Response of wild Mus musculus to baits containing essential oils: I - Cinnamon and clove tested in storage facilities

Abstract

The effect of cinnamon (Cinnamomi zeylanicum) and clove (Eugenia caryophyllata) essential oils on the acceptibility of cereal-based baits to house mice in storage facilities was examined. The effects of three concentrations, 0.5, 0.75 and 1 %, were tested for optimization purposes. The experiments were performed in a mill storage, and in another storage for seeds and seedlings. Attractiveness of the examined concentrations of cinnamon and clove essential oils was statistically significant, compared to placebo bait, within seven days of the experiment. On the seventh day, the average consumption of baits containing 0.75 % and 1 % concentrations of cinnamon oil was 46 % in the mill storage, i.e. 35.5 % more than placebo baits were consumed. Fourteen days after the beginning of the experiment, no significant difference was detected in the consumption of baits offered in the mill storage. On the other hand, a significant difference was revealed regarding the consumption of examined baits in the seed and seedling storage. The average consumption of baits containing 0.75 % and 1 % cinnamon oil was 47 % and 51 % higher after 14 days than the consumption of placebo bait. Compared to the other test baits, those containing cinnamon essential oil at 0.75 % and 1 % concentration demonstrated better attractiveness without mutual statistically significant differences.

Full text

137
Response of wild Mus musculus to baits containing
essential oils: I - Cinnamon and clove tested
in storage facilities
Goran Jokić 1*, Siniša Mitrić2, Dušan Pejin2, Tanja Blažić1, Suzana Đedović1,
Bojan Stojnić3 and Marina Vukša1
1Institute of Pesticides and Enironmental Protection, Banatska 31b, POB 163,
11080 Belgrade, Serbia
2Faculty of Agriculture, University of Banja Luka, Bulevar ojvode Petra Bojoica 1A
78000 Banja Luka, Republic of Srpska, BiH
3Faculty of Agriculture, University of Belgrade, Nemanjina 6, 11080 Belgrade, Serbia
*Corresponding author: jokicg@ptt.rs
Received: 8 June 2018
Accepted: 12 July 2018
SUMMARY
The effect of cinnamon (Cinnamomi zeylanicum) and clove (Eugenia caryophyllata) essential
oils on the acceptibility of cereal-based baits to house mice in storage facilities was examined.
The effects of three concentrations, 0.5, 0.75 and 1 %, were tested for optimization purposes. The
experiments were performed in a mill storage, and in another storage for seeds and seedlings.
Attractiveness of the examined concentrations of cinnamon and clove essential oils was
statistically significant, compared to placebo bait, within seven days of the experiment. On the
seventh day, the average consumption of baits containing 0.75 % and 1 % concentrations of
cinnamon oil was 46 % in the mill storage, i.e. 35.5 % more than placebo baits were consumed.
Fourteen days after the beginning of the experiment, no significant difference was detected in
the consumption of baits offered in the mill storage. On the other hand, a significant difference
was revealed regarding the consumption of examined baits in the seed and seedling storage.
The average consumption of baits containing 0.75 % and 1 % cinnamon oil was 47 % and 51
% higher after 14 days than the consumption of placebo bait. Compared to the other test
baits, those containing cinnamon essential oil at 0.75 % and 1 % concentration demonstrated
better attractiveness without mutual statistically significant differences.
Keywords: Essential oils; Cinnamon; Clove; Storages; House mouse; Attractants
Pestic. Phytomed. (Belgrade), 33(2), 2018, 137–144 UDC 632.693.234:547.913:633.833+633.832:631.24
DOI: https://doi.org/10.2298/PIF1802137J Original scientific paper
INTRODUCTION
The house mouse, Mus musculus, is one of the
most important commensal rodent species in human
environment. The species may cause significant damage
to plant materials and products, as well as to electrical
wiring, machines and electronic devices (Đukić et
al., 2005). The activity of house mice raises the cost
of product storage both through direct damage and
contamination of primary and processed plant products.

