Ocimum gratissimum essential oil and modified montmorillonite clay, a means of controlling insect pests in stored products

Journal of Stored Products Research 52 (2013) 57e62

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Ocimum gratissimum essential oil and modified montmorillonite clay, a means of controlling insect pests in stored products Marie G.M. Nguemtchouin a, b, *, Martin B. Ngassoum a, Pascale Chalier c, Richard Kamga a, Léonard S.T. Ngamo d, Marc Cretin b a

Department of Applied Chemistry, National School of Agro-Industrial Sciences, University of Ngaoundere, P.O. Box 455, Ngaoundere, Cameroon Institut Européen des Membranes, UMR 5635, ENSCM-UMII-CNRS, Place Eugène Bataillon, 34095 Montpellier, France IEM (Institut Europeen des Membranes), UMR 5635 (CNRS-ENSCM-UM2), Universite Montpellier 2, Place E. Bataillon, F-34095 Montpellier, France d Department of Biological Sciences, University of Ngaoundere, P.O. Box 455, Ngaoundere, Cameroon b c

a r t i c l e i n f o

a b s t r a c t

Article history: Accepted 24 September 2012

The insecticidal properties of formulations based on Ocimum gratissimum essential oil and montmorillonite clay have been improved after modifications of the clay. Insecticidal tests have been conducted against the maize weevil Sitophilus zeamais. The mortality of S. zeamais decreased from 100% to 95%, 87% and 0% after 7 days, respectively, for the essential oil adsorbed on modified clay, unmodified clay or used without adsorbent. The formulation prepared with unmodified clay completely lost insecticidal activity after 30 days, whereas the formulation with modified clay lost about 60% of its full insecticidal potency in the same time. The insecticidal effects of the essential oil persisted for about 7, 45 and 80 days respectively for crude essential oil; after adsorption on unmodified and after adsorption on modified clay. The findings suggest that formulations based on essential oils adsorbed on modified clays can be considered as alternatives to synthetic insecticides for use in stored product protection. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Bioinsecticide Montmorillonite Clay modification Maize protection Sitophilus zeamais

1. Introduction The global post-harvest grain losses caused by insect damage and other bio-agents range from 10 to 40% (Papachristos and Stamopoulos, 2002). Small-scale farmers may lose as much as 80% of their stock due to insects after storing for 6e8 months (Kitch et al., 1992; Nukenine et al., 2002). In the northern provinces of Cameroon, where cereals play an essential role as human food, the maize weevil Sitophilus zeamais Motschulsky (Curculionidae) is the main pest in granaries (Ngamo Tinkeu, 2004). Damage due to insects affects mainly the quality, quantity, commercial and agronomic values of the product (Bell et al., 1998). Although synthetic insecticides are commonly used to reduce these losses, there is global concern about their negative effects such as environmental pollution, pest resistance and pesticide residues in food (Ogendo et al., 2008). The continuous application and excessive reliance on chemical pesticides have also resulted in toxicity hazards for nontarget organisms and users (Isman, 2006). In recent years, studies have been focussed on the use of plant essential oils and their * Corresponding author. Department of Applied Chemistry, National School of Agro-Industrial Sciences, University of Ngaoundere, P.O. Box 455, Ngaoundere, Cameroon. Tel.: þ237 77257200, þ237 98306947. E-mail address: [email protected] (M.G.M. Nguemtchouin). 0022-474X/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jspr.2012.09.006

bioactive chemical constituents as possible alternatives to synthetic insecticides (Rajendran and Srianjini, 2008). Research has shown that plant essential oils have a potential use as fumigants; they are locally available, and have low toxicity and rapid degradation (Sha Sha et al., 2010). Although many aromatic plants have recently been proven to have insecticidal effects, the active components are not persistent. Essential oils of Ocimum gratissimum have been tested as grain protectors in Cameroon but due to their high volatility, their insecticidal effect tends to be transient (Ngamo Tinkeu et al., 2007). The challenge is now to develop a formulation that can remain active against insects for a long period. The clay, which is a common constituent of soils and sediments, is used in numerous formulations of insecticides, drugs and cosmetic powders. In crop protection, the clay alone can be used to help control pest infestation as an inert dust (Keita et al., 2001). Insecticidal activity of clay and Xylopia aethiopica essential oil formulations has already been investigated; and shown to increase the persistence of the toxicity and stability of the essential oil (Nguemtchouin et al., 2010). This period of persistence is however insufficient for the protection of stored products. To avoid continuous application and repeated treatment, which could result in development of target pest resistance, the present study aimed to improve the stability of the insecticidal effect of powder formulations.

