Journal of Stored Products Research 43 (2007) 123–128 Fumigant toxicity of essential oil from Artemisia sieberi Besser against three stored-product insects Maryam Negahban a , Saeid Moharramipour a, , Fatemeh Sefidkon b a Department of Entomology, College of Agriculture, Tarbiat Modarres University, P.O. Box 14115-336, Tehran, Iran b Research Institute of Forests and Rangelands, P.O. Box 13185-116, Tehran, Iran Accepted 21 February 2006 Abstract Artemisia sieberi is a widely distributed plant in Iran. Because some species of Artemisia are insecticidal, experiments were conducted to investigate fumigant toxicity of the essential oil. Dry ground leaves were subjected to hydrodistillation using a modified Clevenger-type apparatus and the resulting oil contained camphor (54.7%), camphene (11.7%), 1,8-cineol (9.9%), b-thujone (5.6%) and a- pinene (2.5%). The mortality of 7 days old adults of Callosobruchus maculatus, Sitophilus oryzae, and Tribolium castaneum increased with concentration from 37 to 926 mL/L and with exposure time from 3 to 24 h. A concentration of 37 mL/L and an exposure time of 24 h was sufficient to obtain 100% kill of the insects. Callosobruchus maculatus was significantly more susceptible than S. oryzae and T. castaneum; a second more detailed bioassay gave estimates for the LC 50 of C. maculatus as 1.45 mL/L, S. oryzae 3.86 mL/L and T. castaneum 16.76 mL/L. These results suggested that A. sieberi oil may have potential as a control agent against C. maculatus, S. oryzae and T. castaneum. r 2006 Elsevier Ltd. All rights reserved. Keywords: Stored-product insects; Artemisia sieberi; Botanical insecticides; Fumigant toxicity 1. Introduction Pest control in many storage systems depends on fumigation with either methyl bromide or phosphine. The use of methyl bromide is being restricted because of its potential to damage the ozone layer (Butler and Rodriguez, 1996; MBTOC, 1998). The future use of phosphine could be threatened by the further development of resistant strains (Bell and Wilson, 1995; Daglish and Collins, 1999). Many alternatives have been tested to replace methyl bromide fumigation for stored product and quarantine uses. During recent years, some plants have been receiving global attention and their secondary metabolites have been formulated as botanical pesticides for plant protection since they do not leave residues toxic to the environment, have lower toxicity to mammals and medicinal properties for humans (Duke, 1985). Artemisia species (Asteracae) are widely used medicinal plants in folk medicine. Some species such as A. absinthium L., A. annua L. or A. vulgaris L. have been incorporated into the pharmacopoeias of several European and Asian countries (Proksch, 1992). Many of the substances elaborated by the genus are toxic to pathogens or show other significant physiological activity and may be used in human diets or for animal fodder (Heywood and Humphries 1977; Janssen et al., 1987). For example, the essential oil of A. herba-alba Asso inhibited the asexual reproduction of Aspergillus niger Tiegh, Penicillium italicum Wehmer and Zygorrhychus sp. (Tan- taoui-Elaraki et al., 1993). Moreover, Artemisia species may possess insecticidal, repellent or antifeedent properties (Grainge and Ahmed, 1988; Arnason et al., 1989; Jacobson, 1989; Shakarami et al., 2004a, b, c). Artemisia scoparia Waldst and Kit showed fumigant activity against several stored-product pests (Negahban et al., 2004; Negahban and Moharramipour, 2005). Extracts of A. absinthium L. have been shown to possess a range of biological activities, including insecticidal action as an ARTICLE IN PRESS www.elsevier.com/locate/jspr 0022-474X/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jspr.2006.02.002 Corresponding author. Tel.: +98 21 44196522; fax: +98 21 44196524. E-mail address: [email protected] (S. Moharramipour).
