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Trojstva 3, HR-31000 Osijek, Croatia [ * HYPERLINK "mailto:vrozman@pfos.hr" vrozman@pfos.hr] 2Diatom Research and Consulting Inc., 14 Greenwich Dr., Guelph ON N1H 8B8, Canada Abstract These investigations aimed to determine the effect of wheat grain mass on the effectiveness of the essential oil cineole against Tribolium castaneumn (Herbst.), Rhyzopertha dominica (F.) and Cryptolestes ferrugineus (L.) in an empty space, 50% and 95% spaces occupied with wheat. Concentration of cineole of 50g m-3 in empty space induced 100% mortality in all three tested insect species. However, fumigation in space 50% occupied with wheat was absolutely effective against C. ferrugineus, with 89.5% efficacy against R. dominica, and only 11% against T. castaneum. In space 95% occupied with wheat mortality of C. ferrugineus was 88%, R. dominica 64% and T. castaneum 4.5% only. The price of natural cineole may be a significant barrier to adopt its use for grain fumigation. Keywords: fumigation, cineole, Tribolium castaneumn, Rhyzopertha dominica, Cryptolestes ferrugineus Introduction The primary cause of food contamination and environmental pollution arising from agriculture are chemical pesticides[1]. Also, the pesticide residues in grain arising from postharvest treatments[2] come from their non-selective and uncritical application causing the toxic effects in the food and contamination of the environment[3]. With the growing evidence regarding detrimental effects of many of the conventional pesticides on health and environment, require for safer means of pest management has become very crucial[4]. The use of botanical pesticides is now emerging as one of the safer and prime means to protect crops and their products[5] [6]. Among botanicals the plant volatile essential oils (EO) are the most frequently studied as pesticides for pests and diseases management [6] [7] [8]. However, EO, besides a large scale demonstration of their efficacy and penetration, need a lot of research in order to determine their toxicological and safety data prior to the registration[9]. Also, as with other groups of insecticides, the potential use of the natural EO in stored grain insect pest management depends on many factors. Some of the factors that may greatly prevent the adoption and use of the natural EO in stored grain fumigation are their relatively high concentrations needed for the effective protection of stored grain against insect pests[8] [10], a great difference in the sensitivity of various insect species[10] and current prices of natural essential oils on the market (Korunic and Rozman, unpublished manuscript). One of effective and safe EO is cineole, the active component of many natural EO such as eucalyptus. It is a cyclic ether with empirical formula C10H18O and systematic name 1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane. Sometimes is traded commercially as "eucalyptol". It is readily biodegradable, un-reactive and relatively non-toxic[11]. Also, it inhibits the enzyme acetylcholinesterase[12], it interferes with sonic communication and mating in leafhoppers[13], and it is a mosquito feeding and ovipositional repellent[14]. Several researchers determined a good fumigant activity of cineole against stored-product insects[15] [16] [17] [8] [18] [19] [20]. Significant effect of grain on the effectiveness of cineole and other EO as well, has been determined by Shaaya et al.[21], Lee et al.[8] and Rozman et al.[22]. They found out that cineole was significantly less effective in a space occupied with wheat grain in the comparison with the effectiveness in an empty space. The main objective of this research was a determination of the effectiveness of EO cineole in an empty, 50% and 95% spaces occupied with wheat grains against Cryptolestes ferrugineus (L.), Rhyzopertha dominica (F.) and Tribolium castaneumn (Herbst). Materials and Methods The essential oil used in the experiment was 99% cineole (C10H18O) purchased from “Sigma-Aldrich” (Export Division Grќnwalder Weg 30 D-82041 Deisenhofen, Germany, EC No: 207-431-5). Cultures of the Cryptolestes ferrugineus (L.) - rusty grain beetle, Rhyzopertha dominica (F.) - lesser grain borer and Tribolium castaneum (Herbst.) - red flour beetle were reared in the laboratory under controlled conditions: temperature 30Б1oC, 70Б5% r. h. in darkness. C. ferrugineus and R. dominica were reared on whole wheat grain and T. castaneum on wheat flour containing 10% broken wheat kernels and 5% un-activated yeast. The commodity in the experiment was Canadian Western Hard red wheat, clean, with 14% m.c. The