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Goran Jokić et al.
Storage costs also rise due to mandatory rodent control
measures and compensation for economic damages caused
by rodents (Hrgović et al., 1991; Buckle & Smith, 1994).
Cereals, due to their widespread cultivation, nutritive
value and resulting importance, are the most common
carriers of rodenticide active ingredients for most rodents.
Under dry conditions, such baits are stable over long
periods of time. However, their persistence under humid
and warm conditions depends on the rapidness of mold
development, which spoils the quality of bait, and leads
to ultimate reduction in efficacy (Buckle & Smith, 1994).
Various additives serve to prevent and slow down mold
development, and so improve bait quality and persistence
(Brooks, 1962). On the other hand, despite improving
bait stability, many additives degrade their attractiveness
(Buckle & Smith, 1994).
Essential oils are widely used in human and veterinary
medicine, and for protection against insects, mites and
pathogens. The essential oils of cinnamon (Cinnamoumi
zeylanicum) and clover (Eugenia caryophyllata) are used
against insects (Plata Rueda et al., 2018), mites (Rezaei et
al., 2014) and pathogens (Božik et al., 2017). Antifungal
activity of cinnamon and clover essential oils on mold
has been confirmed ( Ju et al., 2018). Also, laboratory
tests have confirmed the attractiveness of cinnamon
and clover essential oils in food to the laboratory mouse,
Swiss strain ( Jokić et al., 2013).
The present study aimed to test possible improvements of
bait acceptability for the house mouse by adding essential
oils, which is an acceptable solution both from the aspect
of environmental protection, quality and safety of food and
human health protection. The results should be meaningful
to experts in extension services, to manufacturers and to
staff providing rodent control treatments.
MATERIAL AND METHODS
Bait preparation
Test baits were prepared according to the relevant OEPP/
EPPO (2004a) methodology. Non-poisonous, placebo
baits were prepared by mixing coarse grain of wheat,
barley and maize at 30:30:40 ratio. Total ratio of the
cereal mixture, wheat flower and maize oil was 90:5:5.
Baits containing cinnamon or clover essential oils were
prepared by adding specific amounts of essential oil, diluted
in 25 ml alcohol, to placebo baits. The placebo baits in
experiments assessing the effects of essential oils on house
mouse food consumption were supplemented with equal
amounts of pure alcohol. The test baits were prepared
to contain 0.5 %, 0.75 % and 1 % essential oils of either
cinnamon or clover, and the oils used in the testing were
manufactured as commercial products by BeoLab, Serbia.
Experimental design
The attractive activity and effects of essential oils on
bait acceptability, and their optimal concentrations, were
determined in trials conducted in a mill storage (950
m
2
) and a storage for seeds and seedlings (1200 m
2
). The
products were stored in sacks or boxes and elevated on
pallets. In the facility for storing seeds and seedlings, a
small part of the material was stored in bulk.
The experiments were performed according to the
relevant OEPP/EPPO (2004b) methodology. Baits were
given in commercial bait boxes with sideways openings
2 cm in diameter to allow entry to mice while barring
other warm-blooded organisms (Figure 1). The boxes
were positioned in concentration sequence at 1-2 m
distance along mice routes, and in the vicinity of damaged
material. Daily offer included 20 g of bait per box.
Figure 1. Bait boxes
Bait attractiveness
Placebo baits containing only the nutrient carrier, and
baits with different contents of cinnamon and clover
essential oils, were laid in bait boxes in acceptability
(attractiveness) trials (Figure 2). A total of 84 bait boxes
were laid in each storage facility, providing the following
arrangement:
• 12 boxes containing placebo bait;
• three concentrations of cinnamon oil:
3×12 boxes = 36 boxes;
• three concentrations of clover oil:
3×12 boxes = 36 boxes.