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Journal of Stored Products Research 43 (2007) 123–128
www.elsevier.com/locate/jspr
Fumigant toxicity of essential oil from Artemisia sieberi Besser againstthree stored-product insects
aDepartment of Entomology, College of Agriculture, Tarbiat Modarres University, P.O. Box 14115-336, Tehran, IranbResearch Institute of Forests and Rangelands, P.O. Box 13185-116, Tehran, Iran
Accepted 21 February 2006
Abstract
Artemisia sieberi is a widely distributed plant in Iran. Because some species of Artemisia are insecticidal, experiments were conducted
to investigate fumigant toxicity of the essential oil. Dry ground leaves were subjected to hydrodistillation using a modified Clevenger-type
apparatus and the resulting oil contained camphor (54.7%), camphene (11.7%), 1,8-cineol (9.9%), b-thujone (5.6%) and a- pinene
(2.5%).
The mortality of 7 days old adults of Callosobruchus maculatus, Sitophilus oryzae, and Tribolium castaneum increased with
concentration from 37 to 926 mL/L and with exposure time from 3 to 24 h. A concentration of 37 mL/L and an exposure time of 24 h was
sufficient to obtain 100% kill of the insects. Callosobruchus maculatus was significantly more susceptible than S. oryzae and T. castaneum;
a second more detailed bioassay gave estimates for the LC50 of C. maculatus as 1.45 mL/L, S. oryzae 3.86 mL/L and T. castaneum
16.76mL/L. These results suggested that A. sieberi oil may have potential as a control agent against C. maculatus, S. oryzae and
Pest control in many storage systems depends onfumigation with either methyl bromide or phosphine. Theuse of methyl bromide is being restricted because of itspotential to damage the ozone layer (Butler and Rodriguez,1996; MBTOC, 1998). The future use of phosphine couldbe threatened by the further development of resistantstrains (Bell and Wilson, 1995; Daglish and Collins, 1999).
Many alternatives have been tested to replace methylbromide fumigation for stored product and quarantineuses. During recent years, some plants have been receivingglobal attention and their secondary metabolites have beenformulated as botanical pesticides for plant protectionsince they do not leave residues toxic to the environment,have lower toxicity to mammals and medicinal propertiesfor humans (Duke, 1985). Artemisia species (Asteracae) are
ee front matter r 2006 Elsevier Ltd. All rights reserved.
r.2006.02.002
ing author. Tel.: +9821 44196522; fax: +98 21 44196524.
widely used medicinal plants in folk medicine. Some speciessuch as A. absinthium L., A. annua L. or A. vulgaris L. havebeen incorporated into the pharmacopoeias of severalEuropean and Asian countries (Proksch, 1992). Many ofthe substances elaborated by the genus are toxic topathogens or show other significant physiological activityand may be used in human diets or for animal fodder(Heywood and Humphries 1977; Janssen et al., 1987). Forexample, the essential oil of A. herba-alba Asso inhibitedthe asexual reproduction of Aspergillus niger Tiegh,Penicillium italicum Wehmer and Zygorrhychus sp. (Tan-taoui-Elaraki et al., 1993). Moreover, Artemisia speciesmay possess insecticidal, repellent or antifeedent properties(Grainge and Ahmed, 1988; Arnason et al., 1989;Jacobson, 1989; Shakarami et al., 2004a, b, c). Artemisia
scoparia Waldst and Kit showed fumigant activity againstseveral stored-product pests (Negahban et al., 2004;Negahban and Moharramipour, 2005). Extracts of A.
absinthium L. have been shown to possess a range ofbiological activities, including insecticidal action as an
ARTICLE IN PRESSM. Negahban et al. / Journal of Stored Products Research 43 (2007) 123–128124
alcoholic extract against the storage pest Sitophilus
granarius L. (Ignatowicz and Wesolowska, 1994) andnematocidal action against Meloidogyne incognata (Kofoidand White) (Walker, 1995).
Artemisia sieberi Besser (Asteraceae) is a typical desertplant that grows in Iran, Palestine, Syria, Iraq, Turkey,Afghanistan and Central Asia (Podlech, 1986). The presentstudy was conducted to determine the efficiency of theessential oil from A. sieberi as a fumigant in the manage-ment of Callosobruchus maculatus (F.), Sitophilus oryzae
(L.), and Tribolium castaneum (Herbst).