following combinations had been set up: Empty jars 450 ml in volume with 0.5 grams of wheat flour and 10 wheat kernels at the bottom with introduced test insects (control). Uninfested grains 200 grams in 450 ml jars (50% occupied space) with introduced test insects (control). Uninfested grain 360 grams in a 450ml jar (95% occupied space) with introduced test insects (control). Empty jars with 0.5 grams of flour and 10 wheat kernels at the bottom with introduced test insects. A piece of treated filter paper with 0.05 g of cineole (50 g m-3) was put on the bottom of the jar. The jar was tightly closed with metal lid. Uninfested 200 grams of grain in a 450ml jar (50% occupied space) with introduced test insects. A piece of treated filter paper with 0.05 g of cineole (50 g m-3) was put on the surface of grain. The jar was tightly closed with metal lid. Uninfested 360 grams of grain in a 450ml jar (95% occupied space) with introduced test insects. A piece of treated filter paper with 0.05 g of cineole (50 g m-3) was put on the surface of grain. The jar was tightly closed with metal lid. Each combination was repeated 4 times. One hundred (100) adults of mixed sex, 2 to 4 weeks old were introduced into a replication. Each insect species run separately. The experiment was carried out under controlled laboratory conditions: 30 Б 1 0C and 70 Б 5% r.h., 24 hours dark. The results of the experiment were assessed after 2 days (48 hours). All data were subjected to one-way analysis of variance (ANOVA) according to the GLM (general linear model) and LSD test entered in the table. Data processing was conducted by the SAS System for Windows 98. The figures that represent mean values were made by Microsoft Excel 2003. Results and discussion In comparison to the control, fumigation with cineole at the dose of 50 g m-3 proved to be absolutely effective in empty space with achieved mortality of 100% in all three insect species. Fumigation in space 50% occupied with grain was absolutely effective against C. ferrugineus, with obtained mortality for R. dominica of 89.50%, and for T. castaneum 11% only. C. ferrugineus had very good response to cineole fumigation in 95% occupied space (88% mortality), R. dominica showed mortality of 64%, whilst application to T. castaneum proved to be ineffective (4.5%) (Table1). Shaaya[21] were assessed the fumigant activities of a large number of essential oils extracted from various spices and herb plants against T. castaneum, Sitophilus oryzae (L.), R. dominica and Oryzaephilus surinamensis (L.). The highly active Labiatae sp. oil ZP51, in a concentration of 1.4 4.5 Мl L-1 air (1.4 -4.5 g m-3) and exposure time of 24 h caused 90% kill of all insects in space tests. However, in columns 70% filled with wheat, a concentration of 50 Мl L-1 and 7 d exposure were needed to obtain 94 100% kill of the insects. Lee et al.[8] studied the potent fumigant toxicity of 42 essential oils and found out that six of them extracted form Eucalyptus nicholii, E. codonocarpa, E. blakelyi, Callistemon sieberi, Malaleuca fulgens and M. armillary were toxic to S. oryzae, R. dominica and T. castaneum. The fumigant toxicity of five oils in the space 50% filled up with wheat was 3 to 5 times lower and in a case of EO extracted from E. codonocarpa, 9 times lower than in an empty space. Rozman et al.[22] determined bioactivity of cineole essential oil against S. oryzae on stored wheat in spaces differently filled up with wheat (empty space, 50% and 95% filled up). Concentration of 50 g m-3 cineole in empty space induced nearly 100% mortality of S. oryzae. However, fumigation in space 50% filled up with wheat showed 57.5% efficacy and in space 95% filled up with wheat mortality was 34% only. Our results are in a good agreement with the results of Shaaya et al.[21], Lee et al.[8] and Rozman et al.[22] . Table 1. The adults’ mortality (%) after 48 hours induced by application of 50 g m-3 cineole Cryptolestes ferrugineus*Mortality (%) of adults after 48 hoursControlCineole 50 g m -3MeanSD.MeanSD.in empty space0.50b0.57100.00a0.00in 50 % full space1.50b1.73100.00a0.00in 95 % full space1.50b1.7388.00a4.96Rhyzopertha dominica*Mortality (%) of adults after 48 hoursControlCineole 50 g m -3MeanSD.MeanSD.in empty space1.00b0.81100.00a0.00in 50 % full space2.50b1.2989.50a3.69in 95 % full space0.25b0.5064.75a4.50Tribolium castaneum*Mortality (%) of adults after 48 hoursControlCineole 50 g m -3MeanSD.MeanSD.in empty space0.00b0.00100.00a0.00in 50 % full space0.00b0.0011.00b1.41in 95 % full space0.00b0.004.50b1.29*means in the same row followed by the same letters are not significantly (P>0.05) different as determined by the LSD-test. Although, we didn’t study the reasons for such significant effect of wheat grain on the effectiveness, we believe that the probable cause may be in a considerable sorption of cineole in wheat grains and poor permeability of cineole vapours into seed inter-space which largely decreased the fumigation effect. According to Korunic and Rozman[10] to gain as similar results as obtained with phosphine and methyl-bromide cineole concentrations should range from 200-250 g m-3. Champ and Dyte[23] analyzed the concentrations of phosphine and methyl bromide and phosphine dose of 0.03 g m-3 and methyl-bromide dose of 1 g m-3, if applied in airtight space, were found to be enough to gain LD95 for S oryzae, while Lee et al.[8] reported required cineole dose of 42 g m-3 to gain LD95 for S. oryzae in the space 50% filled up with the grain. One (1) kg of phosphine pellets costs approximately US $41.00 US, whilst 1 kg of cineole in packages of 100g reaches about US $236.00. When the highest dosage of phosphine pellets is applied (30 pellets/t) with 1 kg of phosphine it is possible to fumigate approximately 55 tons of grain. It means the cost of phosphine to fumigate one tone of grain is about US $0.74. With 1 kg of 1,8-cineole it is possible to fumigate 4 tons[10] to about 10 tons of grain[8]). It means the cost of 1,8-cineole to fumigate one ton of grain is from US $23.60 to US $59.00. Such a considerable effect of grain on the effectiveness of cineole and relatively high price of cineole and other EO (Korunic and Rozman, unpublished manuscript) may greatly increase the cost of the grain fumigation with cineole and other natural EO and make them to expensive to be adopted for wider use. Conclusions At the applied dose of 50 g m-3 cineole proved to have fumigant effect particularly expressed against C. ferrugineus and only in a certain degree to R. dominica species while its efficacy against T. castaneum did not prove to be significant in practice. Very good results were gained in a fumigation of an empty space, while results of fumigation of space occupied with wheat (50% and 95%) did not prove to be successful and acceptable. Probable cause could be found in considerable sorption of cineole in wheat grains and poor permeability of cineole vapours into seed interspace and into grains, which largely lessen fumigation effect. The effect of grain on significant decreasing of the effectiveness of cineole and relatively high price of cineole and other natural EO make them too expensive to be adopted for wider use. Acknowledgements This study was funded by Croatian Ministry of Science, Education and Sport to the projects No. 079-0790570-0430. References 1 Jolсnkai M, Szentpщtery Zs, Hegedqs Z. Pesticide residue discharge dynamics in wheat grain. Cereal Research Communications, 2006, 34: 505-508 2 Fishwick F B. Pesticide residues in grain arising from postharvest treatments. Aspects of Applied Biology, 1988,17: 37-46 3 WMO. 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Fumigation toxicity of monoterpenoids to several stored product insects. Journal of Stored Products Research, 2003, 39: 77–85 18 Rozman V, Kalinovic I, Liska A. Insecticidal activity of some aromatic plants from Croatia against granary weevil (Sitophilus granarius L.) on stored wheat. Cereal Research Communications, 2006, 34: 705-708 19 Rozman V, Kalinovic I, Korunic Z. Toxicity of naturally occurring compounds of Lamiaceae and Lauraceae to three stored-product insects. Journal of Stored Products Research, 2007, 43: 349-355 20 Rozman V, Kalinovic I, Liska A et al. Toxicity of naturally occurring compounds of Dalmatian (Croatia) Lamiaceae and Lauraceae to maize weevil (Sitophilus zeamais Motsch). Cereal Research Communications, 2007, 35: 1005-1008 21 Shaaya E, Kostjukovski M, Eilberg J. et al. Plant oils as fumigants and contact insecticides for the control of stored-product insects. Journal of Stored Products Research, 1997, 33: 7-15 22 Rozman V, Korunic Z, Kalinovic I et al. Bioactivity of cineole against rice weevil (Sitophilus oryzae L) on stored wheat. VII Alps Adria Scientific Workshop, 2008: Stara Lesna, Slovakia, April 28 – May 3, 2008. 23 Champ B R, Dyte C E. Report of FAO global survey of pesticide susceptibility of stored grain pests. FAO Plant Production and Protection Series No. 5, FAO, 1976, Rome. vƒ†–—ЇЈЉЊABCRSUVЉВйк3GSgq‰ыэŽœКХмш&0;DPНХЧм№ђ  ! / Ѓ І ё є x { 9 < Ж Н G R §љ§љ§љ§љїђъђуђїслжлЮлЮлЮлЦлЮлЮлЮлЮлЮлЮлРлМЖМЖМЖлВЎЉЎЉЎЉЎЉЎЉЎЉ 5H*\5\5] 56CJ5CJ CJ\aJ5CJH*aJ56CJaJCJaJ 5CJaJ\ 0JB*phџjU jUH*H*]]BvЉUЈЉВН! 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