Food and Chemical Toxicology 138 (2020) 111207

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Food and Chemical Toxicology

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Acaricidal activity, mode of action, and persistent efficacy of selected T

essential oils on the poultry red mite (Dermanyssus gallinae)
Mohaddeseh Abouhosseini Tabaria, Arash Rostamib, Aref Khodashenasb, Filippo Maggic,∗,
Riccardo Petrellic, Cristiano Giordanid,e, Léon Azefack Tapondjouf, Fabrizio Papag, Yanting Zuog,
Kevin Cianfaglioneh,i, Mohammad Reza Youssefij,∗∗
a
Faculty of Veterinary Medicine, Amol University of Special Modern Technologies, Amol, Iran
b
Young Research Club and Elite, Babol Branch, Islamic Azad University, Babol, Iran
c
School of Pharmacy, University of Camerino, Camerino, Italy
d
Instituto de Física, Universidad de Antioquia, Calle 70 No. 52-21, Medellín, Colombia
e
Grupo Productos Naturales Marinos, Facultad de Ciencias Farmacéuticas y Alimentarias, Colombia
f
Laboratory of Environmental and Applied Chemistry, Faculty of Science, University of Dschang, Dschang, Cameroon
g
School of Science and Technology, University of Camerino, Camerino, Italy
h
EA 2219 Géoarchitecture, UFR Sciences & Techniques, Université de Bretagne Occidentale, Brest, France
i
School of Biosciences and Veterinary Medicine, University of Camerino, ì Camerino, Italy
j
Department of Veterinary Parasitology, Babol Branch, Islamic Azad University, Babol, Iran

A R T I C LE I N FO A B S T R A C T

Keywords: In this work, the essential oils (EOs) from Litchi chinensis, Clausena anisata, Heracleum sphondylium, Pimpinella
Dermanyssus gallinae anisum, Lippia alba, Crithmum maritimum and Syzygium aromaticum were tested for their contact toxicity against
Essential oils the poultry red mite, Dermanyssus gallinae, a deleterious ectoparasite of aviary systems. In addition, in order to
Acaricides give insights on their mode of action and effectiveness, the vapor phase and residual toxicity tests were also
Food safety
performed. Results showed that amongst all the tested EOs, that of S. aromaticum demonstrated the highest
contact toxicity, with a LC50 value of 8.9 μg/mL, followed by C. maritimum and L. chinensis EOs, with LC50 values
of 23.7 and 24.7 μg/mL, respectively. L. chinensis and C. anisata EOs showed higher vapor toxicity than the other
EOs. L. chinensis and S. aromaticum EOs showed promising toxic effects up to 4 days post-application. Taken
together, these results highlighted L. chinensis and S. aromaticum as two promising sources of biopesticides, able
to cause severe contact, vapor and residual toxicity in the poultry red mites. Given the wide plant cultivation and
uses in foodstuffs, cosmetics, flavour and fragrances, these EOs may be considered cheap and ready-to-use
products as valid, eco-friendly alternatives to pesticides currently used in the aviary systems.

1. Introduction rhusiopathiae, Escherichia coli, Streptomyces sp., Staphylococcus sp., Yer-

sinia, Listeria, Pasteurella sp. and Rickettsia sp.), viruses (Newcastle dis-
Dermanyssus gallinae (De Geer, 1778) (Acari, Dermanyssidae), also ease virus, chickenpox virus, fowl typhoid, and fowl cholera), and
known as the poultry red mite, is the most harmful ectoparasite of parasites such as the genus Hepatozoon (Chirico et al., 2003; Moro et al.,
laying hens in many countries around the world (Sparagano et al., 2005, 2009; Zeman et al., 1982). From an economic point of view, this
2014; Tabari et al., 2017). To a lesser extent, it can also affect broilers, mite is responsible for losses of millions of dollars per year, due to its
aviary birds, wild birds, and in some cases, poultry operators causing higher feed intake causing a lower egg quality and production, high
itching dermatitis (Chauve, 1998; Marangi et al., 2009). This obligatory mortality and expensiveness of the treatment costs (Kilpinen et al.,
blood-sucking ectoparasite causes blood loss leads to anaemia, irrita- 2005; Van Emous et al., 2006). As a chemical control, long term ap-
tion, stress, restlessness, blood-stained eggs, and, in the case of massive plication of pesticides (e.g., organophosphates, organochlorines, car-
infestation, even death (Kirkwood, 1967). Moreover, this mite is likely bamates, amitraz and pyrethroids) induced heritable resistance in
a vector of many pathogenic agents such as bacteria (Erysopelothrix mites, undesirable effects on non-target organisms, threatening both