2. Materials and methods
2.1. Insect cultures
Callosobruchus maculatus, S. oryzae and T. castaneum
were reared on bean grains, whole rice and wheat flourmixed with yeast (10:1, w/w), respectively. Adult insects,1–7 days old, were used for fumigant toxicity tests. Thecultures were maintained in the dark in a growth chamberset at 2771 1C and 6575% r.h.
All experiments were carried out under the sameenvironmental conditions.
2.2. Plant materials
Aerial parts of A. sieberi were collected at full-floweringstage in December, 2003 from Qom province in Iran. TheResearch Institute of Forests and Rangelands, Tehran,Iran confirmed the identity of the plant. The plant materialwas dried naturally on laboratory benches at roomtemperature (23–24 1C) for 5 days until crisp. The driedmaterial was stored at �24 1C until needed and thenhydrodistilled to extract its essential oil.
2.3. Extraction and analysis of essential oil
Essential oil was extracted from the plant samples usinga Clevenger-type apparatus where the plant material issubjected to hydrodistillation. Conditions of extractionwere: 50 g of air-dried sample; 1:10 plant material/watervolume ratio, 4 h distillation. Anhydrous sodium sulphatewas used to remove water after extraction. Oil yield (2.9%w/w) was calculated on a dry weight basis. Extracted oilwas stored in a refrigerator at 4 1C.
Gas chromatographic (GC) analysis was performed witha Shimadzu GC-9A with helium as a carrier gas with alinear velocity of 30 cm/s on a DB-5 column (30m �
0.25mm i.d, 0.25 mm film thickness). The oven wasprogrammed to rise to a 60 1C (3min) isotherm, and thento 210 1C at a rate of 3 1C/ min. Injector and detectortemperatures were 300 and 270 1C, respectively. The GCmass analysis was carried out on a Varian 3400 equippedwith a DB-5 column with the same characteristics as theone used in GC. The transfer line temperature was 260 1C.The ionization energy was 70 eV with a scan time of 1 s and
mass range of 40–300 amu. Unknown essential oilcomponents were identified by comparing their GCretention times to those of known compounds and bycomparison of their mass spectra, either with knowncompounds or published spectra.
2.4. Fumigant toxicity
To determine the fumigant toxicity of the A. sieberi oil,filter papers (Whatman No. 1, cut into 2 cm diameterpieces) were impregnated with oil at doses calculated togive equivalent fumigant concentrations of 37–926 mL/L inair. The impregnated filter papers were then attached to thescrew caps of glass vials with volumes of 27mL. Caps werescrewed tightly on the vials, each of which containedseparately 10 adults (1–7 days old) of each species of insect.Each concentration and control was replicated five times.Mortality was determined after 3, 6, 9, 12 and 24 h fromcommencement of exposure. When no leg or antennalmovements were observed, insects were considered dead.Percentage insect mortality was calculated using theAbbott correction formula for natural mortality inuntreated controls (Abbott, 1925).Another experiment was designed to assess 50% and
95% lethal doses. A series of dilutions was prepared toevaluate mortality of insects after an initial dose-settingexperiment. Ten adult insects were put into 280mL glassbottles with screw lids, which were dosed as described inthe first experiment above. Concentrations of the oil testedon C. maculatus were 0, 0.70, 1.07, 1.43, 1.79, 2.14, 2.50,2.86 and 3.21 mL/L air. Sitophilus oryzae was evaluated at0, 1.43, 1.74, 2.14, 2.86, 3.57, 4.29, 5.36, 6.07, 7.14 and8.93 mL/L air, and Tribolium castaneum at 0, 10.71, 14.29,17.86, 21.43, 25.00, 28.57 and 32.14 mL/L air. Controlinsects were kept under the same conditions without anyessential oil. Each dose was replicated five times. Thenumber of dead and live insects in each bottle was counted24 h after initial exposure to the essential oil. The mortalitywas determined as described in the earlier experiment. Thetreatment bottles were monitored for at least 48 h afterrecording the data and no affected insects recovered. Probitanalysis (Finney, 1971) was used to estimate LC50 andLC95 values.