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2. Materials and methods

2.5. Preparation of modified clay

2.1. Essential oil distillation

All chemicals of analytical grade and water were obtained from a Milli-Q system (resistivity 18.2 Um). For organic modifications, fresh solutions of organic solutions were obtained by solution of an appropriate quantity of cethyltrimethylammonium chloride (CTMAC) 25% in ultra pure water. Organic solutions prepared at 0.1 mol L1 were immediately used for clay modification. Organo-clays were synthesized according to procedure described by Bouras, Unaobonah, Reddy and co-workers (Bouras et al., 2007; Unuabonah et al., 2008; Reddy et al., 2009). A given mass of montmorillonite-Na previously obtained was dispersed in ultra pure water in a proportion of 0.5% (W/W) and 500 mL of CTMA solution was added (8 mL min1 using a peristaltic pump) to the suspension previously stirred for 1 h. The resulting suspension was aged for 24 h at room temperature. After reaction, the clay sample was separated by centrifugation and washed repeatedly with ultra pure water. The washing and centrifugation steps were repeated until complete removal of the foam formed due to the surfactant. The resulting clay sample was dried at 40  C for 24 h, ground in an agate mortar to a fine powder and identified as Mont-CTMA and fractions of Mont-CTMA less than 50 mm were obtained by passing the final modified sample through a sieve of 50 mm mesh.

The O. gratissimum plants used as source of essential oil were collected from the Ngaoundere Adamaoua region (Cameroon) in July 2010. Fresh whole plant samples were placed in labelled bags and transported to the laboratory. Fresh leaves of plant were hydrodistilled in a modified Clevenger-type apparatus for 4 h, extracted and then dried over anhydrous sodium sulphate. The essential oil was then stored in airtight containers in a refrigerator at 4  C until its use. 2.2. Essential oil analysis Quantitative and qualitative analysis of O. gratissimum essential oil were performed using gas chromatography and mass spectroscopy. Concerning quantitative analysis, essential oil was analysed by a 14B Shimadzu Co. apparatus fitted with a flame ionisation detector, an HP-3395B integrator and a fused silica capillary column [30 m  0.25 mm ID, 0.25 mm film thickness coated with a 5% phenyl 95% dimethylsiloxane stationary phase (HP-5MS from Agilent) at 230  C]. The detector temperature was 250  C. Nitrogen was used as carrier gas at 1.5 mL min1, hydrogen at 30 mL min1 and air at 250 mL min1 (FID detector). The column temperature programme was: an initial temperature of 40  C (hold 5 min), a ramp at 5  C min1 from 40  C to 200  C and at 10  C min1 from 200  C to 230  C with a final hold at 230  C for 10 min. The sample (1 mL) was injected with a split ratio of 1:10. The identification of essential oil components was undertaken firstly by comparing their mass spectra with those stored in NIST 05 and Wiley 275 libraries or with mass spectra from the literature after analysis with GCeMS in the same conditions as used in GCeFID analyses, and. secondly comparing their retention indices with those in the literature or with those of authentic compounds available in the laboratory. The retention indices were determined in relation to a homologous series of n-alkanes (C8eC24) under the same operating conditions. 2.3. Insect rearing The used maize weevil S. zeamais was of the strain 01Z/LN/01 in vivo collection of insect grain pests of Storeprotect Laboratories at the University of Ngaoundere. This strain has been in collection since 2008 in an incubator monitored at local temperature (28  4  C). Adult insects used for tests were three weeks old. 2.4. Preparation of clay powder A local montmorillonite was collected in Maroua locality in the far North of Cameroon. After collection, stones and other heavy particles were manually removed from the sample, which was then kept dispersed in ultra pure water for several hours. Fractions less than 50 mm were obtained by using an appropriate sieve. This specific small particle size was used to improve the adsorption capacity of our clay material. Prior to use, clay was firstly converted to montmorillonite-Naþ form (Mont-Na) to prepare for treatment with cethyl trimethyl ammonium. The preparation of Mont-Na was performed by dispersing raw montmorillonite (<50 mm) in a sodium chloride solution (1 mol L1) over 24 h to replace all exchangeable cation content in the clay by Naþ. The product was then washed with deionised water until free of chloride as indicated by the AgNO3 test. The Na-clay was dried at 70  C and ground to pass through a 50 mesh sieve. The treated Na-clay was designated Mont-Na.