∗
Corresponding author.
∗∗
Corresponding author.
E-mail addresses: fi[email protected] (F. Maggi), youssefi[email protected] (M.R. Youssefi).

https://doi.org/10.1016/j.fct.2020.111207
Received 26 November 2019; Received in revised form 7 February 2020; Accepted 14 February 2020
Available online 16 February 2020
0278-6915/ © 2020 Elsevier Ltd. All rights reserved.
M.A. Tabari, et al. Food and Chemical Toxicology 138 (2020) 111207

animals and human health and environmental issues (Flamini, 2003; 2.2. Hydrodistillation
Naqqash et al., 2016).
On the above, there is an urgent need to substitute these chemicals The plant material cut into small pieces was put into glass flasks of
through the adoption of safer alternative control strategies. Plant-de- 6–10 L capacity equipped with Clevenger-type modules and subjected
rived essential oils (EOs) are liquid mixtures usually made up of small, to hydrodistillation for 3 h. The oil yield (w/w) was determined on a
lipophilic and bioactive compounds of terpenoid or phenylpropanoid dry weight basis; it was 5.6, 0.3, 1.6, 2.9, 0.4 and 2.0% for S. ar-
nature, with many of them being used as natural acaricides devoid of omaticum, L. chinensis, C. maritimum, P. anisum, H. sphondylium, and C.
detrimental effects on non-target organisms and environment (Benelli, anisata essential oils, respectively.
2015).
Although botanical pesticide research has been mainly focused on 2.3. GC-FID and GC-MS analyses
mosquito and ticks (Pavela et al., 2016), studies on other arthropods of
medical and veterinary importance are still in the preliminary phase. An Agilent 4890D gas chromatograph coupled with an ionization
Several botanical pesticides are currently used in arthropod pest man- flame detector (FID) was used. The separation was achieved on HP-5
agement. For instance, products based on the neem tree are quite capillary column (5% phenylmethylpolysiloxane, 25 m, 0.32 mm i.d.;
common. Neem extracts are reported to have toxic effects against 200 0.17 μm f.t.) (J & W Scientific, Folsom, CA). The oven temperature was
species of arthropod pests (Choi et al., 2004). Plants can produce a taken 5 min at 60 °C, then raised to 220 °C at 4 °C/min up, finally up to
broad range of secondary metabolites such as terpenoids, poly- 280 °C at 11 °C/min. The essential oils were diluted in hexane (1:100)
acetylenes, sugars, flavonoids and alkaloids which may act as anti- and 1 μL of the solution was injected with a split ratio of 1:34. The
feedants and repellents. In addition, they can also suppress the acet- temperature of the injector and detector was 280 °C. A mixture of n-
ylcholinesterase activity leading to nervous system intoxication (Isman, alkanes (C8-C30) (Supelco, Bellefonte, CA) was used under the above
2006; Masoumi et al., 2016; Tabari et al., 2015; Vigan, 2010). Besides, conditions to calculate the linear retention index (RI) of peaks. The
they may act on other types of targets in the nervous system like ni- quantitative values of peaks, as relative peak areas, were obtained by
cotinic acetylcholine receptors (nAChR), octopamine receptors, tyr- FID peak area normalization.
amine receptors, sodium channels and γ-aminobutyric acid (GABA)- GC-MS analysis was carried out by an Agilent 6890N gas chroma-
gated chloride channels (Coats et al., 1991; Kostyukovsky et al., 2002; tograph equipped with 5973N mass spectrometer (MS). A HP-5MS ca-
Nesterkina et al., 2018). pillary column (5% phenylmethylpolysiloxane, 30 m, 0.25 mm i.d.,
On the above, we decided to document the potentially toxic effects 0.1 μm f.t.) (J & W Scientific) was used for separation. The oven tem-
of some plant EOs, notably those from Litchi chinensis Sonn., Clausena perature was set to 60 °C for 5 min, then raise to 220 °C at 4 °C/min,
anisata (Willd.) Hook.f. ex Benth., Heracleum sphondylium L., Pimpinella finally up to 280 °C at 11 °C/min, held for 15 min, and to 300 °C at
anisum L., Lippia alba (Mill.) N.E.Br. ex Britton & P.Wilson, Crithmum 11 °C/min, held for 5 min. The carrier gas was helium at a flow rate of
maritimum L., and Syzygium aromaticum (L.) Merr. & L.M.Perry on D. 1.0 mL/min. The temperatures of the injector and transfer line were
gallinae. The selection of the above EOs was based on previous reports 280 and 250 °C, respectively. The essential oils were diluted in hexane
documenting their effectiveness against insect vectors and pests of (1:100) and 2 μL of the solution injected into GC-MS with a split ratio of
public health and agricultural importance (Park and Shin, 2005; Benelli 1:50. The scan time was 75 min; the acquisition mass range was 29–400
et al., 2018; Kamte et al., 2018; Pavela et al., 2017, 2018). Therefore, m/z. The peak assignment was based on the correspondence of MS and
we evaluated the toxicity of these EOs through contact and fumigant RI of peaks with those stored in the commercial libraries ADAMS 2007
assays on adult mites. Also, their duration of action was assessed (Adams, 2007), NIST 17, FFNSC2 and WILEY 275 using the ChemSta-
through residual toxicity bioassays. tion software. In addition, whenever possible the co-injection of au-
thentic standards (Sigma-Aldrich, Milan, Italy) available in the authors’
laboratory was used as an additional criteria.
2. Material and methods

