Proceedings of the 7th Intenuiiumal Worki1/{JConference on Stored-product Protectwn - Yolume 1

The use of high pressure carbon dioxide (20 bar) to controlsome insects of foodstuff

Dana Patnzia Locatelli", Luciano Sussl and MaunZIO Frati2

Abstract

The efficaciousness of high pressure COz(20 bar) on Plodiamierpunctella (Hbn.) , Oryzaephtlus euruuunensis(L ) and Sitophilus oryzae (L ) was studied. Tests werecarried out III a pilot plant (volume 100 htre) at 25 ± 1°CThe treatments lasted respectively for 5, 10, 15, 30 and 45mm At the end of each treatment the gas pressure wasdecreased to atmosphenc pressure m 20" A mortality over95% was venfied after 30 nun for all the species m thedifferent stages

Introduction

The use of carbon dIOXIdein pest control requires on theaverage exposure times longer than 1 week III relation tospecies, developmental stages and temperature (Annis,1990; Jay et al ,1990) Also combmmg low pressures WIthvacuum, exposure times are generally longer than 24 - 48hours (Locatelh and Daoho, 1993; Suss and Locatelli ,1994) In the food mdustnes rapid penods to controlfoodstuff pests are frequently required: before bemg storeddned and desiccated frurt, ingredients of manyconfectionery, are dismfested m order to avoid thatarthropods reach manufactunng departments. The nce, thatgenerally IS not vacuum packed, IS treated WIth methylbromIde or carbon dIOXIdeIII vacuum cells Many researchessuggested that exposure times can be reduced by usmg hIghpressure COz(Stahl et al ,1985; ReIchmuth. 1990; Sabmeand ReIchmuth, 1991; Le Torc'h and Fleurat-Lessard,1992; Prozell and ReIchmuth, 1991; CalIbosoet al , 1994;ReIchmuth and Wohlgemuth. 1994; Ulnchs, 1995; Ulnchsand ReIchmuth, 1997; Ulnchs et al , 1997; ReIchmuth,1997). Nowadays the mechamsm of action on the msect ISnot yet completely known The lethal effect probablydepends on the phySIOlogIcalaction of COzas the solubIlIty ofgases mcreases III the msect hemolymph at hIgher pressures

1 Istltuto dl Entomologta agrana, Umverslta Degh Studl, Via Celona2 20123 Mtlano, Italy

2 fur Llqwde Itaha sri, Via E Femu 16 50019 Sesto FJorentmo(FI), Italy

WIth a consequent decrease of pH (Stahl et al., 1985;Gerard et al., 1988; Prozell and Reichmuth, 1991;Reichmuth, 1991) Besides the physical action of highpressure gas m tissues of the msects, causing expansion asthe gas IS suddenly released to the atmosphere, causes therupture of cell membranes (Caliboso et al , 1994, Nakakitaand Kawashima, 1994; Reichmuth and Wohlgemuth, 1994).The decompression time had a strong influence on themortality achieved. It IS speculated that this effect can beImked to appearance of gas bubbles and to mechamcaldestruction of cell membranes and of organs due to quickexpansion of the gas (Ulnchs and Reichmuth, 1997).In this paper the efficaciousness of the use of high

pressure COzon some insects of foodstuff was valued.

Materials and Methods

The test was carried out on each stage of Oryzaephtlussurnuunensis (L ), Suophuus oryzae (L ) and eggs,larvae and pupae of Plodui interpunctella (Hbn.).Insects were taken from reanngs of the Istituto di