2.6. Bioassay procedures with crude essential oil Bioassays were conducted with Whatman No 1 filter paper disks treated with essential oil diluted in acetone. The filter paper was placed in a 9 cm diameter Petri dish. Three mL of different concentrations of O. gratissimum solutions were obtained by diluting 0, 100, 200, 300, 400, 500, 600, 700, 800, 900 and 1000 mL of pure essential oil in acetone and 350 mL of each solution sample was uniformly flowed on a 9 cm diameter disk of filter paper previously introduced into separate Petri dishes. The treated filter papers were left to dry at room temperature for 5 min after which 10 adult S. zeamais were introduced into each Petri dish and enclosed. A control without essential oil was run. Each treatment was replicated two times with four sub-replicates for each Petri dish along with control sets. After 24 h, insect mortality was assessed. The mortality rate was corrected for control response using Abbott’s formula. The experimental set up used for the crude essential oil test was also used to assess the remnant effect of the oil on grain. In this test 200 g of maize was mixed with 6 mL of O. gratissimum essential oil solution diluted in acetone. Concentrations used here corresponded to the LC95 found for crude essential oil. After the grain coating, acetone was left to evaporate for 15 min. The control included grain treated with acetone alone. Every 2 days, 20 g of maize grain was introduced in glass vials (diameter 2.5 cm, height 9.5 cm, volume 40 mL). The vials containing insects were kept under conditions used for testing the whole essential oils. Survival was recorded in four replicates, 24 h after treatment. 2.7. Preparation of clay-O. gratissimum essential oil formulation Formulation used in this study was prepared with modified montmorillonite (Mont-CTMA) and unmodified one (Mont-Na) by using the following ratio

mEO ¼ 0; 1 mclay with mEO: mass of essential oil; mclay: mass of clay. To prepare 10 g of each formulation, 10 g of clay powder (MontNa or Mont-CTMA) were transferred in a 100 mL flask and the

M.G.M. Nguemtchouin et al. / Journal of Stored Products Research 52 (2013) 57e62

appropriate quantity of O. gratissimum diluted in 10 mL of acetone, was added. After 5 min of manual shaking, the mixture was placed in a water bath set at 30  C for 90 min to complete the evaporation of acetone. The aromatized powders obtained (Mont-NaeO. gratissimum and Mont-CTMAeO. gratissimum) were kept in coloured vials tightly closed using aluminium foil. 2.8. Bioassay procedures with formulations Ingestion-contact bioassays were carried out with 20 young adult S. zeamais per Plexiglas box mixed with 20 g of maize and aromatized formulations (Mont-NaeO. gratissimum and MontCTMAeO. gratissimum) sealed with canvas cloths held in place with rubber bands. Tests were carried out on S. zeamais previously starved for 48 h. MalagrainÒ (Malathion 5%), a synthetic insecticide, was used at 0.01 g per 20 g of maize as a positive control (reference) with acetone-treated grain as the blank control. The mass of formulation able to kill a given number of S. zeamais was determined. For each formulation (i.e. prepared with modified and unmodified clay), 20 g of maize was mixed in 7 boxes, with increasing mass of formulation (0.50; 0.75; 1.00; 1.25; 1.50; 1.75; 2.00 g) and 20 insects. Each experiment was repeated five times. Then these boxes were covered with canvas cloths and maintained with rubber bands. Five days later, mortality was estimated. The mortality rate was corrected for control mortality using Abbott’s formula and the results were plotted on log/probability paper. 2.9. Formulation stability To determine the stability of the powder formulations, 1.75 g of the formulation with unmodified clay and 1.50 g of the formulation with modified clay were added to 60 boxes (180 boxes for the two clay formulations plus MalagrainÒ). Of the 60 boxes for each formulation, 30 were tightly closed and sealed with rubber bands and the others were just covered with canvas cloths secured by rubber bands. Then, after 1, 8, 15, 21 or 30 days, for each of the two essential oil formulations 20 g of maize were mixed with the previously stored formulation and 20 insects were added. As in the previous test, mortality was estimated 5 days after the mixing of the formulation and insects. 2.10. Formulation remnant effect on maize The remnant effect of insecticidal activity of formulation was further studied for boxes containing maize mixed with formulation (amounts as for the stability tests) and covered with canvas cloths (the usual method employed for product storage in Central African markets) by noting the S. zeamais mortality when beetles were added to maize previously treated and conserved for the periods of 1, 8, 15, 21, 30, 45, 60, 75, 90 and 105 days (4 boxes for each). Again in each case mortality was estimated after 5 days. 2.11. Statistical analysis In order to determine whether there was a statistically significant difference among the results obtained for insecticidal activity assays, one-way analysis of variance (ANOVA) and the least significant (LSD) multiple range tests were performed on the data (P < 0.05) with Statgraphic Plus 5.0. Probit analysis (Finney, 1971) was conducted to estimate the mean lethal concentration of crude essential oil (LC50), lethal mass (LM50) and storage time (ST50) for 50% mortality of powder formulations, respectively by Microsoft Excel software package (Microsoft Corp.) and XLstat add-on.