3. Results
3.1. Fumigant toxicity
In all cases, considerable differences in mortality ofinsects to essential oil vapour were observed with differentconcentrations and times.From the graph in Fig. 1 it can be seen that, A. sieberi oil
was relatively more toxic to C. maculatus than to S. oryzae
and T. castaneum. The lowest concentration (37 mL/L) ofthe oil yielded 100% mortality of C. maculatus after a 12 hexposure but the mortalities of S. oryzae and T. castaneum
at the lowest concentration were 76% and 60% after 12 h,
Fig. 1. Percentage mortality of Callosobruchus maculatus, Sitophilus oryzae and Tribolium castaneum exposed for various periods of time to essential oil
from Artemisia sieberi impregnated on filter paper discs and held at 27 1C and 65% r.h.
M. Negahban et al. / Journal of Stored Products Research 43 (2007) 123–128 125
respectively. Total mortality of all three species wasachieved with the lowest concentration after 24 h ofexposure. At 444 mL/L air A. sieberi oil against C.
maculatus caused about 50% mortality with a 3 h exposureand 100% mortality after 6 h. At this concentration 100%mortality was achieved after 12 h for S. oryzae and T.
castaneum. At the highest concentration (926 mL/L air),kills of C. maculatus reached 80% with a 3 h exposure. Bycontrast only about 20% mortality was achieved for S.
oryzae and T. castaneum at the same time exposure. The oilat 556 mL/L air caused 100% mortality for S. oryzae and T.
castaneum with 9 and 12 h exposure, respectively.Probit analysis showed that C. maculatus was more
susceptible (LC50 ¼ 1.453 mL/L air) to A. sieberi oil than S.
oryzae (LC50 ¼ 3.861 mL/L air) and T. castaneum (LC50 ¼
16.757 mL/L air). The corresponding LC95 were 7.95, 15.55and 57.32 mL/L air, respectively (Table 1). The estimate ofthe LC95 for T. castaneum was higher than that implied bythe 100% mortality attained at 37 mL/L in the earlierexperiment and reflects experimental variability. Theestimate of the LC95 in the later experiment was based ona larger number of test insects.
3.2. Chemical constituents of Artemisia sieberi
The oil from A. sieberi contained camphor (54.7%),camphene (11.7%), 1,8-cineol (9.9%), b-thujone (5.6%)and a- pinene (2.5%) (Table 2).
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Table 1
Fumigant toxicity of Artemisia sieberi oil against Callosobruchus maculatus, Sitophilus oryzae and Tribolium castaneum
Insect species LC50a,b LC95
a,b Slope7SE Degrees of freedom Chi square (w2)
C. maculatus 1.45 7.95 2.2370.32 6 6.11
(1.23–1.66) (5.57–14.75)
S. oryzae 3.86 15.55 2.7270.26 8 2.29
(3.49–4.28) (12.23–21.89)
T. castaneum 16.76 57.32 3.0870.46 5 2.70
(14.64–18.61) (44.27–90.64)
aUnits LC50 and LC95 ¼ mL/L air, applied for 24 h at 27 1C.b95% lower and upper fiducial limits are shown in parenthesis.
Table 2
Chemical constituents of the essential oil from Artemisia sieberi
Compound Retention index % Composition A. sieberi
a-Thujene 920 0.59
a-Pinene 932 2.50
Camphene 942 11.73
Sabinene 972 0.69
b-Pinene 976 1.05
3-Octanol 990 0.68
a-Terpinene 1015 0.26
P-Cymene 1019 0.92
1,8-Cineole 1027 9.91
Lavender lactone 1049 0.15
g-Terpinene 1057 0.32
Artemisia alcohol 1081 0.13
a-Thujone 1103 0.56
b-Thujone 1114 5.64
Myrcenol 1123 0.37
Camphor 1139 54.68
Cis-chrysanthenol 1160 0.85
Pinocarvone 1162 1.57
Borneol 1165 1.29
P-Cymen-8-ol 1176 1.05
Myrtenol 1190 0.26
Cis-piperitol 1194 0.64
Trans-piperitol 1207 0.62
Piperitone 1260 1.15
M. Negahban et al. / Journal of Stored Products Research 43 (2007) 123–128126
4. Discussion
In this study, the essential oil of A. sieberi demonstratedfumigant toxicity to C. maculatus, S. oryzae and T.