Entomologia agrana- University of MIlano The biologicalmatenal was obtained by mass reanng in thermostaticallycontrolled incubators at 26 ± rc; 65% ± 5 r h Eachinsect m the different stages was placed m polyethyl Jar(03 .Scm; h == Scm), sealed with a stopper provided WithWIrenet (20 mesh). Each Jar was loaded WIth the specificsubstratum for every considered speCIes: nee for S.oryzae; bran, malze-meal, soft wheat meal, ground hazels,mIlk chocolate, bISCUItSand dned frmt m the same volumesfor 0 sunna nwnsts; 2 volumes (v) of bran, 1v of groundhazels, 1/2 v of soft wheat meal, 1/2 v of maIze meal, 1/2v of germ of wheat, 1/4 v of brewers' yeast for Pm te'rpunctella 20 larvae, 20 pupae and 20 adults wereused for the speCIes hvmg outSIde the food For Pinterpunctella 100 eggs were tested. For eggs of O.sunnamenSLS 20g samples of mIxed food (200g),preViouslymfested by 200 adults (20 - 30 days old) for 7days, were used To obtam eggs, larvae and pupae of S.oryzae, 25g samples of nee-kernels (200g), prevIOuslymfested by 200 adults (10 - 20 days old) for 7 days wereused. 4 rephca tes were conducted for each speCies andstage.The pIlot plant used for expenments was a chamber

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Proceedmq« of the 7th International Working Conference an Stored-product Protectuni - Volume 1

suitable to carry out tests at high pressure (volumes 100htres}, provided WIth a COz delivery device and system oftemperature control by water in Jacket COz flux wasregulated by a pressure reducer on the feed pipe.Pressurizmg time was regulated by a valve on the feed pipe,after the pressure reducer Decompression time wasregulated by a valve placed on COz discharge pipe of thechamber.Temperature was kept at 25 ± 1°C inside the chamber.

After placing samples m the chamber, COz at atmosphencpressure was injected for 30 minutes m order to ehmmateair. Thus, when COz was mcreased till to 20 bar in 3minutes, the gas concentration was higher than 99%. Atthe end of each treatment the gas pressure was decreased toatmosphenc pressure in 20".At the end of the exposure times larvae and adults of o.

surinamensis and larvae of P. mierpuncieila wereobserved immediately and after 48 hours (possIbleanaesthetic effect of treatment); pupae were controlled atmtervals of 2 days till to 15 days to venfy possible emergmgof adults. S oryzae was controlled every 2 days for amaximum of 45 days in the case of eggs. The biologicalsample was kept at a temperature of 25 ± 1°C and 65 ± 5%r h , and with 12 hrs of hght alternatmg With 12 hrs ofdarkness. Percentage mortahty was corrected from controlmortahty by Abbott's (1925) formula.

Results

Tables 1 - 3 show the mortahty for the different testedspecies and stages of P . uuerpuncieila , o.suruuimensis and S. oryzae 100% of mortality of larvaeand pupae of P interpunctella was observed after 5mmutes while 15 minutes at 20 bar (99% COz) wererequired to prevent eggs hatchmg.

Eggs of O. suruuumeneis are more tolerant as 100% ofmortality IS venfied after 45 minutes. after 5 mmutes amortahtyover 95% IS observed for young larvae, after 10minutes for larvae while mortality IS 100% after 5 and 10mmutes for pupae and adults. The complete mortahty of S.oryzae adults IS obtained after 15 minutes wlule 45 mmutesare required for the other stages

Discussion

Moth proved less tolerant to the treatment conditions thanbeetles; m fact complete mortahty was obtamed after 15minutes for all the tested stages of P. mterpunctella andafter 45 minutes for 0 sunnarnensU5 and S oryzaeThere was a significant reduction of time required to obtammortahty of the species compared to the use of COz atatmosphenc pressure (Annis, 1987).Eggs were the most tolerant stage; m fact they are

charactenzed by a low water content and by very few cellmembranes; besides they are most stable being spheres(Reichmuth and Wohlgemuth, 1994).The most tolerant species was S. oruzae , as the eggs,

larvae and pupae are inside kernels and were protected,even if partially, by the effect of high pressure COz Cahbosoet al (1994) earned out tests on different stages of S.oryzae, R. dommica , T. casianeum and C.cephalonica exposed to carbon dIOXIdeat the pressure of 5,10, 20 and 30 kg/crrr' for 5 or 10 mmutes m the chamberand venfied that, in general, beetle species were moredifficult to control than moths, and eggs were the mosttolerant stage. Obtamed results showed that the preventivemjection of COz for 30 mmutes, pressunzmg to 20 bar m 3mmutes and the restauration of atmosphenc pressure WIth asudden discharge of COz m 20" helped to reduce timerequired to obtam the mortality of the species.