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3. Results and discussion 3.1. Ocimum gratissimum analysis On the basis of fresh plant of O. gratissimum weight of extract part, the hydro-distillation yielded about 0.9  0.1%. Thirty-two compounds, representing about 95.30% of the essential oils have been identified; their retention indices and percentage composition, listed in order of elution are given in Table 1. Percentages yield and the identified volatile constituents from O. gratissimum show a composition rich in thymol and g-terpinene (53.9% and 17.8% respectively). Five chemotypes of O. gratissimum are currently identified and are represented by the types eugenol, thymol, citral, ethyl cinnamate, and linalool (Cortez et al., 1998). Essential oils can contain more than 50% of any one of these chemotypes (Cortez et al., 1998). The O. gratissimum essential oil used in this study is clearly a thymol chemotype. In contrast, Ogendo et al. (2008) showed that the chemical composition of O. gratissimum obtained from Kakamega forest in western Kenya is a methyl eugenol/ocimene chemotype with 64.28% and 10.40% methyl eugenol and b-(Z)-ocimene, respectively. These results concur with Kothari et al. (2005), in which the related Ocimum tenuiflorum L.f. had methyl eugenol as the major constituent accounting for 65.2e83.7% depending on the plant part analysed. However, essential oils from leaves of O. gratissimum obtained from different locations in Brazil showed that eugenol (35e60%) and ocimene (0.49e4.1%) were the major and minor constituents, respectively (Vasconcelos-Silva et al., 2004). It has been reported that O. gratissimum from eastern Kenya was different and contained 68.81% and 7.47% eugenol and cis-ocimene, respectively (Matasyoh et al., 2007). Essential oil obtained from leaves of a chemotype of O. gratissimum in Gabon Table 1 Ocimum gratissimum essential oil composition.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

Components

Formula

Percentage (%)

KI

a-thujene a-pinene

C10H16 C10H16 C10H16 C10H16 C10H16 C10H16 C10H16 C10H16 C10H16 C10H16 C10H16 C10H16 C10H16 C10H14O C10H14 C10H16O C10H16O C10H16O C10H16O C10H16O C10H16O C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24O

1.5 3.2 0.1 0.2 0.6 5.5 0.3 2.0 3.9 2.0 0.6 0.2 17.8 0.2 0.1 0.1 0.3 0.6 0.4 53.9 0.9 0.1 0.7 0.2 0.2 2.8 0.3 0.1 0.6 0.2 0.4 0.4 4.7

926 933 947 976 978 990 1003 1015 1023 1033 1038 1050 1062 1096 1107 1111 1125 1175 1180 1298 1305 1355 1377 1389 1391 1418 1456 1483 1495 1517 1523 1582

Camphene Sabinene b-pinene Myrcene a-phellandrene a-terpinene P-cymene Limonene Z-b-ocimene E-b-ocimene g-terpinene Linalool 1,3,8-p-menthatriene Trans-thujone Neo-allo-ocimene Terpinen-4-ol p-cymen-8-ol Thymol Carvacrol a-cubebene a-copaene b-cubebene b-elemene b-caryophyllene a-humulene Germacrene a-selinene 7-épi-a-selinene d -cardinene Caryophyllene oxide Unidentified components

KI: Kovats index as determined on an HP-5 MS column using the homologous series of n-hydrocarbons. In bold: Main compounds.

