article on grain storage 1

8/2/2019 Article on Grain Storage 1

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AIR-TIGHT STORAGE O F GRAIN; ITS EFFECTS ON INSECT PESTSI. CALANDRA GRANARIA L. (COLEOPTERA,URCULIONIDAE)

By S. W. BAILEY"( M a n u s c r i p t received J u l y 15, 1 9 5 4 )

S u m m a r yThe responses of adu lt and immature C a l a n d r a g r a n a ~ i aL. to high

concentrations of carbon dioxide and low concentrations of oxygen have beendetermined. To bring about 100 per cent. mortality of all stages requires anincrease of carbon dioxide t o 40 per cent. or a decrease of oxygen to 2 percent. The respira tory quotient of the species has been measured an d i t isshown tha t the death of the insects, under air- tight gr ain storage conditions,is due to the depletion of oxygen caused by the respiration of the insects a ndthe gra in an d not to the accumulation of carbon dioxide.

The adult insects are the most resistant stage and the first instar larvaethe most susceptible.

Estimates for the time required for the insects to die and for the amountof damage they cause before death are given.

I. INTRODUCTIONThe storage of grain in sealed containers has been practised in manyparts of the world for a very long time. For example, Pruthi and Singh

(1950) describe a number of types of pits and receptacles used by Indianfarmers. Other types have been reported as being used in Malta, Italy,Africa, South America, and elsewhere. Reports on the condition of grainstored in these containers have generally been favourable, the absence ofinsect attack frequently being stressed. I t has generally been assumedthat the control of any insects present a t the time of sealing was due tothe accumulation of carbon dioxide caused by the respiration of the insectsand the grain. Some authors have disagreed with this, Cole (1906) con-sidering that high carbon dioxide and low oxygen even favoured thedevelopment of weevils. Dendy (1918) reviews the conflicting evidence;and his work during World War I, on behalf of the Grain Pests Committeeof the Royal Society, appears to include the first carefully executed seriesof experiments designed to elucidate the role that gas exchange plays inkilling the insects. Dendy, and Dendy and Elkington (1918, 1920) workedwith most of the economically important insect and mite pests of grain.

In spite of Dendyk evidence and the obvious advantages of a methodwhich gives freedom from losses due to pests without the need for fumiga-tion or other applied treatments, no deliberate large-scale adoption of thesystem followed for two decades. I t is of interest to note, however, thatAustralia in 1918 enclosed large stacks of infested grain in gas-proofmaterial and fumigated it by pumping in carbon dioxide. Experiments

" Division of Entomology, C.S.I.R.O., Canberra, A.C.T.


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34 S. W. BAILEYcarried out by Spafford and Hargreaves (Winterbottom 1922) hadindicated that a high concentration of carbon dioxide would kill the insects.I t was subsequently found tha t the addition of carbon dioxide from outsidewas unnecessary and that the gas exchange due to the respiration of theinsects alone was. sufficient to give a satisfactory control if the stackswere covered and made gas-tight. In addition some experimental pitswere sunk at Walleroo, S.A., in 1918 and filled with infested wheat(Winterbottom 1922 ; Spafford 1939). However, the construction of thesepits was such that i t is very doubtful if they were gas-tight and the resultswhich were assumed to be due to hermetic sealing were much more likelydue to heat produced by the insects.

In 1942 Argentina was faced with the problem of storing very largestocks of grain which could not be shipped owing to war-time conditions.A serious loss due to insect attack was feared and a low cost storage methodhad to be found that would accommodate very large quantities of bulkgrain and would not require the use of fumigants or insecticides, as thesewere not manufactured in the Argentine. A consideration of possiblemethods led to practical field experiments on the storage of grain inhermetically sealed underground silos. The success of these experimentsled to a large-scale adoption of the method and by 1948 1540 silos with acapacity of approximately 850,000 tons had been built (Argentine Republic1949). These appear to have been very successful, providing adequatestorage and obviating both losses due to insect attack and expensesassociated with conventional methods of insect control. A large fund ofpractical knowledge concerning the construction, gas- and waterproofing,and covering and sealing of the pits was acquired, thus overcoming theengineering problems that may have been partly responsible for thefailure to adopt the system elsewhere.

Since the results of the work in Argentina have become known, freshinterest in the subject has been aroused and similar silos have been builtelsewhere, notably in other parts of South America (Shellenberger andFenton 1952) and in east Africa (Swaine, unpublished data) .