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Pestic. Phytomed. (Belgrade), 33(2), 2018, 137–144
Consumed bait was measured daily per box, and fresh
bait was added to each as needed. The baits were offered
over a total period of 14 days. The effects were assessed
1, 2, 3, 7 and 14 days after the baits were first offered.
Figure 2. Baits laid
Data processing
The results were processed according Sokal and Rohlf
(1995) using StatSoft software (1997). One-way analysis
of variance (ANOVA) was employed to assess the effects
of different concentrations of cinnamon and clover
essential oils on house mouse feeding in the mill and in
the facility for storing seeds and seedling material. Bait
consumption means were compared using Duncan’s test
at P=0.05 significance level.
The house mouse populations were assessed at the
beginning and the end of each experiment using a method
proposed by Hrgović et al. (1991). Mice numbers were
calculated based on daily consumption of placebo baits
over several consecutive days using the formula:
H
N = ––––––
h × n
where
N = assessed number of animals;
H = total amount of food (g) consumed over the
entire experimental period (n);
h = average daily food requirement of the rodent
species per animal (g), i.e. 6 g/day (six, h=6) for the
house mouse, according to Hrgović et al. (1991);
n = duration of baiting period (days).
RESULTS
Table 1 shows the daily amounts of eaten placebo baits
over a five-day baiting period in the mill storage. House
mouse population was assessed to be 40 animals at the
beginning, and 39 animals at the end of the experiment.
Table 1. Daily placebo bait consumption in the mill storage
(facility I)
Baiting
duration
Placebo bait consumption per day (g)
1st day 2nd day 3rd day 4th day
5
th
day
Beginning
of experiment 192.5 255.8 273.2 237.4 245.6
End of experiment
237.8 241.3 232.6 229.8 240.8
Figures 3-7 show the average mouse consumption of
placebo bait and baits containing different concentrations
of cinnamon and clover essential oils. The symbols
used in the figures should read: Pl= Placebo; C0.5=
Cinnamon 0.5 %; C0.75= Cinnamon 0.75 %; C1=
Cinnamon 1 %; K0.5= Clover 0.5 %; K0.75= Clover
0.75 %; K1= Clover 1 %. The data are means for eaten
(consumed) bait per box, according to Duncan’s test
(p=0.05). Symbols marked with the same letter indicate
non-significant statistical difference.
Figure 3. Average consumption of placebo bait and baits
with different contents of cinnamon and clover
essential oils by house mice on the 1st (first) day
of experiment in the mill storage
Figure 4. Average consumption of placebo bait and baits
with different contents of cinnamon and clover
essential oils by house mice 2 (two) days after
the beginning of experiment in the mill storage

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Goran Jokić et al.
Figure 5. Average consumption of placebo bait and baits with
different contents of cinnamon and clover essential
oils by house mice 3 (three) days after the beginning
of experiment in the mill storage
Figure 6. Average consumption of placebo bait and baits with
different contents of cinnamon and clover essential
oils by house mice 7 (seven) days after the beginning
of experiment in the mill storages
Figure 7. Average consumption of placebo bait and baits with
different contents of cinnamon and clover essential
oils by house mice 14 (fourteen) days after the
beginning of experiment in the mill storage
Daily consumption of placebo baits in the facility
for storing seeds and seedling material, assessed over
five days, is presented in Table 2. Mouse presence was
assessed to be 59 animals at the beginning, and 61
animals at the end of the experiment.
Table 2. Daily consumption of placebo baits in the seed and
seedling storage (facility II)
Baiting
duration
Placebo bait consumption per day (g)
1st day 2nd day 3rd day 4th day
5
th
day
Beginning
of experiment 324.4 334.7 374.5 361.1 389.7
End of experiment
355.6 394.2 367.7 354.4 370.4
Figures 8-12 show the average consumption of
placebo baits and baits supplemented with different
concentrations of cinnamon and clover essential oils
by house mice in the storage for seeds and seedlings.
Figure 8. Average consumption of placebo baits and baits
supplemented with different concentrations of
cinnamon and clover essential oils by house mice
on the 1st (first) day of experiment in the seed and
seedling storage
Figure 9. Average consumption of placebo baits and baits
supplemented with different concentrations of
cinnamon and clover essential oils by house mice
after 2 (two) days of experiment in the seed and
seedling storage