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contained 75.5% and 4.0% eugenol and (Z)-b-ocimene, respectively (Tchoumbougnang et al., 2006; Agnaniet et al., 2005). Ocimum gratissimum essential oil contains 77.2% monoterpenes, 60.7% of which are oxygenated. Documented information from Asian, African and Latin American countries has shown the existence of significant intra-species variations in chemical composition with up to nine different chemotypes for O. gratissimum. Its chemical composition varies with season, time and stage of harvest and geographical origins (Kothari et al., 2005; Vasconcelos-Silva et al., 2004; Tchoumbougnang et al., 2006; Jirovetz et al., 2005; Madeira et al., 2005).

100

a

100

83

b

75

80

c

65 Mortality (%)

60

3.2. Ocimum gratissimum toxicity

d

60

40

20

The insecticidal activity of O. gratissimum essential oil on S. zeamais varied according to the quantity of essential oil used (Fig. 1). Concentrations of 32.8 mg mL1 and 225 mg mL1 of O. gratissimum essential oil led respectively to 45% and 100% mortality of S. zeamais and calculated LC50 and LC95 values were 37.9 mg mL1 and 152.3 mg mL1 respectively. These results compared favourably with other investigations in which essential oils and their bioactive chemical constituents from Lamiaceae (Labiatae) family plants produced significant repellent activity against stored-product insects (Pavela, 2009). Kordali et al. (2008) also showed that essential oil isolated from Turkish Origanum acutidens was inhibitory against fungi, and the mycelial growth of all tested fungi was completely inhibited at 25 mg/Petri dish dose of the oil. 3.3. Ocimum gratissimum remnant effect on maize The toxicity of O. gratissimum to S. zeamais declined as the time before exposure to essential oil-treated maize increased (Fig. 2). Bioassays results showed that O. gratissimum essential oil quickly lost its insecticidal activity after being impregnated on maize. After 6 days the mortality effect decreased from 96% to 65% and from the 8th day all insecticidal activity was lost. Nevertheless, compared to direct exposure toxicity test results, the insecticidal effect of O. gratissimum essential oil was improved for about 6 days by being impregnated on maize. 3.4. Formulation efficiency For each of the two formulations (Mont-Na/O. gratissimum and Mont-CTMA/O. gratissimum), mortality of S. zeamais significantly

0

0 0

2

4

6

e

8

e

e

0

10

12

0

Essential oil-maize contact time (days) Fig. 2. Evaluation of the toxicity of O. gratissimum on S. zeamais as a function of the contact time between essential oil and maize. Mortality values followed by the same letters are not significantly different at level of P < 0.05.

increased (P < 0.05) with the mass of formulation introduced in the boxes containing maize and insects (Fig. 3). Insect mortality varied from 27% to 35% with 0.5 g of formulation to 100% with 2 g and 1.5 g respectively for the formulation with unmodified clay (Mont-Na) and that with modified clay (Mont-CTMA). The same quantity of formulation with modified clay thus induced more insect mortality than that with unmodified clay. This was confirmed by a multiple range test which showed a statistical difference (P < 0.05) between the two formulations. The amount of formulation required to kill 50% of S. zeamais adults (LM50) as carried out by Probit analysis was 1.01 g and 0.69 g for Mont-NaeO. gratissimum and Mont-CTMAeO. gratissimum respectively. Thus the formulation based on O. gratissimum with modified clay proved to be more toxic than the same formulation with unmodified clay, indicating that more insecticidally active compounds (i.e. terpenic components) were incorporated. This is due to the improvement of clay adsorption capacity induced by the initial treatment with cethyl trimethyl ammonium chloride solution which modified the crystallographic structure of the material.