In spite of the large amount of information provided by the work inthe Argentine, the only reliable data of the effects of such an environmenton insect pests of grain appear to be those given by Dendy and Elkington(1918, 1920). They worked in the main by sealing insects, grain, androom air in flasks and subsequently examining for mortality and analysingthe gaseous composition of the atmosphere within the flasks. A smallernumber of experiments were carried out in which the insects were sealedup in a predetermined mixture of gases. However, in all their experimentsthe atmosphere surrounding the insects varied during the course of theexperiments owing to the respiration of the insects and to a lesser extenttha t of the grain. Their work deals predominantly with the effects ofcarbon dioxide accumulation, and i t is not known whether it is this factoror the depletion of oxygen, or a combination of both, that is the important


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AIR-TIGHT STORAGE OF GRAIN. I 35controlling mechanism. This paper describes the results of experimentsto answer this and other questions ar ~s ing rom this method of grainstorage.

11. METHODSIn order to investigate the concentrations of carbon dioxide and

oxygen that the insects are able to tolerate the changes due to their respira-tion must be eliminated. This is best done by arranging for the insectsto be exposed to a constant stream of gases which sweep away allrespiratory products and maintain the atmosphere a t the desired values.

Fig. 1.-Diagram showing details of insect containers and th eir arr angement within anexposure chamber.

A constant flow gas-mixing apparatus was designed (Bailey l954),capable of giving a number of gas streams each of which could be set toany desired mixture of nitrogen, oxygen, and carbon dioxide. Thisapparatus was fed with gases obtained from normal commercial sources,the oxygen being of medical quality. The suppliers' analyses of thesegases indicated slight impurities but as all results are based on analysisof the mixtures passing over the insects the presence of these impuritieswas unimportant. All gas analyses, including check analyses carried outduring the course of each experiment, were made on a Sleigh gas analysisapparatus (Sleigh 1937) and are expressed as percentages of dry gas.

After mixing, the gases were led to a constant temperature water-bath in which conditioning equipment and the exposure chambers holdingthe experimental and control insects were submerged (Fig. 1) . The bathwas fitted with. a lid to exclude light. The gas temperature was broughtup to that of the water-bath, 25"C, by passing i t through a coil of suitabletubing and the relative humidity adjusted to approximately 72 per cent.by passing the gas stream over a saturated solution of sodium nitrate.The gas then passed over the insects and bubbled to waste against 4 n.


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36 S . W. BAILEYof water pressure. This method of exhausting provided an easy checkon the gas flow and eliminated any chance of back diffusion by atmosphericgases.

In order to determine mortality the insects had to be examined inroom air and subsequently incubated fcr 48 hr to detect any delayedeffects; consequently survivors could not be returned to the experimentalatmosphere for fu rther exposure. This necessitated dividing the insectmaterial into a number of replicates, successive replicates being removedfor examination and the remainder left in the gas stream. The insectsand grain were therefore held in small plastic and wire gauze containersof 15 ml capacity designed to permit maximum gas exchange. Theseare illustrated in Figure 1. Ten were placed end to end in an exposurechamber made from 60 cm of 3.5 cm bore Pyrex tube, the gas mixturebeing admitted a t one end and exhausted from the other. This arrange-ment resulted in a small gradient of carbon dioxide and oxygen along thelength of the exposwe chamber owing to the respiration of the insects,but this was minimized by the rate of gas flow and the number of insectsused. Variations in the composition of the gas mixtures due to thisgradient and to the method employed for mixing the gases were measured,and showed a maximum absolute error of 1 .6 per cent. This exceedederrors in gas analysis by a factor of 20. The control insects were suppliedvi t h 10 C.C. of conditioned air per min.One hundred insects of undetermined sex were used fo r each experi-ment, these being divided into 10 replicates of 10 each. The insectsoriginated from field-collected specimens that had been bred throughseveral generations in the laboratory at 25OC and 70 per cent. R.H. Stockcultures were sieved a t regular intervals to provide material of knownage. Owing to the habit of newly emerged adult CaZundra g r u n a k L.of sometimes staying within the gra in for a few days (Richards 1947)some slight increase in the indicated age is possible. Ignoring this allinsects were 0-7 days old a t the s tart of each experiment.

The grain was F.A.Q. wheat supplied by the Australian Wheat Board.Before use it was sieved to remove small and broken grains and sterilizeda t 60C for 6 h r to kill any insects or mites present. A calculated amountof water was then added and the grain stored over saturated sodium nitratesolution until equilibrium was attained. When ready fo r use the moisturecontent averaged 14.2 per cent. (wet weight basis) , as determined on aCarter-Simon Rapid Moisture Tester. Each container held approximately11 g of wheat (= 240 grains).At half-weekly intervals one container u7as removed from eachexposure chamber and the contents examined. The 10 replicates there-fore allowed each experiment to run for 5 weeks, which approximates theduration of the life cycle of C. gramria under the experimental conditionsof temperature and relative humidity. The removal of the containers wascarried out very rapidly to minimize the amount of room ai r entering


