AIR-TIGHT STORAGE O F GRAIN; ITS EFFECTS ON INSECT PESTS

I. CALANDRA GRANARIA L.

(COLEOPTERA, CURCULIONIDAE)

By S. W. BAILEY"
(Manuscript received July 15, 1954) Summary The responses of adult and immature Calandra g r a n a ~ i a L. to high concentrations of carbon dioxide and low concentrations of oxygen have been determined. To bring about 100 per cent. mortality of all stages requires an increase of carbon dioxide t o 40 per cent. or a decrease of oxygen to 2 per cent. The respiratory quotient of the species has been measured and i t is shown that the death of the insects, under air-tight grain storage conditions, is due to the depletion of oxygen caused by the respiration of the insects and the grain and not to the accumulation of carbon dioxide. The adult insects are the most resistant stage and the first instar larvae the most susceptible. Estimates for the time required for the insects to die and for the amount of damage they cause before death are given.

I. INTRODUCTION The storage of grain in sealed containers has been practised in many parts 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 Indian farmers. Other types have been reported as being used in Malta, Italy, Africa, South America, and elsewhere. Reports on the condition of grain stored in these containers have generally been favourable, the absence of insect attack frequently being stressed. I t has generally been assumed that the control of any insects present a t the time of sealing was due to the accumulation of carbon dioxide caused by the respiration of the insects and the grain. Some authors have disagreed with this, Cole (1906) considering that high carbon dioxide and low oxygen even favoured the development of weevils. Dendy (1918) reviews the conflicting evidence; and his work during World War I, on behalf of the Grain Pests Committee of the Royal Society, appears to include the first carefully executed series of experiments designed to elucidate the role that gas exchange plays in killing the insects. Dendy, and Dendy and Elkington (1918, 1920) worked with most of the economically important insect and mite pests of grain. In spite of Dendyk evidence and the obvious advantages of a method which gives freedom from losses due to pests without the need for fumigation or other applied treatments, no deliberate large-scale adoption of the system followed for two decades. I t is of interest to note, however, that Australia in 1918 enclosed large stacks of infested grain in gas-proof material and fumigated it by pumping in carbon dioxide. Experiments

" Division

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

34

S. W. BAILEY

carried out by Spafford and Hargreaves (Winterbottom 1922) had indicated that a high concentration of carbon dioxide would kill the insects. I t was subsequently found that the addition of carbon dioxide from outside was unnecessary and that the gas exchange due to the respiration of the insects alone was. sufficient to give a satisfactory control if the stacks were covered and made gas-tight. In addition some experimental pits were sunk a t Walleroo, S.A., in 1918 and filled with infested wheat (Winterbottom 1922 ; Spafford 1939). However, the construction of these pits was such that it is very doubtful if they were gas-tight and the results which were assumed to be due to hermetic sealing were much more likely due to heat produced by the insects. In 1942 Argentina was faced with the problem of storing very large stocks 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 method had to be found that would accommodate very large quantities of bulk grain and would not require the use of fumigants or insecticides, as these were not manufactured in the Argentine. A consideration of possible methods led to practical field experiments on the storage of grain in hermetically sealed underground silos. The success of these experiments led to a large-scale adoption of the method and by 1948 1540 silos with a capacity of approximately 850,000 tons had been built (Argentine Republic 1949). These appear to have been very successful, providing adequate storage and obviating both losses due to insect attack and expenses associated with conventional methods of insect control. A large fund of practical knowledge concerning the construction, gas- and waterproofing, and covering and sealing of the pits was acquired, thus overcoming the engineering problems that may have been partly responsible for the failure to adopt the system elsewhere. Since the results of the work in Argentina have become known, fresh interest in the subject has been aroused and similar silos have been built elsewhere, notably in other parts of South America (Shellenberger and Fenton 1952) and in east Africa (Swaine, unpublished data). In spite of the large amount of information provided by the work in the Argentine, the only reliable data of the effects of such an environment on insect pests of grain appear to be those given by Dendy and Elkington (1918, 1920). They worked in the main by sealing insects, grain, and room air in flasks and subsequently examining for mortality and analysing the gaseous composition of the atmosphere within the flasks. A smaller number of experiments were carried out in which the insects were sealed up in a predetermined mixture of gases. However, in all their experiments the atmosphere surrounding the insects varied during the course of the experiments owing to the respiration of the insects and to a lesser extent that of the grain. Their work deals predominantly with the effects of carbon dioxide accumulation, and it is not known whether it is this factor or the depletion of oxygen, or a combination of both, that is the important

AIR-TIGHT STORAGE O F GRAIN. I

35

controlling mechanism. This paper describes the results of experiments to answer this and other questions a r ~ s i n gfrom this method of grain storage. 11. METHODS In order to investigate the concentrations of carbon dioxide and oxygen that the insects are able to tolerate the changes due to their respiration must be eliminated. This is best done by arranging for the insects to be exposed to a constant stream of gases which sweep away all respiratory products and maintain the atmosphere a t the desired values.