2.1. Plant material 2.4. Mites

Fresh leaves of C. anisata were gathered in the village of Bafou (N 5° Red mite (D. gallinae) colonies used in this study were collected from
32′ 30.0′′; E 10° 06′ 13.7″), Western Cameroon, in December 2016. A a laying hen farm near Quchan, Khorasan Razavi province, Iran, in June
voucher specimen was archived in the Cameroon National Herbarium, 2019. The collected specimens were transferred to a sealable glass
Yaoundé, under the codex 44242/HNC. Schizocarps of P. anisum were container and held at 25 ± 1 °C and 55 ± 5% relative humidity
obtained from a farm sited in Castignano (N 42°56′10″, E 13°35′00″, (R.H.). Mites were stored for 24 h before the experiment phase.
496 m a.s.l.), central Italy, in September 2014. Schizocarps of H.
sphondylium were obtained from plants growing in Montelago (N 2.5. Contact toxicity
43°06′30”; E 12°58′25″), central Italy, in July 2015. A voucher spe-
cimen was deposited in the Herbarium Universitatis Camerinensis The toxicity caused by direct contact was assayed based on the
(CAME), School of Bioscience and Veterinary Medicine, University of protocol designed by Tabari et al. (2015). We prepared different dilu-
Camerino with the code CAME 25673. The flowering aerial parts of C. tions of EOs in ethanol (50 μL) corresponding to concentration of 5, 10,
maritimum were collected in Le Conquet (N 48°20′49.31″, O 20, 50, 100 and 200 μg/mL. Whatman No. 1 filter papers were im-
4°46′12.04″), Brittany, France in September 2016 and a voucher spe- pregnated with corresponding dilutions of EOs, and after 3 min drying
cimen was stored in the Herbarium of Géoarchitecture, Université de in a fume cupboard were added to Petri dishes (4.8 cm dia-
Bretagne Occidentale with the reference BRECK9. For L. alba, we di- meter × 1.4 cm). Then, alive mites of all motile stages (n = 30) were
rectly bought the relative EO from Centro de Investigación de transferred into each Petri dish, and all were sealed with parafilm™.
Excelencia – CENIVAM, Bucaramanga, Santander, Colombia. Cloves Controls were treated with only 50 μL of ethanol. All assays were car-
buds S. aromaticum were purchased from the herbal company Minardi & ried out in the same aforementioned conditions of temperature and
Figli (https://www.minardierbe.it) in July 2013. The fruits of L. chi- humidity. Three replicates were performed at the same time for all the
nensis (origin: Madagascar) were purchased from a market in Rome, tested groups of mites. Observations were made by stereomicroscope,
Italy, in July 2013. 24 h after treatment and the number of dead mites was recorded.
Permethrin was used as a positive control.