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AIR-TIGHT STORAGE O F GRAIN. I 37the exposure chambers. After sieving out, the insects were examinedfor mortality, any insects showing any sign of movement being countedas alive. They were then placed in a muslin covered tube with a littlefresh grain, incubated in room air a t 25OC and 70 per cent. R.H., andre-examined at 24 and 48 hr to determine any delayed mortality or revival,the same criterion of death being applied. All results recorded here arebased on the examination a t 48 hr. The grain from the experiment wasplaced in separate muslin covered tubes and incubated under the sameconditions. I t was sieved twice. weekly and the number of F, generationinsects and their distribution in time recorded. In those instances where,

I , I I I I I0 5 10 15 2 0 25 30 35

TIME (DAYS)Fig . 2.-The effect of time on the mor ta lit y of adul tC. granaria exposed to differ ent concentrations of carbondioxide. Oxygen as in ai r. Carbon dioxide concentrations:B, 0, 20, and 30.1 per cent.; X , 34.2 per cent.; @, 38.2 per

cent.; A, 1.7 per cent.; 0,4.9 per cent.after incubation, there was little or no emergence of F, generation insectsthe grain was examined for egg plugs in order to determine the effectof oxygen and carbon dioxide on oviposition behaviour. To facilitate thisthe plugs were stained with acid fuchsin (Frankenfeld 1948) or berberinesulphate (Milner, Barney, and Shellenberger 1950) but neither method wascompletely satisfactory, possibly owing to the age of the egg plugs. Theroutine finally adopted was to stain with acid fuchsin, carry out a roughsorting by eye, and then examine the selected grains under a binocularmicroscope. Positive ones were then dissected to determine the extentof development of the immature stages. Considerable difficulty wasexperienced, as the eggs or larvae had been dead for 5-12 weeks and werein a very shrivelled condition.


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S. W. BAILEY111. RESULTS

( a ) The E f f e c t s o n A d u l t I ns ec tsThree series of experiments were run to determine:( i ) The effect of increasing concentrations of carbon dioxide, oxygenbeing maintained a t 21 per cent."(ii) The effect of decreasing oxygen concentrations, carbon dioxidebeing absent."

Fig . 3.-The effect of time on th e mor tal ity of adult C. yranariuexposed to different concentrations of oxygen. Carbon dioxidea s in air. Oxygen concentrations: 0,0, 5, and 3.1 per cent.;X , 2.05 per cent.; 9, 1.3 per cent.

(ii i) The combined effects of increasing concentrations of carbondioxide and decreasing concentrations of oxygen.The response of the insects is due to the environment and to the length

of time they are exposed to it. In Figures 2 and 3 time is plotted againstmortality due to exposure to carbon dioxide and oxygen respectively. Inspite of considerable scatter due to the biological variation of the material,there is a clear trend indicating that the full effect is reached not laterthan 17 days after the st art of the experiment, insects surviving this periodliving for the full 5 weeks' duration of the experiment. Results aretherefore presented on the basis of a 17-day exposure period, as mortalitiesachieved in a shorter period are of no interest here.

" Normal a i r contains approx imatel y 20.9 per cent. oxygen and 0.03 pe r cent.carbon dioxide.


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AIR-TIGHT STORAGE O F GRAIN. I 39Figures 4 and 5 show the independent effects of carbon dioxide and

oxygen. It will be seen that the response is very sharp but that it doesnot occur until very substantial variations from normal atmosphericconditions occur.

The combined effect of the two gases was investigated in two seriesof experiments. In the first, insects were exposed to different concentra-tions of carbon dioxide, oxygen being maintained a t a dilution close to buta little above that which would cause mortality. In the second seriesoxygen was varied whilst carbon dioxidebelow a lethal concentration.

was kept close to but a little

CARBON DIOXIDE (%)Fig. 4.-The niortality of adult C. gramzria due toexposure to carbon dioxide f o r 17 days. Oxygen a s inair.

The results are shown in Tables 1 and 2. With a low oxygen tension,over 25 per cent. carbon dioxide is necessary before any mortality occurs.On the other hand with high carbon dioxide some mortali ty is evident withoxygen as high as 9 per cent. Nevertheless oxygen has to drop to as lowas 3 per cent. to bring about 100 per cent. kill. This is only 1 per cent.higher than the concentration required in the absence of carbon dioxide.

In all experiments causing full or partial mortality there was verylittle recovery or delayed mortality during the 48 hr the insects were keptunder observation. A number of the insects that survived conditionscausing a high mortality were tested for fertility. All were found to be


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40 S. W . BAILEYnormal in this respect. The effect of the age of the adults was notexamined, all insects being 0-7 days old. Howe and Oxley (1952) found

I I0 2

I4

I I6 8 1 0 12

OXYGEN (%)Fig . 5.-The m or ta l i ty of ad ul t C. granuria d u e t o e x p o s u r e t or educed oxygen concen t r a t ions fo r 17 days . Ca rbon d iox ide a si n a i r .

no change in the rate of carbon dioxide production due to the age of theadults and it does not seem likely that an increase in age will cause themto be more tolerant to an unfavourable atmosphere.