Fig. 1.-Diagram

showing details of insect containers and their arrangement within a n exposure 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 to any desired mixture of nitrogen, oxygen, and carbon dioxide. This apparatus was fed with gases obtained from normal commercial sources, the oxygen being of medical quality. The suppliers' analyses of these gases indicated slight impurities but as all results are based on analysis of the mixtures passing over the insects the presence of these impurities was unimportant. All gas analyses, including check analyses carried out during the course of each experiment, were made on a Sleigh gas analysis apparatus (Sleigh 1937) and are expressed as percentages of dry gas. After mixing, the gases were led to a constant temperature waterbath in which conditioning equipment and the exposure chambers holding the experimental and control insects were submerged (Fig. 1 ) . The bath was fitted with. a lid to exclude light. The gas temperature was brought up to that of the water-bath, 25"C, by passing it through a coil of suitable tubing 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 in.

36

S . W. BAILEY

of water pressure. This method of exhausting provided a n easy check on the gas flow and eliminated any chance of back diffusion by atmospheric gases. In order to determine mortality the insects had to be examined in room air and subsequently incubated fcr 48 hr to detect any delayed effects; consequently survivors could not be returned to the experimental atmosphere for further exposure. This necessitated dividing the insect material into a number of replicates, successive replicates being removed for examination and the remainder left in the gas stream. The insects and grain were therefore held in small plastic and wire gauze containers of 15 ml capacity designed to permit maximum gas exchange. These are illustrated in Figure 1. Ten were placed end to end in an exposure chamber made from 60 cm of 3.5 cm bore Pyrex tube, the gas mixture being admitted a t one end and exhausted from the other. This arrangement resulted in a small gradient of carbon dioxide and oxygen along the length 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 insects used. Variations in the composition of the gas mixtures due to this gradient and to the method employed for mixing the gases were measured, and showed a maximum absolute error of 10.6 per cent. This exceeded errors in gas analysis by a factor of 20. The control insects were supplied v i t h 10 C.C. of conditioned air per min. One hundred insects of undetermined sex were used for each experiment, these being divided into 10 replicates of 10 each. The insects originated from field-collected specimens that had been bred through several generations in the laboratory a t 25OC and 70 per cent. R.H. Stock cultures were sieved a t regular intervals to provide material of known age. Owing to the habit of newly emerged adult CaZundra g r u n a k L. of sometimes staying within the grain for a few days (Richards 1947) some slight increase in the indicated age is possible. Ignoring this all insects were 0-7 days old a t the start 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 sterilized a t 60C for 6 h r to kill any insects or mites present. A calculated amount of water was then added and the grain stored over saturated sodium nitrate solution until equilibrium was attained. When ready for use the moisture content averaged 14.2 per cent. (wet weight basis), as determined on a Carter-Simon Rapid Moisture Tester. Each container held approximately 1 g of wheat ( = 240 grains). 1 At half-weekly intervals one container u7as removed from each exposure chamber and the contents examined. The 10 replicates therefore allowed each experiment to run for 5 weeks, which approximates the duration of the life cycle of C. gramria under the experimental conditions of temperature and relative humidity. The removal of the containers was carried out very rapidly to minimize the amount of room air entering

AIR-TIGHT STORAGE O F GRAIN. I

37

the exposure chambers. After sieving out, the insects were examined for mortality, any insects showing any sign of movement being counted as alive. They were then placed in a muslin covered tube with a little fresh grain, incubated in room air a t 25OC and 70 per cent. R.H., and re-examined a t 24 and 48 h r to determine any delayed mortality or revival, the same criterion of death being applied. All results recorded here are based on the examination a t 48 hr. The grain from the experiment was placed in separate muslin covered tubes and incubated under the same conditions. I t was sieved twice. weekly and the number of F, generation insects and their distribution in time recorded. In those instances where,

,
10

15

20

25

30

35

TIME (DAYS)

