No. 2377/90, Ofcial Journal of
the EU Nr.224 from 18 August 1990, 1990.
16. Bogdanov, S., Kilchenmann, V., Fluri, P., Bühler, U. and Lavanchy,
P.: Infuence of organic acids and components of essential oils on
honey taste. Am. Bee J. 139: 61-63, 1999.
17. Pasay, C., Mounsey, K., Stevenson, G., Davis, R., Arlian, L., Mor-
gan, M., Vyszenski-Moher, D., Andrews, K. and McCarthy, J.:
Acaricidal activity of eugenol based compounds against scabies
mite. PlosOne. 5: e12079, 2010.
18. Kafe, L. and Shih, C.J.: Toxicity and repellency of compounds
from clove (Syzygium aromaticum) to red imported fre ants So-
lenopsis invicta (Hymenoptera: Formicidae). J. Econ. Entomol.
106: 131-135, 2013.
19. Omidbeygi, M., Barzegar, M., Hamidi, Z. and Naghdibadi, H.:
Antifungal activity of thyme, summer savory and clove essential
oils against Aspergillus xavus in liquid medium and tomato paste.
Food Control. 18: 1518-1523, 2007.
20. Fernandez, M., Pico, Y. and Manes, J.: Analytical methods for
pesticide residue determination in bee products. J. Food Protect.
65: 1502-1511, 2002.
Research Articles
DECEMBER Book.indb 196 04/12/2014 10:57:07

Published under a Creative Commons License By attribution, non-commercial