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Pestic. Phytomed. (Belgrade), 33(2), 2018, 137–144
Figure 10. Average consumption of placebo baits and baits
supplemented with different concentrations of
cinnamon and clover essential oils by house mice
after 3 (three) days of experiment in the seed and
seedling storage
Figure 11. Average consumption of placebo baits and baits
supplemented with different concentrations of
cinnamon and clover essential oils by house mice
after 7 (seven) days of experiment in the seed and
seedling storage
Figure 12. Average consumption of placebo baits and baits
supplemented with different concentrations of
cinnamon and clover essential oils by house mice
after 14 (fourteen) days of experiment in the seed
and seedling storage
The average consumption of placebo baits and
baits supplemented with different concentrations of
cinnamon and clover essential oils by house mice in the
mill storage (facility I), and seed and seedling storage
(facility II) showed significant statistical differences
after 1, 2, 3 and 7 days. No significant difference was
detected in daily bait consumption in the mill storage
after 14 days (F
6.21
=0.688; P=0.6611). In the seed
and seedling storage, bait consumption data for the
same time period also revealed significant differences
(Table 3).
On the first day of baiting in facility I, the highest
daily consumption was noted for baits containing 0.75
and 1 % cinnamon essential oil, and for the highest
concentration of clover essential oil. In facility II, the
highest level of initial attractiveness was demonstrated
by baits with 0.75 % cinnamon oil, and the average daily
consumption was 3.2 g.
On the second day of baiting in facility I, the highest
attractiveness was shown by baits with 0.75 and 1 %
concentration of cinnamon oil, and the average daily
consumption was 6.8-7.3 g/day. Excepting placebo
baits, which achieved the lowest attractiveness for house
mice, there were no significant statistical differences
among any of the other baits. In facility II, the highest
daily consumption was found for baits containing
1 % cinnamon oil concentration and for the lowest
concentration of clover oil.
Comparing data for facility I on the 3rd day of
experiment w ith the other test baits, cinnamon
oil concentrations of 0.75 and 1 % showed the best
attractiveness for house mice. The average daily
consumption of those baits was steadily at around 9.3
g/day. Daily consumption of all other baits, including
placebo bait, was not significantly different. In facility
II, baits with 0.75 and 1 % cinnamon oil concentrations,
as well as baits with the highest clover oil concentration,
showed attractiveness for the house mouse. Daily
consumption of those baits showed no mutual difference
on average in statistical terms.
Seven days after the experiment started in facility I,
the highest level of attractiveness for the house mouse
was demonstrated by cinnamon oil bait with 0.75 %
concentration as its average daily consumption was 20.1
g. The average daily consumption of baits with the lowest
concentration of clover oil and placebo baits was at the
same level and showed no statistical difference over the
given period. In facility II, the average daily consumption
of baits containing the two top concentrations of
cinnamon oil was 20.8 g on average and higher than
consumption of all other baits.