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M.A. Tabari, et al. Food and Chemical Toxicology 138 (2020) 111207

2.6. Vapor phase toxicity 3.2. Contact toxicity

To find out whether the mode of action of the EOs on red mite was Toxicity data of the seven EOs tested on adult D. gallinae by direct
by contact toxicity or vapor toxicity, the protocol described by Tabari contact are shown in Table 2. Based on the LC50 values, the S. ar-
et al. (2015) was used. In brief, 20 mites were put in a 2 mL container omaticum EO was the most toxic, showing a LC50 value of 8.9 μg/mL,
and two ends of the container were closed by using a mesh barrier followed by C. maritimum and L. chinensis EOs, with LC50 values of 23.7
which limited the direct contact of mites with the tested EOs. These and 24.7 μg/mL, respectively. Permethrin as positive control resulted in
containers were placed in 15 mL vessels containing EO treated filter a LC50 value of 35.1 μg/mL, which was significantly higher than that of
papers with concentrations equal to their contact LC50 values in 50 μL S. aromaticum and L. chinensis, but not C. maritimum. Treatment with
of ethanol calculated based on 15 mL volume of the vessel container. EOs of P. anisum, C. anisata and L. alba resulted in a LC50 value of 47.5,
Mite containing vessels were either closed with a layer of parafilm™ 59.0, and 77.7 μg/mL, respectively. Finally, the EO of H. sphondylium
(Closed) or left uncovered (Open). Mortality rates were determined had a weak effect on D. gallinae, resulting in 50% lethality at con-
after 24 h by pushing mites with a pin. If no reaction was recognized centrations higher than 200 μg/mL. When comparing the LC90 values of
under a loop, mites were regarded as dead. the tested EOs, S. aromaticum was the most potent acaricidal agent
against D. gallinae, with a value of 19.7 μg/mL. P. anisum and L. chi-
nensis were the next two efficacious acaricidal EOs though their LC90
2.7. Residual toxicity values were significantly higher (78.0 and 121.9 μg/mL, respectively).
Permethrin LC90 value was 210.2 μg/mL.
The assay was performed according to a previously described
method (Masoumi et al., 2016). In brief, aluminum tray surfaces were 3.3. Vapor toxicity
sprayed with each tested compound at its contact LC50 values. After
different time periods, i.e. 1, 2, 4, and 6 days post spraying, mites were All the tested EOs displayed significant toxic effects in the closed
exposed to treated surfaces, and 24 h mortality rates were recorded. vapor phase in comparison with the control vessels (P < 0.05, Fig. 1).
There was also a significant reduction of toxicity in EOs tested when
2.8. Statistical analyses using the open methods (P < 0.05). The L. chinensis and C. anisata EOs
showed higher fumigant toxicity in comparison with the other EOs with
Mite mortality data were subjected to Probit analysis to determine moratlity rates of 78 ± 3.66 and 68 ± 2.8%, respectively. There was
LC50 and LD90 values. Tukey's HSD was used to test the significant no significant difference between the vapor phase toxicity of EOs of P.
difference between EOs in vapor toxicity assays. Values of P < 0.05 anisum, L. alba, C. maritimum and S. aromaticum (P > 0.05) (see Fig. 2).
were considered significant. All the statistical analyses were carried out
using SPSS software, version 18 (Chicago, IL). 3.4. Residual toxicity