castaneum. The insecticidal activity varied with insectspecies, concentrations of the oil and exposure time. Theresults showed higher mortality rates in C. maculatus thanin S. oryzae and T. castaneum. The slopes of the mortalitycurve were very steep from 6 to 12 h and after this time theslope leveled off. At 444 mL/L air the mortality was 100%after 6 h for C. maculatus and 12 h for S. oryzae and T.
castaneum.Studies have not been reported previously concerning
the activity of A. sieberi as a fumigant on insect pests. The
fumigant activity of essential oils from other Artemisia
species has been evaluated against a number of stored-product insects including oils from A. annua against T.
castaneum and C. maculatus (Tripathi et al., 2000), andfrom A. tridentata Nutt. against some stored-grain insects(Dunkel and Sears, 1998). Artemisia aucheri Boiss hadfumigant activity against C. maculatus, S. oryzae and T.
castaneum (Shakarami et al., 2004a,b,c), and A. scoparia oilagainst S. oryzae and T. castaneum (Negahban et al., 2004;Negahban and Moharramipour, 2005). Repellent activityof oil from A. verlotiorum Lamotte has been demonstratedfor T. castaneum (Novo et al., 1997), and from A.
saissanica (Krasch.) Filatova for Sitophilus granarius
(Adekenov et al., 1990).The A. sieberi oil described here appears to have greater
fumigant toxicity than the oils of related species and plantfamilies. Compared with our data, Artemisia tridentata wasless effective against S. oryzae (Weaver et al., 1995). Theessential oil from Labiatae species (ZP51) resulted in85–100% mortality in T. castaneum, S. oryzae, Rhyzo-
pertha dominica (F.) and Oryzaephilus surinamensis (L.)within 4 days exposure at 70 mL/L air (Shaaya et al., 1997).The total mortality of A. sieberi oil, however, was achievedat 37 mL/L air within 24 h.The insecticidal constituents of many plant extracts and
essential oils are monoterpenoids. Due to their highvolatility they have fumigant activity that might be ofimportance for controlling stored-product insects (Coatset al., 1991; Konstantopoulou et al., 1992; Regnault-Roger and Hamraoui, 1995; Ahn et al., 1998). The toxiceffects of A. sieberi could be attributed to majorconstituents such as camphor (54.7%), camphene(11.7%), 1,8-cineol (9.9%) and a-pinene (2.5%). Themonoterpene camphor might have broad insecticidalactivity against stored-product insects and act as thefumigant in A. sieberi oil. In a detailed study, it has beenreported that camphor from A. tridentata (Dunkel andSears, 1998), and 1,8-cineol from Ocimum kenyense
(Ayobangira) (Obeng-Ofori et al., 1997) are toxic andrepellent against some stored-product beetles. Ojimelukweand Adler (1999) found a-pinene was toxic to Tribolium
ARTICLE IN PRESSM. Negahban et al. / Journal of Stored Products Research 43 (2007) 123–128 127
The genus Artemisia is a member of the large andevolutionary advanced plant family Asteracae (Composi-tae). More than 300 different species comprise this diversegenus which is mainly found in arid and semi-arid areas ofEurope, America, and North Africa as well as in Asia(Heywood and Humphries, 1977). Artemisia is a genus thatgrows in many areas of Iran. We have collected A. sieberi
from dry lands located in the vicinity of Qom Lake, and asthe results showed this genus is highly toxic to stored-product insects. Iran is situated in arid and semi-arid areasand has many endemic aromatic plants from differentfamilies. It therefore seems very worthwhile to mount acomprehensive screening program to determine the in-secticidal efficacy of such plants.
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