Table 1. Mean percent mortahty of different stages of Plodui mierpuncieila exposed to high pressure (20 bar) carbondIOXIde.

Exposure Time Stage

(rmnutes) Eggs Young larvae Larvae Pupae

5 98.0 ± 0.6 100 100 100

10 99.4 ± 0.4 100 100 100

15 100

30 100

Mean of 4 rephcatesThe age (days) of the different stages was as follows: eggs 2 - 4; young larvae 6 - 8; larvae 12 - 15; pupae 20 - 22Means of control were 86 0 ± 1 7, 19 2 ± 0 5, 19 6 ± 0 3, 19 6 ± 0 3 for eggs, young larvae, larvae and pupae respectively

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Proceedmqs of the 7th International Worktng Conference on. Stored-product Protection - Volume 1

Table 2. Mean percent mortahty of different stages of Oryzaephilus surinamensie exposed to high pressure (20 bar) carbondioxide.

Exposure Time Stage

(rrunutes) Eggs Young larvae Larvae Pupae Adults

5 86.7 ± 1.1 97.5 ± 1.5 89.9 ± 3.5 100 73.1 ± 5.2

10 95.8 ± 0.7 100 98.8 ± 1.3 100 100

15 97.9 ± 0.6 98.8 ± 1.3 100 100

30 98.6 ± 0.6 100 100

45 100 100

Mean of 4 rephcates.The age (days) of the different stages was as follows: eggs 1 - 8; young larvae 15 - 20; larvae 24 - 28; pupae 32 - 34; adults 15 - 30Means of control were 72.0 ± 3.7, 19.4 ± 0.3, 19.5 ± 0 4, 18.8 ± 0.4, 19 4 ± 0.4 for eggs, young larvae, larvae, pupae anq adultsrespectively

Table 3. Mean percent mortality of different stages of Sitophtlus oryzae exposed to high pressure (20 bar) carbon dioxide.

Exposure TIme Stage

(rrunutes) Eggs Young larvae Larvae Pupae Adults

5 46.5 ± 4.1 82.9 ± 1.2 90.3 ± 0.7 89.3 ± 1.0 90.8 ± 1.3

10 72.0 ± 2.0 94.2 ± 0.7 96.6 ± 0.7 96.7 ± 0.9 94.9 ± 2.1

15 93.3 ± 1.7 99.2 ± 0.5 98.6 ± 0.9 97.5 ± 0.4 100

30 97.3 ± 0.5 99.3 ± 0.5 99.1 ± 0.6 99.0 ± 0.6 100

45 100 100 100 100

Mean of 4 rephcatesThe age (days) of the different stages was as follows: eggs 1 - 8; young larvae 10 -14; larvae 18 - 22; pupae 26 - 28; adults 10 -15.Means of control were 76.4 ± 3 5, 68.4 ± 2.6, 56.6 ± 2.7, 53 3 ± 2.0, 19 5 ± 0 3 for eggs, young larvae, larvae, pupae, and adultsrespectively

Reichmuth and Wohlgemuth (1994) observed that eggs ofP. interpunctella after being exposed to 20 bar with asubsequent decompression within 1 nun for 15 min, wereunable to hatch. On the contrary Prozell (1997) observed intests carried out m a 3 rrr' chamber a 20'C, 20 bar ofpressure with a subsequent decompression Within 8 minutesthat mortality of all stages of O. surinamensie and P.interpunctella was obtained after 3 hours. In thisexpenment the species were placed inside the substratum totreat as the rate of distribution of the gas depended onnature and density of the product. Another variable toconsider m order to reduce exposure times IS temperature atwluch the treatment IS carried out. Prozell et al. (1997) intests carried out on 12 species at different developmentalstages placed in 1 ton of bagged foodstuff in a 3m3 chamber,observed that, at a temperature of about lOoe under 20 barof COz, the lethal exposure period for treatment was 3 hoursand this was about 1/5 longer than at 20°C.Comparing the observed mortahty for the species in the