EOs were tested for the persistence of their toxic effects on D. gal-
3. Results linae at 1, 2, 4, and 6 days. Considering the high LC50 value in the
contact toxicity assay, the H. sphondylium EO was not further tested for
3.1. Chemical profiles of EOs residual toxicity. As can be seen in Table 2, all tested EOs showed
significant toxic effects at 1 and 2-days post spraying in comparison
The chemical compositions of the seven EOs tested in this work are with the control (P < 0.05). EOs of L. chinensis, C. anisata, and S.
presented in Table 1. aromaticum demonstrated a prolonged toxicity in comparison with the
The S. aromaticum EO chemical profile was dominated by the phe- other EOs at day 2 post spraying. Notably, the toxic effects of L. chi-
nylpropanoid eugenol (81.0%) with a minor contribution by its acetate nensis and S. aromaticum EOs lasted for up to 4 days post spraying
ester (eugenol acetate, 15.5%). The sesquiterpene (E)-caryophyllene (P < 0.05). At a 6-day time point, none of the EOs was able to keep its
was another component occurring at a noteworthy level (2.6%) in this toxicity on the poultry red mite.
EO.
The L. chinensis EO chemical profile was made up of sesquiterpene 4. Discussion
hydrocarbons (91.4%), with α-zingiberene (38.7%), ar-curcumene
(16.9%) and β-selinene (16.2%) as the main constituents. Other note- Arthropod pests are increasingly developing resistance to synthetic
worthy components were β-sesquiphellandrene (5.9%) and (E)-car- pesticides due to the extensive application of these products during the
yophyllene (3.6%). last decades (Naqqash et al., 2016; Tabari et al., 2017). Some of these
The L. alba EO was characterized by monoterpenes (75.7%), with pesticides can contaminate food commodities, soil, water, and even air
carvone (35.2%) and limonene (32.0%) as the most abundant con- by their residues which may impact human health and other life forms
stituents. Germacrene D (14.8%) was the most abundant compound in (Aktar et al., 2009). Growing demands for residue-free food products
the sesquiterpene fraction. has led market trend toward alternative pesticides, such as nature-de-
The major groups dominating the C. maritimum EO composition rived pesticides (Pavela and Benelli, 2016; Pavela et al., 2016; Semmler
were the phenylpropanoids (60.4%) and monoterpenes (39.5%), with et al., 2011). Plant-derived EOs have distinct advantages over synthetic
dill apiole (55.7%), γ-terpinene (14.0%) and carvacrol methyl ether chemicals. For instance, their residues are easily biodegradable in foods
(11.8%) as the main components. and the environment. Besides, thanks to their diverse action sites, re-
The EO of H. sphondylium was almost all made up of aliphatic sistance to these substances is not easy to occur (de Oliveira Monteiro
compounds (94.8%), with esters (89.9%) as the major fraction. Octyl et al., 2010; Pavela and Benelli, 2016; Pavela et al., 2016). Despite
acetate (61.0%) was the most abundant compound followed by octyl these advantages, there are some challenging facts, including EOs sta-
butyrate (9.4%) and octyl hexanoate (8.3%). bility and persistence in the field and determination of their exact
The P. anisum and C. anisata EOs were characterized by phenyl- chemical composition (Turek and Stintzing, 2013).
propanoids (98.8 and 84.0%, respectively), with (E)-anethole (94.8 and Plant-EOs were found to exert repellent, antifeedant, ovicidal, and
64.6%, respectively) as the predominant compound. Methyl chavicol toxic effects using both fumigation and filter paper assays. In contact
(2.6%) and (E)-methyl isoeugenol (16.1%) were other noteworthy toxicity testing 0.07 mg/cm2 of clove (S. aromaticum) bud and leaf EOs,
compounds in these EOs, respectively. 100% mortality has been reported; however, reduction of concentration

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M.A. Tabari, et al. Food and Chemical Toxicology 138 (2020) 111207

Table 1
Chemical composition of the selected essential oils assayed against the poultry red mite Dermanyssus gallinae.a
Componentb RI exp.c RI lit.d Peak area percentage (%)e IDf