TABLE1MORTALITY OF ADULT C. GRANARIA DUE TO INCREASING CONCEIiTRATIONSOF CARBON DIOXIDE WI TH OXYGEN HELD A LITTLE ABOVE A LETHAL

DILUTIONE x p o s u r e p e r io d 1 7 d a y sE x p er im e n t C a rb o n D io xid e O xy ge n ~ i r t a l i t yNo. ( 7 % ) ( % I (%)

1 10.9 4.3 02 25.2 4.4 03 29.4 3.8 604 35.3 4.1 20

The response of the insects to low oxygen (Fig. 5 ) was much sharperthan expected, and resulted in the oxygen gradient along the length of the


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AIR-TIGHT STORAGE OF GRAIN. I 4 1exposure chamber having a noticeable effect in some experiments. Theseeffects were, however, covered by the limits of accuracy given for the gasmixtures.

TABLEMORTALITY OF ADULT C. GRANARIA DUE TO DECREASING COKCENTRATIONSOF OXYGEN WITH CARBON DIOXIDE HELD A LITTLE BELOW A LETHAL

CONCENTRA4TIONExposure period 17 days

Experiment Oxygen Carbon Dioxide Mortal ityNo. ( % I (a) (%)

( b ) The E f e c t s o n Inz?nature S tagesThe determination of the total number of F, generation insects

emerging from the grain after its removal from the experimental gasI

CARBON DIOXIDE (%)Fig. 6.-The mortal ity of immature stages of C.gramria due t o exposure to carbon dioxide. Oxygena s in air. Mortality based on emergence comparedwith concurrent controls.

mixture and subsequent incubation gave information on the grcss effectsonly as the age composition of the immature stages was mixed. Forexample, the grain in the first replicate could contain no stages older than3 days, whereas, if the weevils had been laying eggs throughout the


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42 S. W. BAILEYexperiment, the last might contain individuals whose ages varied from0 to 35 days. These gross effects are shown in Figures 6 and 7 respectively.

The fact that no emergence of F, generation insects takes place, evenat the end of the incubation period, at concentrations that are not lethalto the adults suggests either that the immature stages were killed earlyi11 life or that the adults failed to oviposit. Owing to the difficulty ofidentifying egg plugs and the possibility that some oviposition may havetaken place during the first few hours of an experiment, the results ofegg plug counts and dissection of the grain can only indicate the probablecauses. Table 3 shows the effects of carbon dioxide and oxygen on ovi-position and is based on the 28th day replicate.

Fig. 7.-The mor ta lity of imma tu re sta ges of C.granaria due to exposure to reduced oxygen concen-trations. Carbon dioxide as in air. Mortality basedon emergence compared with concurrent controls.Table 3 indicates that the ra te of egg laying was much reduced but

not entirely suspended in gas concentrations that did not permit develop-ment of immature stages. Dissection of the grain showed that in mostcases the eggs had hatched and the larvae died in the first instar. Thissuggested tha t under some conditions eggs laid between the day of removaland approximately 4 days earlier survived. This was in fact so; all F,insects that survived 30.2 per cent. carbon dioxide ( a total of 29 for allreplicates) emerged not earlier than 5 weeks after their removal from


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AIR-TIGHT STORAGE O F GRAIN. I 43the gas mixture. All these, therefore, had been eggs or very young larvaewhen removed.

TABLE3E F F E C T O F O X YG E N A N D C A R B O N D IO X ID E O K O V I P O S I T IO N B E H AV I O UR O F C. G R A N A R I A

Carbon No. of No. of F1 No. of No. of F1Dioxide* Ge ner ation Oxygen't % ) Genera t ion( % ' C ) Egg ' lugs Emerging Egg ' lugs Emerging0 $ 1718 20.9 $ 1718

19.5 $ 77 10.9 t 15830.2 47 2 5.0 t 10334.2 19 0 3.1 2 035.9 0 0 2.1 0 037.8 0 041.7 0 0* P e r c en t. o xy ge n a s i n a i r .Jy P e r c e n t. c a r b o n d io x id e a s i n a i r .$ E g g p lug counts no t poss ib le owing to ho led condit ion of gra in .8 Mean of controls.Additional evidence that first instar larvae were the most susceptible

was obtained by exposing immature stages of known age to gas concen-trations causing partial mortality. A suitable quantity of conditioned

TABLEE F F E C T O F OXYGEN A N D C A RB O N D I O X I DE O N I M M A T U R E S T A G E S O F C. G R A N A R I AT h e n u m b e r s i n t h e b od y of t h e t a b l e r e p r e s e n t t h e F1 genera t ion adu l t s emerg inga f t e r i nc u ba ti onA g e 31.1 P er cen t . Carbon D ioxide* 4.4 Per cen t . Oxygen?G r o u p s - -D a y s ' 14 D a y s ' 7 Days ' 14 Days ' Contro ls