110 110

100

90

90

80

80

70

Mortality (%)

Mortality (%)

100

60 50 40 30

MontNa MontCTMA

70 60 50 40

20

30

10

20

0 0

50

100

150

200

250

300

350

-1

O. gratissimum concentration (µg.mL ) Fig. 1. Evaluation of the toxicity of O. gratissimum essential oil on S. zeamais.

0,6

0,8

1,0

1,2

1,4

1,6

1,8

2,0

Formulation mass (g) Fig. 3. Effect of quantity of powder formulations with different clays on S. zeamais.

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3.5. Stability of formulations Figure 4 is a representation of mortality of S. zeamais after 5 days due to contact with formulations left for different periods of time in open and closed boxes before the insects and maize were added. For all formulations the rate of decline of activity was faster in open boxes (Fig. 4a) than in closed ones (Fig. 4b). There was a significant difference between the two oil/clay formulations applied in both open and closed boxes (P < 0.05), the level of kill achieved by Mont-NaeO. gratissimum declining more rapidly with formulation age and losing all activity in open boxes at 30 days. In the closed boxes (Fig. 4b); mortality caused by the Mont-CTMA formulation based on O. gratissimum essential oil was slightly higher than the mortality due to the positive control (MalagrainÒ) on the first day, but in the open boxes (Fig. 4a) the mortality due to the synthetic insecticide (MalagrainÒ) decreased slightly less rapidly than that caused by the essential oil formulation as formulation age increased. The stability of organophosphorus formulations such as malathion and pirimiphos-methyl is well established (Collins and Cook, 1998). The formulation half-life time, namely the formulation storage time (ST50) at which it was still possible to achieve 50% mortality of S. zeamais, was calculated

61

from the mortality data by Probit analysis, and confirmed that formulating the oil with modified montmorillonite significantly improved stability. For all formulations tested, the previous storage or holding time at which each formulation could induce 90% or 50% mortality of S. zeamais (ST90 or ST50) (i.e. corresponding to a loss of insecticidal potency of about 10% and 50% of mortality) was also determined by Probit analysis using a logarithmic transformation of days (Table 2). It was found that formulations conserved in open boxes lost 50% of their insecticidal potency after about 7 days, 16 days and 18 days respectively for formulations with unmodified clay, modified clay and finally MalagrainÒ. The difference between formulations in the period losing 10% efficacy (inducing 90% mortality) was much smaller, in the region of 4 days for all formulations. In contrast, formulations conserved in closed boxes lost 10% of their insecticidal potency (ST90) after 6 days and 12 days 17 h respectively for Mont-Na and Mont-CTMA MalagrainÒ, while after 30 days no formulation had lost very much more of its insecticidal power and as a result very high periods were calculated for ST50 values (Table 2). The apparent small loss of active components in formulations in tightly closed boxes could have been caused by sorption into Plexiglas.

3.6. Formulations remnant effect

a 100

Open boxes

Mortality (%)

80

Mont-NA Mont-CTMA Malagrain®

60

40

20

0 5

10

15

20

25

30

Formulations preservations (days)

b

100

Closed boxes

Mortality (%)

95

Mont-Na Mont-CTMA Malagrain®

90

85

Each formulation previously mixed with grain at day 1 and infested 8, 15, 21, 30, 45, 60, 75, 90 and 105 days later, was found to be active on S. zeamais for a longer period than the same formulation mixed with maize and tested immediately. As shown in Fig. 5, for both essential oil formulations and MalagrainÒ, mortality progressively decreased in the order: Mont-Na > MontCTMA > synthetic insecticide formulations (P ¼ <0.05). Duration of formulation conservation before infestation on mortality (P > 0.05). At the end of the 30th day, Mont-Na induced less than 25% of S. zeamais mortality, while Mont-CTMA induced 75%. These remnant bioassay results indicated that insecticidal effect of O. gratissimum essential oil persisted for about 80 days after its adsorption on Mont-CTMA. The poorer retention of activity by the Mont-Na formulation was a consequence of less terpenic components being adsorbed initially, and a higher release rate of these components, compared to Mont-CTMA. The latter formulation was benefited firstly by the capacity for hydrogen bonds to form between the clay adsorbent and thymol, which is the major compound in O. gratissimum, and secondly between the CTMA molecules incorporated in the layers of clay and hydrocarbon monoterpenes such as p-cymene, limonene and a-pinene. These compounds are known for their synergic insecticidal effect when they occur together (Prates et al., 1998; Ngamo Tinkeu et al., 2001; Tapondjou et al., 2002; Kouninki et al., 2007).