S. aromaticum L. chinensis C. maritimum P. anisum H. sphondylium L. alba C. anisata

isopropyl-2-methyl butyrate 884 880 1.4 ± 0.3 RI,MS

α-pinene 926 932 2.3 ± 0.2 0.2 ± 0.0 Trg 0.2 ± 0.0 RI,MS,Co-I
sabinene 966 969 4.7 ± 0.6 Tr 0.6 ± 0.1 RI,MS,Co-I
n-octanal 1005 998 Tr 2.4 ± 0.5 RI,MS,Co-I
p-cymene 1022 1020 3.5 ± 0.4 0.1 ± 0.0 Tr 2.9 ± 0.6 RI,MS,Co-I
limonene 1025 1024 tr 0.2 ± 0.0 32.0 ± 3.0 0.4 ± 0.1 RI,MS,Co-I
γ-terpinene 1056 1054 14.0 ± 1.8 0.1 ± 0.0 Tr 2.4 ± 0.5 RI,MS,Co-I
n-octanol 1071 1071 1.3 ± 0.3 RI,MS
methyl chavicol 1196 1195 2.6 ± 0.5 2.0 ± 0.4 RI,MS
(3Z)-3-octenyl acetate 1200 1200 3.0 ± 0.6 RI,MS
octyl acetate 1218 1219 61.0 ± 3.1 RI,MS,Co-I
thymol, methyl ether 1235 1232 11.8 ± 2.0 RI,MS
carvone 1245 1239 35.2 ± 2.8 RI,MS,Co-I
piperitone 1254 1249 1.1 ± 0.2 RI,MS
(E)-anethole 1287 1282 94.8 ± 2.1 64.6 ± 2.9 RI,MS,Co-I
piperitenone 1337 1340 2.1 ± 0.4 RI,MS
eugenol 1360 1356 81.0 ± 3.4 Tr RI,MS
β-bourbonene 1377 1387 Tr 1.8 ± 0.4 0.1 ± 0.0 RI,MS
β-elemene 1385 1389 Tr 1.0 ± 0.2 0.1 ± 0.0 RI,MS,Co-I
7-epi-sesquithujene 1387 1391 1.3 ± 0.3 RI,MS
octyl butyrate 1392 1394 9.4 ± 1.5 RI,MS,Co-I
(E)-caryophyllene 1410 1417 2.6 ± 0.5 3.6 ± 0.7 0.2 ± 0.0 0.3 ± 0.1 0.8 ± 0.2 RI,MS,Co-I
α-himachalene 1442 1449 1.0 ± 0.2 Tr RI,MS
amorpha-4,11-diene 1457 1451 1.3 ± 0.3 RI,MS
γ-himachalene 1470 1481 1.2 ± 0.3 0.8 ± 0.2 RI,MS
germacrene D 1472 1484 0.2 ± 0.0 0.2 ± 0.0 14.8 ± 2.2 2.2 ± 0.5 RI,MS
β-selinene 1478 1477 16.2 ± 1.1 RI,MS
ar-curcumene 1479 1479 16.9 ± 0.9 Tr RI,MS
α-zingiberene 1492 1493 38.7 ± 2.4 Tr Tr Tr 0.1 ± 0.0 RI,MS
(E)-methyl isoeugenol 1499 1491 16.1 ± 2.6 RI,MS
β-bisabolene 1505 1505 1.1 ± 0.2 Tr 0.1 ± 0.0 0.3 ± 0.1 RI,MS
myristicin 1519 1517 4.4 ± 0.9 0.6 ± 0.2 RI,MS,Co-I
β-sesquiphellandrene 1520 1521 5.9 ± 0.8 0.2 ± 0.0 Tr RI,MS
eugenol acetate 1530 1521 15.5 ± 1.9 RI,MS
octyl hexanoate 1585 1575 8.3 ± 1.4 RI,MS
agarospirol 1620 1632 1.4 ± 0.3 RI,MS
dillapiole 1627 1622 55.7 ± 3.8 RI,MS
β-acorenol 1638 1637 2.0 ± 0.4 RI,MS
α-bisabolol 1672 1685 1.3 ± 0.2 RI,MS
xanthorrhizol 1754 1753 1.0 ± 0.2 RI,MS
(E)-pseudoisoeugenyl 2- 1833 1841 1.3 ± 0.3 RI,MS
methylbutyrate