( d ay s ) E x p o s u r e E x p o s u r e E x p o s u r e E x p o s u r e0-7 6 3 8 2 287-14 42 42 34 35 44

14-21 56 36 52 42 4121-28 0 0 0 1 128-35 11 8 8 6 10* P e r c e n t. o x y ge n a s i n a i r ..F P e r cent . ca rbon d iox ide a s in a i r .

grain was exposed to ovipositing adults for 7 days, the adults then beingmoved on to fresh grain. This was repeated every 7 days until five batchesof grain were obtained, each containing different known age groups. Afterthorough mixing each batch was divided into five equal replicates of 180grains each, two of each batch being exposed to high carbon dioxide andtwo t o low oxygen. The fifth replicate was exposed to a stream of roomair and used as a control. One replicate from each experimental batch


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44 S. W. BAILEYwas removed after 7 days, the other replicates and the controls after 14days. All were incubated in room air a t 25OC and 70 per cent. R.H. andthe number of F, adults recorded. The results are shown in Table 4.

For each age group, excluding 21-28 days in which the low numberswere believed to be due to a breakdown in the temperature control of theincubator during the preparation of the grain, a X"est was made of thevariability between treatments in the proportion of grains from which

TABLE5RESPIRATORY QUOTIEKT* O F C. GRANARIA AT 250C A N D 70 PER CENT. R.H.

ReplicateNo.

Conditions Carbon Oxygen RespiratoryDioxide(%) (%) Quotient

Approx. 150 mixed age adultsonly; no grain1 Af ter : 5 days 16.57 0.37 0.812 8 days 16.72 0.18 0.81

0- to 21-day-old immature s tagesin grain1 Af ter : 13 days 13.73 0.114 0.682 20 days 5.03 12.72 0.623 23 days 13.42 0.19 0.654 28 days 14.24 0.11 0.69

0- to 21-day-old immature stagesin g ra in and associated adults1 After : 24 h r 12.1 4.5 0.742 28 hr 15.9 0.57 0.783 2 days 15.9 0.17 0.794 13 days 15.9 0.11 0.775 23 days 16.1 0.13 0.78* Respiratory quotient = 72 CO2/ (20.88- 0 2 ) .adults emerged. Only for the 0-7 day old age group were the differences

significant. The difference between the 7 and 1 4 days' exposure to theexperimental concentrations of carbon dioxide and oxygen did not reachsignificance, and the overall significance is due wholly or almost whollyto the difference between the applied treatment and the control. Forolder age groups the results do not indicate significant differences betweentreatments although the 14-day exposure tended to give lower emergencethan the 7-day.

( c ) The Respira tory Quot ien t o f C . granariaThe combined concentrations of oxygen and carbon dioxide that occur

in infested hermetically stored grain is governed by the respiratoryquotient (R.Q.) of the insects, that of the grain making only a small


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AIR-TIG HT STORAGE OF GRAIN. I 45contribution (Lindgren 19% ; Oxley 1945). Three series of experimentswere therefore set up to determine the R.Q. of the adult insects, of theimmature stages, and of a mixed population. In each series the materialwas placed in 100-ml flasks containing room air, which were then sealed.After a suitable time had elapsed the gaseous contents of the flasks wereanalysed. In the second and third series the flasks were completely filledwith the grain, thus containing about 40 ml of room air (Page and Lubatti1937; Jones 1943). A number of replicates were set up in order to avoidany effects which might have been caused by the reduction in atmosphericpressure due to the extraction of successive gas samples from a singleflask. I t was to be expected that the indicated R.Q. would be a little lowerin the presence of grain as some of the carbon dioxide produced by theinsects might have been absorbed by it (Howe and Oxley 1952). Theresults are shown in Table 5 .

In order to determine the contribution due to the respiration of thegrain a further 100-ml flask was filled with grain only. This produced1.71 per cent. of carbon dioxide in 35 days.

The age structure of an infestation in stored grain is inevitablycomplex. Here it is sufficient to take a respiratory quotient of 0.8 as abasis for the estimation of carbon dioxide and oxygen concentrations thatmight be found in practice. Dendy and Elkington (1918) using an entirelydifferent method, determined the R.Q. of adult C. granaria and obtainedvalues ranging from 0.78 to 0.86 with a mean of 0.815.

Figure 8 shows the combined concentrations of the two gases thatoccur on the basis of an R.Q. of 0.8.

IV. DISCUSSIONThe maximum variations tha t can occur in the components of the

intergranular air in wheat infested witn C. granaria under the physicalconditions of these experiments are shown in Figure 8. This allows amaximum concentration of less than 17 per cent. carbon dioxide, evenassuming th at the insects are able to make use of all the oxygen initiallypresent. The experimental results on the tolerance of C. granaria tocarbon dioxide show that this concentration does not kill any of the adultsor prevent oviposition and that a large proportion of the immature stagessurvive and complete their development. On the other hand the oxygentension drops to a very low value, and it seems clear that in practice it isthe depletion of oxygen and not the build-up of carbon dioxide that is theimportant factor. When the oxygen tension is low the carbon dioxidepresent has little additional effect.