various developmental stages, ummportant differences m

mortahty percentages, except for S oryzae, are recordedby changing exposure times The effect of high pressureCOz on the insect takes place very rapidly and tlus seems todepend not only on COz pressure but mostly on theconcentration and on a very rapid discharge of this gas at theend of the treatment.ThIS method could be largely used as it seems that it

doesn't modify foodstuff quality according to Prozell's(1997) researches on cocoa beans.

References

Abbot, W. C. 1925. A method for computing theeffectiveness of an insecticide. Journal of EconomicEntomology, 18, 265 - 267.Annis, P. C. 1987. Towards rational controlled atmospheredosage schedules: a review of current knowledge. In:Donahaye E. , Navarro S. , ed. , Proceedings of the FourthInternational Working Conference on Stored-ProductProtection, Tel AVIV, Israel, 1986. 128 -148.

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Annis, P. C. 1990. Requirements for fumigation andcontrolled atmospheres as options for pest and qualitycontrol m stored grain In: Champ, B. R. , Highley, E. ,Banks, H. J, ed., Proceedmgs of InternationalConference on Fumigation and Controlled AtmosphereStorage of Grain, Singapore, February 1989 ACIARProceedmgs No. 25, 20 - 28Caliboso, F. M , Nakakita, H , Kawashima, K. 1994. Apreliminary evaluation of carbon dioxide under highpressure for rapid fumigation. In: Highley, E., Wright,E. I., Banks H. J., Champ, B. R., ed., Proceedings ofthe 6th International Working Conference on Stored-product Protection, Canberra, Australia, October 1994. I,45 - 47.

Gerard, D., Kraus, J., Qumn, K. V and Wohlgemuth,R. 1988 Anwendung von Kohlendioxid (CCh) unter Druckzur Bekampfung vorratsschadlicher Insekten und Milben.Pharrnazeutische Industne , 50, 1298 - 1300.Jay, E. G., Banks, H. J. , Keever, D. W., 1990 Recentdevelopments in controlled atmosphere technology In:Champ, B. R., HIghley, E., Banks, H J , ed.,Proceedmgs of International Conference on Fumigation andControlled Atmosphere Storage of Gram, Singapore,February 1989. ACIAR Proceedmgs No. 25, 134 -143Le Torc'h, J. M. , Fleurat-Lessard, F 1991. Effet de fortespressions sur l'efficaci te insecticide des atmospheresmodifiees par CCh contre Sitoplulu» grana nus (L ) etSitophiius oruzae In: Fleurat-Lessard, F , Ducom, P ,ed. , Proceedings 5th International Workmg Conference onStored-product Protection, Bordeaux, France, September1990. II, 847 - 855.Locatelh, D. P. , Daoho, E 1993 Effectiveness of carbondioxide under reduced pressure agamst some msectsmfesting packaged nee. Journal of Stored ProductsResearchs, 29, 81 - 87.Nakakita, H., Kawashima, K. 1994. A new method tocontrol stored-product msects using carbon dIOXIde Withhigh pressure followed by sudden pressure loss In:Highley, E , Wright, E. I , Banks H. J. , Champ, B R ,ed., Proceedings of the 6th International WorkmgConference on Stored-product Protection, Canberra,Austraha, October 1994 I, 126 - 129.Prozell, S. , Reichmuth, C. 1991 . Effets des atmospheresmodifiees a haute pression sur le charancon des grams,Sitoplulu» grananus (1.) et le Coleoptere xylophage,Lyctus brumneus (Stephens). In: Fleurat-Lessard, F.,Ducom, P. , ed., Proceedmgs 5th International WorkmgConference on Stored-product Protection, Bordeaux,France, September 1990. II, 919.Prozell, S. , Reichmuth, C. 1991 Response of the granaryweevil Sitopluiu« grana nus (1.) (Col.: Curcuhomdae)to controlled atmospheres under high pressure. In:Fleurat-Lessard, F., Ducom, P , ed , Proceedmgs 5th