Oil yield (%, w/w) 5.6 0.3 1.6 2.9 0.4 NDh 2.0
Total identified (%) 99.8 99.2 100.0 99.9 97.5 99.2 99.6
Grouped compounds (%)
Monoterpene hydrocarbons 26.5 Tr 0.7 32.9 9.6
Oxygenated monoterpenes 13.0 0.3 42.8 0.3
Sesquiterpene hydrocarbons 2.9 91.4 Tr 1.2 0.9 21.9 5.2
Oxygenated sesquiterpenes 0.2 7.5 1.3 0.2
Phenylpropanoids 96.6 60.4 98.9 0.6 84.0
Others 0.1 0.3 Tr 94.9 0.3 0.2

a
For sake of clarity, only components occurring at percentages above 1% were included.
b
Compounds are listed in order of their elution from a HP-5 column.
c
Linear retention index on HP-5 column, experimentally determined using homologous series of C8-C30 alkanes.
d
Linear retention index taken from Adams, 2007, or NIST 17 and FFNSC2.
e
Relative percentage values are means of three determinations ± SD.
f
Identification methods: Co-I, based on co-injection with authentic compound (Sigma-Aldrich); RI, based on comparison of calculated RI with those reported in
ADAMS, FFNSC 2 and NIST 17; MS, based on comparison with WILEY, ADAMS, FFNSC2 and NIST 17 MS databases.
g
Tr, % below 0.1%.
h
ND, not declared by manufacturer.

to 0.02 mg/cm2 caused a significant decrease in the lethal activity (Kim be concluded that the toxicity of clove EO is due to direct contact of
et al., 2004). In the present study, clove EO showed significant toxicity mites with the EO, and this was limited in the vapor phase assay. On the
against the poultry red mite with LC50 value of 8.9 μg/cm3 appearing as other hand, the high mortality rate of mites in litchi treated vessels can
the most potent EO tested in contact toxicity assay. On the other hand, clarify the presence of toxic compounds from litchi EO which can exert
in the vapor phase toxicity assay, the EO from litchi (L. chinensis), was their toxicity without direct contact with mites.
the most potent acaricidal agent, resulting in nearly 80% mortality. In The effectiveness of S. aromaticum EO against D. gallinae can be
this case, the mortality rate of clove EO was less than 50%. Thus, it can attributed to its main component eugenol (Lee et al., 2019). This

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M.A. Tabari, et al. Food and Chemical Toxicology 138 (2020) 111207

Table 2
Contact toxicity of the seven essential oils on the poultry red mite, Dermanyssus gallinae, based on 24 h exposure time.
EO LC50a CI95 LC90a CI95 Chi p-value Df

L. chinensis 24.7 ± 1.6 21.7–28.2 78.0 ± 8.0 65.0–97.4 2.228 (ns) 0.693 4
C. anisata 59.0 ± 3.7 52.1–66.9 170.0 ± 17.7 141.9–213.4 4.259 (ns) 0.372 4
H. sphondylium ˃200
P. anisum 47.5 ± 5.2 34.8–51.3 121.9 ± 21.1 85.9–139.1 7.174 (ns) 0.126 4
L. alba 77.7 ± 7.7 64.7–95.6 468.9 ± 19.3 358.9–623.8 1.572 (ns) 0.813 4
C. maritimum 23.7 ± 3.8 18.2–33.7 290.5 ± 65.8 258.1–515.3 0.922 (ns) 0.631 3
S. aromaticum 8.9 ± 0.4 7.9–9.9 19.7 ± 1.7 16.9–24.2 4.001 (ns) 0.405 4
Positive control
Permethrin 35.1 ± 3.1 29.5–41.7 210.2 ± 15.8 157.8–304.3 6.178 (ns) 0.186 4

a
Lethal concentrations (LC50 and LC90) values in μg/mL and CI95–95% confidence intervals; essential oils activity is considered significantly different when the 95%
CI fail to overlap. Chi-square value, not significant (ns) at P > 0.05 level.