The great importance of reduced oxygen tension is at variance withthe generally kield belief that the effectiveness of hermetic storage is dueto the accumulation of carbon dioxide. I t explains the failure of attemptsto fumigate grain by the simple addition of carbon dioxide, generally in


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46 S. W. BAILEYthe form of dry ice (Johnston and Sorenson 1952; Shellenberger andFenton 1952). Concentrations as high as 40 per cent. fo r 3 weeks, andpossibly longer a t lower temperatures than 25OC, would be necessary andthese would be very difficult to maintain in normal concrete silos orsheeted stowages owing to leakage and diffusion. Attempts to reduce thepractical difficulties by maintaining a higher concentration for a shorterperiod would be ineffective, as some experiments run to check this pointgave survivals of up to 20 per cent. of adult C. yrwnaria exposed toapproximately 100 per cent. carbon dioxide for 5 days.

CARBON DIOXIDE (%)Fig. 8.-The concentrations of carbon dioxide andoxygen occurring in the intergranular air amonggrain infested with C. granaria. Based on a respira-

tory quotient of 0.8.

The greater susceptibility to unfavourable gas concentrations shownby the immature stages may be, in part , only apparent. All immaturestages live within the individual grains whose walls will act as a partialbarrier to the diffusion of oxygen and carbon dioxide. As a larva growsit hollows out a larger cavity, thus increasing the internal surface areathrough which diffusion of gases must take place. I t is likely, therefore,that the younger the larvae the greater will be the difference betweengas concentrations occurring within the cavities and in the intergranular


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AIR-TIGHT STORAGE O F GRAIN. I 47air. In the experiments on immature stages the gases in the intergranularair only were analysed and the immature stages may have been exposedto lower concentrations of oxygen than those indicated by analyses.Nevertheless for the interpretation of results obtained from stored grain,values based on the analyses of intergranular air a re the ones required.

I t has been shown that a maximum concentration of approximately17 per cent. carbon dioxide can be expected; nevertheless higher concen-trations have been reported. Analyses of the intergranular ai r in theArgentine pits have given values as high as 27 per cent., representingan R.Q. of 1.3, and the reasons for this require investigation. Possibleexplanations include the presence of other species of insects, and thesemay have higher respiratory quotients though it is unlikely that any willexceed 1.0. The physical conditions of temperature and moisture contentmay also play a part. A more probable reason is the existence of someanaerobic respiration by microflora infesting small damp areas in thebulk of the grain or against the floor or walls of the pits. A furtherexplanation is suggested by the results obtained from early attempts todetermine the R.Q. of C. yranaria. In these early experiments infestedgrain was placed in conical flasks which were then carefully sealed withcorks and wax. Results were very variable, R.Q.'s ranging up to justabove unity. It was not until flasks with ground glass stoppers were usedthat consistent results (Table 5) were obtained, and it seems likely thatdiffusion between the contents of the flasks and room a ir occurred in theearly experiments. Owing to the different physical properties of oxygenand carbon dioxide, diffusion past the waxed corks might produce a highapparent R.Q. The rate of diffusion is directly proportional to the pressureand inversely proportional to the square root of the density of the gas,and under these conditions oxygen will diffuse into the flask more rapidlythan carbon dioxide will diffuse out. This additional oxygen will be usedby the insects to produce more carbon dioxide. Equilibrium will be reachedwhen the effect of the increased carbon dioxide pressure within the flaskequals the effect due to the different densities. Calculation shows thatequilibrium will be reached at approximately 26 per cent. carbon dioxide,representing an apparent R.Q. of 1.27.

Another point of interest in Table 5 is the very low oxygen tensionshown in many experiments, similar values being reported by Dendy andElkington (1920). If, as has been shown, death of all stages occurs whenthe oxygen has dropped to 2 per cent. some other mechanism must beresponsible for the further reduction in oxygen tension. The value of 2 percent. was determined by exposing the insects to a constant flow of gasin which the concentration of oxygen was maintained a t the predeterminedfigure, death occurring after several days. In the flasks, however, anyrespiration occurring during the period that the insects were dying wouldbe at the expense of the remaining oxygen. As death is not a rapidresponse and as the oxygen tension dropped comparatively rapidly, it is .


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48 S. W. BAILEYreasonable to assume that the insects would not die as soon as the oxygenfell to 2 per cent.; instead they would be motionless and anaesthetizedbut would continue to respire a t the expense of the remaining oxygen.Even after death occurs the respiration of the tissues may continue as longas the cytochrome system remains intact. Bacterial activity on the deadtissues is another explanation (Davidson 1941).