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Prozell, S. , Reichmuth, C. , Ziegleder, G. , Schartmann,B , Matissek, R., Kraus, J , Gerard, D and Rogg, S.1997. Control of pests and quahty aspects in cocoa beansand hazelnuts and dIffUSIOn expenments m compressedtobacco With carbon dIOXIde under high pressure. In:Donahaye, E J , Navarro, S. and Varnava, A., ed.,Proceedings of International Conference on ControlledAtmosphere and Fumigation m Stored Products, NiCOSia,Cyprus, Apnl 1996, 325 - 333Reichmuth, c., 1991 New techniques in fumigationresearch today. In: Fleurat-Lessard, F., Ducom, P.,ed. , Proceedmgs 5th International Workmg Conference onStored-product Protection, Bordeaux, France, September1990. 11,709 -724.Reichmuth, C. 1997. There is no resistance of stored-product moths agamst treatment With carbon dIOXIdeunderhigh pressure! In: Donahaye , E. J , Navarro, S. andVarnava, A., ed, Proceedmgs of InternationalConference on Controlled Atmosphere and Furrugation mStored Products, Nicosia, Cyprus, Apnl 1996, 519 - 525.Reichmuth, C., Wohlgemuth, R., 1994. Carbon dioxideunder high pressure of 15 bar and 20 bar to control theeggs of the Indianmeal moth Plodia interpunctella(Hubner) ( Lepidoptera: Pyrahdae ) as the most tolerantstage at 25°C. In: Highley E. , Wright, E I. , Banks HJ., Champ, B R, ed, Proceedmgs of the 6thInternational Workmg Conference on Stored-productProtection, Canberra, Austraha, October 1994. I, 163-172.Sabme , P. , Reichmuth, C , 1991. Response of the granaryweevil Sitoplulu« granarw.3 (1.) (Col. : Curcuhorndae)to controlled atmosphers under high pressure. In: Fleurat-Lessard, F, Ducom, P., ed., Proceedmgs 5thInternational Workmg Conference on Stored-productProtection, Bordeaux, France, September 1990 II, 911-918

Stahl, E., Rau, G., Adolphi , H. 1985. Entwesung vonDrogen durch Kohlendioxide-druchbehandlung.Pharmazeutische Industne, 47, 528 - 530.Suss, 1., Locatelh, D. P 1994. Effectiveness of carbondIOXIde under reduced pressure agamst some msectsmfestmg dned fruit In: Highley E , Wnght, E I ,Banks H. J., Champ, B R. , ed., Proceedings of the 6thInternational Workmg Conference on Stored-productProtection, Canberra, Austraha, October 1994 I, 194-200.Ulnchs, C. 1995. Emfluss der Temperatur auf dieWirksamkeit emer Bekampfung des Tabakkafers nut CChunter Hochdruck Mitteilungen der Deutschen Gesellschaftfar Allgememe und Angewandte Entomologie , 10, 263-

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264Ulnchs, C. , Reichmuth, C. 1997 Emfluss physikalischereParameter auf die Bekampfung des TabakkafersLasuxierma serricorne Fab. (Col.: Anobudae ) mitKohlenstoffdioxid unter Hochdruck. Anzeiger furSchadlmgskunde, Pflanzenschutz, 70, 81 - 86Ulnchs, C., Reichmuth, C and Rassmann, W. 1997.

Carbon dioxide under high pressure to control the tobaccobeetle Lasioderma serricorne, In: Donahaye , E J.,Navarro, S. and Varnava, A., ed., Proceedmgs ofInternational Conference on Controlled Atmosphere andFumigation m Stored Products, Nicosia, Cyprus, Apnl1996, 335 - 341.

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