phenylpropanoid has demonstrated potent acaricidal effects on mites, aromaticum, C. maritimum, P. anisum and C. anisata, respectively, were
where the position of the double bond in the side chain of the molecule active against D. gallinae. As reported in literature (Afshar et al., 2017),
plays a pivotal role for the bioactivity (Pasay et al., 2010; Sparagano although the mechanism of action of phenylpropanoids is not com-
et al., 2013). Eugenol is also effective to treat bee products against pletely understood, it is assumed that they act as inhibitors of cyto-
varroosis (Girisgin et al., 2014). Regarding the mode of action, it has chrome P450 detoxicative enzymes. S. aromaticum and C. maritimum
been assumed that its functional group may interfere with mitochon- revealed to be rich of eugenol and dillapiole whose acaricidal properties
drial respiration of the target mite (Tewary et al., 2006). were highlighted above. Regarding (E)-anethole-containing EOs,
The most active EOs after S. aromaticum were those from C. mar- namely P. anisum and C. anisata, the higher was the content of the
itimum and L. chinensis. The former was characterized by the phenyl- active compound (94.8 vs 64.6%, respectively), the higher was the
propanoid dill apiole which was previously shown to display contact toxicity of the EO on the poultry red mite (LC50 of 47.5 and 59 μg/mL,
toxicity similar to that of eugenol on the two-spotted spider mite respectively, Table 2). Notably, (E)-anethole was previously found quite
Tetranychus urticae C.L. Koch. (Araújo et al., 2012). In addition, dill active against the larvae of Rhipicephalus microplus Canestrini and Der-
apiole was also capable of affecting the food preference of mites. γ- macentor nitens Neumann (Senra et al., 2013).
Terpinene, another C. maritimum EO main component, is a mono- Determining the mode of action of any chemicals using in this field
terpene hydrocarbon occurring at noteworthy percentages in several is necessary for red mite control since it will give us valuable knowledge
plant EOs that showed effectiveness as acaricidal agents (Cetin et al., on the proper formulation and delivery means. Concerning the life cycle
2010; Lima et al., 2018; Villarreal et al., 2017). The L. chinensis EO was and feeding behavior of D. gallinae, it prefers feeding during dark hours
characterized by sesquiterpenes such as α-zingiberene and ar-curcu- for short period and most of the time it hides in poultry house cracks
mene. These compounds were reported as two main factors affecting and clefts. This is a favorable property for an acaricide to exert its ef-
plant resistance against mites and other arthropod pests (Antonious and ficacy by vapor toxicity without direct contact with mites (George et al.,
Kochhar, 2003; Lima et al., 2016; Maluf et al., 2001). This EO may be 2009). The fumigant activity against D. gallinae has also been reported
interesting as a new source of acaricidal agents since it is obtained from for EOs of Artemisia sieberi Besser, Thymus vulgaris L., Leptospermum
a part (peel) which is a by-product obtained during the litchi proces- scoparium J.R.Forst. & G.Forst., and Mentha pulegium L. (George et al.,
sing. 2009; Tabari et al., 2017). These findings revealed that the mode of
Our study put in evidence that some EOs containing phenylpropa- action for some EOs was mostly through the vapor form, although the
noids such as eugenol, dill apiole and (E)-anethole, namely S. final effect is the result of their multitarget action.

Fig. 1. Fumigant toxicity of essential oils in the vapor form in closed and open containers. Data are presented as Mean ± SEM. Different superscript letters show a
significant difference. ANOVA, post hoc Tukey P < 0.05.

5
M.A. Tabari, et al. Food and Chemical Toxicology 138 (2020) 111207

Fig. 2. Mortality rates of Dermanyssus gallinae after

application of the essential oils at different time
points post spraying. Data are displayed as
Mean ± Standard error; Capital letters indicate
significant difference between different treatments,
Small letters indicate significant difference among a
treatment (P ≤ 0.05, ANOVA, post hoc Tukey).

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