The high tolerance shown by C. g ramr ia to carbon dioxide build-upand oxygen deficiencies cannot in any way be regarded as an adaptationto its normal environment, as it has been shown by Oxley and Howe (1944)that the intergranular air among infested grain stored in orthodox con-tainers rarely contains more than 1 per cent. of carbon dioxide, owing toleakage or diffusion. Howe (1943) does describe an infestation in a smallopen-top metal bin where carbon dioxide reached 20 per cent. at one point,but as he points out this is exceptional. Powning (1947), analysing theintergranular air in huge bulks of Australian wheat heavily infested withRhixopertha clonzinica F., found a maximum concentration of 2.4 per cent.carbon dioxide and a minimum of 18.5 per cent. oxygen.

Two points of great importance in practice are: ( i ) the time takenfo r an infested bulk of grain that has been hermetically sealed to becomecompletely sterilized, and (ii) the amount of damage that is likely to bedone by the insects between the times of sealing and sterilization. 011the first of these will depend the effectiveness of air-tight storage as amethod to be used for short-term as well a s long-term storage of grain,and its possible use as an alternative to fumigation before grain is usedor exported. The time (i) will be dependent on the degree of infestationpresent, the temperature, and possibly the moisture content of the grain.Howe and Oxley (1944) describe a method of determining the degree ofinfestation of a sample of grain, the measure being expressed a s thepercentage of carbon dioxide present in the intergranular air in a flaskthat has been completely filled with grain, sealed, and incubated a t 25OCfor 24 hr. This method is now widely used and is clearly very appropriateto this study. Figure 9 shows the time taken for the oxygen in theintergranular air in flasks completely filled with 2 lb of grain infested with120 adult C. gramr ia to drop to 2 per cent., the grain having an initialcarbon dioxide value of 5.8 per cent. Extrapolating from this value, lightlyinfested grain with a carbon dioxide value of 0.5 per cent., equivalent toabout six weevils per lb of grain, will take 39 days for the oxygen to dropto 2 per cent. To this is added a fur ther 17 days (Fig. 3 ) , giving a totalof 56 days. Heavily infested grain with an initial carbon dioxide value of5 per cent. would require a total of 21 days. These estimates of time canbe regarded as maxima, as in practice the oxygen will continue to dropto a lower value and this will reduce the 17-days component of the total.In the above experiments all insects were dead in 7 days. The estimatesare based on the physical conditions of these experiments, namely, 25OCand 14 per cent. moisture content. Different conditions will cause varia-


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AIR-TIGHT STORAGE O F GRAIN. I 49tions in the time factor, the effect of temperature being particularlyimportant. Lindgren (1935) determined the rate of respiration of C.gramr ia a t various temperatures, but his findings must be used withcaution here a s the limits of tolerance of the insects to oxygen depletionmay also vary with temperature and moisture. A further possible causeof small variations in the response of the insects, applying to all sets ofphysical conditions, may be the rate at which oxygen depletion takes place.

I I I I I 0 8 10 10 20 30 4 0 50 60 70 80 90 100

TIME (HR)Fig. 9.-Reduction in oxygen concentrations due toan infestation of C. granaria. producing a carbondioxide value of 5.8 per cent. Mean of three experi-ments.

Lindgren believed that the respiration rate of the adult insects wasdepressed by carbon dioxide concentrations in excess of 9 per cent. Figure9 indicates a progressive reduction, but in fact very considerable variationwas found. In some experiments respiration continued a t a constant rateuntil oxygen had dropped as low as 3.5 per cent., equivalent to 14 per cent.carbon dioxide ; in others the rate decreased from the s ta rt of the experi-ment. Howe and Oxley (1952) report a similar situation and found thatthe rate was depressed by concentrations of carbon dioxide between 4 and14 per cent.

The estimates of the time taken for lethal conditions to be broughtabout also serve to illustrate the degree of advantage to be gained bycompletely filling the silo. As, in any given set of conditions, the time isdirectly proportional to the amount of oxygen present, the existence of a


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50 S. W. BAILEY10 per cent. air space above the grain will increase the time by 15 per cent.,a 50 per cent. air space causing a 75 per cent. increase.

Direct measurement of the amount of damage caused by the insectsbefore they die is difficult owing to a number of factors, but a usefulestimate can be made, again based on the amount of oxygen present a t thetime of sealing. One ton (2240 lb) of grain occupies approximately47 cu. ft . Of this 40 per cent. or 18.8 cu. ft . is intergranular a ir containing3.9 cu. f t . of oxygen. This is equivalent to 158 g of oxygen and, assumingfor the sake of simplicity that starch (R.Q. = 1) is being utilized by theinsects, this oxygen is sufficient to oxidize 133 g of food. Thus for everyton of grain stored just under 5 oz will be consumed by the insects. Thisamount is negligible, representing 0.013 per cent., and is independentof either the number of insects present or the physical conditions. However,i t is dependent on the amount of free space above the grain, the absoluteamount consumed bearing the same relation to this factor as does time.In a half-filled bin the quantity consumed will be 133 X 175/100 = 233 gper half-ton of grain. The effect of a 50 per cent. air space will thereforebe to increase the loss from 4.7 to 16.4 oz/ton. This is still negligible, andit is clear that in practice the amount of loss will be controlled entirelyby the degree of perfection of the sealing.