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At the end of the experiment (14 days), the average
daily bait consumption in facility I ranged from 22.2 g
(clover oil concentration 0.75 %) to 27.4 g (cinnamon
oil concentration 0.75 %). In facility II, baits with
all three concentrations of cinnamon oil and the
highest concentration of clover oil showed the highest
attractiveness.
Table 3. Effects of different concentrations of cinnamon and
clover essential oils on bait consumption by house
mice in the mill, and in the facility for storing seeds
and seedling material (one-way ANOVA)
Assessment
intervals
Facility I*Facility II†
F6.21 P F P
1st day 2.786 0.0372 9.678 0.0001
2nd day 3.019 0.0275 6.643 0.0001
3rd day 2.972 0.0290 9.307 0.0001
7th day 3.514 0.0141 3.917 0.008
14th day 0.688 0.6611 11.728 0.0001
* Facilit y I = mill storage; † Facilit y II = seed and seedling stora ge
DISCUSSION
The approximate counts of house mice in the facilities
where the attractiveness of baits with different contents
of cinnamon and clover essential oils were evaluated
indicate stable populations which were not exposed to
any other harmful activity.
All storages were isolated units in which no
rodenticides had been used for at least six months prior
to the experiment.
Buckle and Smith (1994) reported that cinnamon
essential oil may be used as an attractant for rodents,
but neither a formulation nor a rate were suggested.
In our earlier studies, the predominant constituent
of cinnamon essential oil with 96.7 % content was
cinnamaldehyde (IUPAC: (E)-3-phenyl-2-propenal),
known synonymously also as: cinnamic aldehyde,
3-phenyl acrolein, 3-phenyl propenal, benziliden
acetaldehyde, cinamal, 3-phenyl acrilaldehyde, and under
its CAS identifier 104-55-2. Eugenyl acetate (2.2 %)
and eugenol (0.5 %) were found as minor ingredients.
Eugenol is known to have been used for trapping rodents
(Safety Data Sheet, 2016). Eugenol (CAS: 97-53-0),
an organic compound with the chemical formula
C
10
H
12
O
2
, is the main component of clover essential
oil (75 %), while eugenyl acetate content is lower, 4.4 %.
Data analysis showed that all test concentrations of
cinnamon and clover oils increased bait acceptability
as early as 24 h after the experiment started. Regarding
the 2-7 days period of baiting, cinnamon essential oil
was found to have positive effect on bait acceptability,
especially its 0.75 and 1 % concentrations. The highest
concentration of clover oil was also found to influence
bait consumption by house mice over the period, only
less significantly than the cinnamon oil concentrations
mentioned. After seven days, the average consumption of
baits containing cinnamon oil concentrations 0.75 % and 1
% was up to 46 %, i.e. 35.5 % higher than the consumption
of placebo baits. This level of contribution of clover and
cinnamon essential oils to bait attractiveness is consistent
with laboratory data (Jokić et al. 2013).
No significant difference was observed between the
effects of the test concentrations of essential oils and placebo
baits in the mill storage at the end of the experiment, i.e.
after 14 days of assessment. Over the interval between
the last two assessments, average bait consumption was
approximate in the test facility, ranging from 7 to 11 g.
Based on the results, as well as the fact that essential oils
are highly volatile, the test baits were assumed to have lost
their attractiveness to the house mouse over the trial period.
Furthermore, dust may also have spoiled the baits and
decreased their attractiveness. As manufacturers recommend
that baits for rodent control be placed for periods of 7-10
days, the formulated products at concentrations tested
in this study may be expected to achieve full utilization.
Significant statistical differences in bait consumption
were revealed in the seed and seedling storage in all
assessment intervals. The average consumption of baits
containing 0.75 % and 1 % essential oil of cinnamon was 47
% and 51 % higher, respectively, compared to placebo baits.
Analysing the results, baits containing 0.75 and 1 %
cinnamon essential oil were found to possess feeding
attractiveness for the house mouse. As the data showed
steady average daily consumption of baits with those two
concentrations of cinnamon, the lower concentration
of 0.75 % was chosen for further optimization tests for
bait formulation improvement.
ACKNOWLEDGEMENT
The study was funded by the Serbian Ministry of
Education, Science and Technological Development
(Grant III 46008).

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Uticaj etarskih ulja na ishranu jedinki
domaćeg miša: I - cimet i karanfilić
ispitivani u skladištima
REZIME
Utvrđivan je uticaj etarskih ulja cimeta (Cinnamomi zeylanicum) i karanfilića (Eugenia
caryophyllata) na prihvatljivost mamaka na bazi žita za domaćeg miša u skladišnim uslovima.
U ciju optimizacije sadržaja etarskog ulja, ispitivan je efekat tri različite koncentracije, 0.5,
0.75 i 1 %. Eksperimenti su izvedeni u skladišnim objektima mlina i objektima za skladištenje
semenskog i sadnog materijala.
Atraktantno delovanje ispitivanih koncentracija etarskih ulja cimeta i karanfilića u odnosu
na placebo mamac bilo je statistički značajno od početka pa do sedmog dana eksperimenta.

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Goran Jokić et al.
Sedmog dana, u objektima mlina, prosečna konzumacija mamaca sa sadržajem etarskog ulja
cimeta u koncentracijama od 0.75 % i 1 % bila je do 46 %, odnosno 35.5 % viša u odnosu na
placebo mamac. Četrnaestog dana nije zabeležena statistički značajna razlika u konzumaciji
ponuđenih mamaca u objektima mlina. U skladišnim objektima semenskog i sadnog materijala,
zabeležena je statistički značajna razlika u konzumaciji ispitivanih mamaca. Prosečna konzumacija
mamaka sa sadržajem od 0.75 % i 1 % etarskog ulja cimeta, četrnaestog dana od početka
eksperimenta bila je za 47 %, odnosno 51 % viša u odnosu na placebo mamac. U odnosu na
druge ispitivane mamce, mamci sa sadržajem etarskog ulja cimeta u koncentracijama od
0.75 % i 1 % ispoljili su atraktantnije delovanje u odnosu na druge mamce i međusobno se
nisu statistički značajno razlikovali.
Ključne reči: Etarska ulja; Cimet; Karanfilić; Skladišta; Domaći miš; Atraktanti