Table 2 ST50 and ST90 values (days) of formulations based on O. gratissimum essential oil adsorbed on Mont-Na or Mont-CTMA, and MalagrainÒ.

80

75 5

10

15

20

25

30

Formulations preservation (days) Fig. 4. Stabilities of insecticidal effect of formulations based on O. gratissimum essential oil adsorbed on Mont-Na, Mont-CTMA and MalagrainÒ and preserved in open boxes (a) and closed boxes (b).

ST90a Slope  SEb

Formulations

ST50a

Open Mont-Na boxes Mont-CTMA Malagrain Closed Mont-Na boxes Mont-CTMA Malagrain

4.751 1.63 2.76 13.80 1.85 1.47 18.00 1.69 1.24 (519.25) 6.00 0.66 (508.87) 12.17 0.79 (1445.42) 8.59 0.57

a b c

     

Interceptc

c2

df

0.23 6.86  0.26 12.55 18 0.06 6.74  0.07 7.33 18 0.06 6.56  0.05 7.00 18 0.04 6.794  0.04 6.08 18 0.05 7.14  0.05 6.23 18 0.05 6.81  0.05 5.99 18

Number of days at which formulation could induce 50% or 90% mortality. Slope of mortality-day regression line. Intercept of regression line.

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100 90 80

Mortality (%)

70 60 50 40 30 20

Malagrain Mont-Na Mont-CTMA

10 0 0

10

20

30

40

50

60

70

80

90

Duration of formulation conservation before infestation (days) Fig. 5. Insecticidal effect of formulations previously mixed with maize.

4. Conclusion The present study is a contribution in research of environmentally safe ways of pest management through use of a formulation based on modified clay and essential oil. The use of bioinsecticides based on such materials could become important components of integrated pest management strategies because potentially they are widespread, easy to use, and environmentally benign. Many phytochemicals which showed pest control properties are now known to possess deleterious and toxicity hazards to non-target organisms and although this is unlikely to be the case for essential oils it is recommended that some studies are carried out for information purposes. Acknowledgements The authors are grateful to IFS (International Foundation for Sciences) and SCAC (Service de Coopération et d’Action Culturelle of Coopération Française) for their financial and material support. References Agnaniet, H., Anguille, J.J., Bessiere, J.M., Menut, C., 2005. Aromatic plants of tropical central Africa. Part XLVII. Chemical and biological investigation of essential oils of Ocimum species from Gabon. Journal of Essential Oil Research 17, 466e470. Bell, A., Mück, O., Schneider, H., 1998. La protection intégrée des denrées stockées est une affaire rentable! GTZ, Eschborn, Germany, 42 pp. Bouras, O., Bollinger, J.C., Baudu, M., Khalaf, H., 2007. Adsorption of diuron and its degradation products from aqueous solution by surfactant-modified pillared clays. Journal of Applied Clay Science 37, 240e250. Collins, D.A., Cook, D.A., 1998. Periods of protection provided by different formulations of pirimiphos-methyl and etrimfos, when admixed with wheat, against four suspection storage beetle pests. Crop Protection 17, 6e10. Cortez, D.A.G., Cortez, L.E.R., Pessini, G.L., Doro, D.L., Nakamura, C.V., 1998. Analysis of essential oil of alfavaca Ocimum gratissimum L. (Labiatae). Orquivos de Ciencias da Saude da UNIPAR 2 (2), 125e127. Finney, D.J., 1971. Probit Analysis, third ed. Cambridge University Press, Cambridge, UK. Isman, M.B., 2006. Botanical insecticides, deterrents, and repellents in modem agriculture and an increasingly regulated world. Annual Review of Entomology 51, 45e66. Jirovetz, L., Buchbauer, G., Ngassoum, M.B., Ngamo Tinkeu, L.S., Adjoudji, O., 2005. Combined investigation of the chemical of essential oils of Ocimum gratissimum and Xylopia aethiopica from Cameroon and their insecticidal activities against stored maize pest Sitophilus zeamais. Ernahrung 29, 55e60. Keita, M.S., Vincent, C., Schmit, J.P., Arnason, T.J., Bélanger, A., 2001. Efficacy of essential oil of Ocimum basilicum L. and O. gratissimum L. applied as an

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