The author wishes to express his thanks to Mr. J. B. McCabe forassistance in this work and to Mr. L. A. Marshall, who drew the diagramsand figures.

VI. REFERENCESARGENTINE EPUBLIC,INISTERIODE AGRICULTURAGANADERIA1949) .-Conservationde granos y almacenamiento en silos subterraneos. (Buenos Aires.)BAILEY,. W. (1954) .-An ap pa ra tu s for producing a number of different g as mixturesfor a long period of time. J. Sci. I n s t n u n . 31: 93.COLE,. J. (1906).-The bionomics of gra in weevils. J. Econ. Biol. 1: 63.DAVIDSON,J. (1941).-Wheat sto rage problems in South Aust ralia . Bull. Dep. Agric.S. Aust. No. 366.DENDY,. (1918).-Report on the effect of air -ti gh t storage upon grain insects. Pa r tI. Rep. Grain Pests Comni. Roy. Soc. No. 1.DENDY,., and ELKINGTON,. D. (1918).-Report on th e effect of ai r- ti gh t sto rag eupon grain insects. Pa r t 11. Rep. Grain Pests Comm. Roy. Soc. No. 3.DENDY,., and ELKINGTON,. D. (1920).-Report on th e effect of air -ti ght sto rag eupon gra in insects. P a r t 111. Rep. Grain Pests Comm. Roy. Soc. No. 6.FRANKENFELD,. C. (1948).-Staining methods fo r detecting weevil infestat ion in

grain. Rep. U.S. Bur . Ent. No. ET-256.HOWE,R. W. (1943).-An inves tigation of th e changes in a bin of sto red wheatinfested by insects. Bull. Ent. Res . 34: 145.HOWE,R. W., and OXLEY,. A. (1944).-The use of carbon dioxide production as ameasure of infestation by grain insects. Bull. Ent. Res . 35: 11.HOWE,R. W., and OXLEY,. A. (1952).-Detection of insects by thei r carbon dioxideproduction. (H.M.S.O.: London.)


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AIR-TIGHT STORAGE OF GRAIN. I 51JOHNSTON, H. G., and SOREESON, . W. (1952).-Prog. Rep. Tex. Agric. Exp. Sta.

No. 1488.JONES, J. D. (1943).-Intergranular spaces in some stored foods. Food 12: 325.LINDGREN,D. A. (1935).-The respiration of insects in relation to the heat ing and

fumigation of grain. Tech. Bull. Minn. Agric. Exp. Sta. NO. 109.MILNER, M., BARNEY,D. L., and SHELLENBERGER,. A. (1950).-Use of selectivefluorescent stains to detect insect egg plugs on grain kernels. Science 112: 791.OXLEY,T. A. (1945).-The spontaneous heating of stored cereals. Sci . J. R. Coll . Sci .15: 71.OXLEY,T. A., and HOWE,R. W. (1944).-Factors influencing th e course of a n insectinfestation in bulk wheat. A n n . A p p l . B i o l . 31: 76.PAGE,. B. P., and LLBATTI,0. F. (1937) .-Determination of fumigants. VIII. Sampl-ing from small spaces. J. Soc. Chem. I n d . 56: 54T.POWNING,. F. (1947).-The sub-surface atmosphere of whea t infested with

R l ~ i x o p e r t h a o m i n ic a F. J . C o u n . S c i. I n d u s t r . Res . A u s t . 20: 475.PRUTHI,H. S., and SINGH,M. (1950) .-Pests of stored grai n and the ir control. I?zdianJ . A g r i c . S c i . 18 ( 4 ) (spec. no.).RICHARDS, . W. (1947) .-Observations on grain weevils, C a l a n d r a (Col., Curculioni-dae). 1. General biology and oviposition. Proc. 2001. o c . Lo n d . 117: 1.SHELLENBERGER,. A., and FEETON,F. C. (1952) .-Underground gra in storage. N o w e s lMi l l e r (Mi l l i n g Pr o d . S ec . ) 248 (2) : 3a.SLEIGH,S. W. (1937) .-Portable appar atus fo r precise ga s analysis. J. Soc. Cl tein . Ind .56: 430T.SPAFFORD,W. J. (1939).-Storing loose gr ai n in South Aust ralia. Bull. Dep. Agric.

S. Aust. No. 352.WINTERBOTTOM,. C. (1922).-Weevil in whea t an d storage of grain in bags. (Govt.Printer : Adelaide.)

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