Fig. 2.-The effect of time on t h e mortality of adult C. granaria exposed to different concentrations of carbon dioxide. Oxygen as in air. Carbon dioxide concentrations: B, 10, 20, and 30.1 per cent.; X , 34.2 per cent.; @, 38.2 per cent.; A, 41.7 per cent.; 0, per cent. 44.9

after incubation, there was little or no emergence of F, generation insects the grain was examined for egg plugs in order to determine the effect of oxygen and carbon dioxide on oviposition behaviour. To facilitate this the plugs were stained with acid fuchsin (Frankenfeld 1948) or berberine sulphate (Milner, Barney, and Shellenberger 1950) but neither method was completely satisfactory, possibly owing to the age of the egg plugs. The routine finally adopted was to stain with acid fuchsin, carry out a rough sorting by eye, and then examine the selected grains under a binocular microscope. Positive ones were then dissected to determine the extent of development of the immature stages. Considerable difficulty was experienced, as the eggs or larvae had been dead for 5-12 weeks and were in a very shrivelled condition.

S. W. BAILEY

1 1 RESULTS 1. ( a ) The E f f e c t s o n A d u l t Insects Three series of experiments were run to determine: ( i ) The effect of increasing concentrations of carbon dioxide, oxygen being maintained a t 21 per cent." (ii) The effect of decreasing oxygen concentrations, carbon dioxide being absent."

Fig. 3.-The effect of time on the mortality of adult C. yranariu exposed to different concentrations of oxygen. Carbon dioxide 10, a s in air. Oxygen concentrations: 0, 5, and 3.1 per cent.; X, 2.05 per cent.; 9, 1.3 per cent.

(iii) The combined effects of increasing concentrations of carbon dioxide 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 against mortality due to exposure to carbon dioxide and oxygen respectively. In spite of considerable scatter due to the biological variation of the material, there is a clear trend indicating that the full effect is reached not later than 17 days after the start of the experiment, insects surviving this period living for the full 5 weeks' duration of the experiment. Results are therefore presented on the basis of a 17-day exposure period, as mortalities achieved in a shorter period are of no interest here.

" Normal a i r contains approximately 20.9 per cent. oxygen and 0.03 per cent. carbon dioxide.

AIR-TIGHT STORAGE O F GRAIN. I

39

Figures 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 does not occur until very substantial variations from normal atmospheric conditions occur. The combined effect of the two gases was investigated in two series of experiments. In the first, insects were exposed to different concentrations of carbon dioxide, oxygen being maintained a t a dilution close to but a little above that which would cause mortality. In the second series oxygen was varied whilst carbon dioxide was kept close to but a little below a lethal concentration.

C A R B O N D I O X I D E (%)

Fig. 4.-The niortality of adult C. gramzria due to exposure to carbon dioxide f o r 17 days. Oxygen a s i n air.

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 mortality is evident with oxygen as high as 9 per cent. Nevertheless oxygen has to drop to as low as 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 very little recovery or delayed mortality during the 48 h r the insects were kept under observation. A number of the insects that survived conditions causing a high mortality were tested for fertility. All were found to be

40

S. W. BAILEY

normal in this respect. The effect of the age of the adults was not examined, all insects being 0-7 days old. Howe and Oxley (1952) found

6
OXYGEN

10

12

(%)

Fig. 5.-The mortality of adult C. granuria due to exposure to reduced oxygen concentrations f o r 17 days. Carbon dioxide a s in air.

no change in the rate of carbon dioxide production due to the age of the adults and it does not seem likely that an increase in age will cause them to be more tolerant to a n unfavourable atmosphere.
TABLE1
MORTALITY O F ADULT C. GRANARIA DUE TO INCREASING CONCEIiTRATIONS OF CARBON DIOXIDE W I T H OXYGEN HELD A LITTLE ABOVE A LETHAL DILUTION

Exposure period 17 days Experiment No.

1 2 3 4

Carbon Dioxide (7%) 10.9 25.2 29.4 35.3

Oxygen

(%I
4.3 4.4 3.8 4.1

~irtality (%) 0 0 60 20

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

AIR-TIGHT STORAGE OF GRAIN. I

41

exposure chamber having a noticeable effect in some experiments. These effects were, however, covered by the limits of accuracy given for the gas mixtures.
TABLE 2
MORTALITY OF ADULT C. GRANARIA DUE TO DECREASING COKCENTRATIONS OF OXYGEN W I T H CARBON DIOXIDE HELD A LITTLE BELOW A LETHAL CONCENTRA4TION

Exposure period 17 days Experiment No.

Oxygen

Carbon Dioxide

(%I

(a)

Mortality (%)

( b ) The Efects o n Inz?nature Stages The determination of the total number of F, generation insects emerging from the grain after its removal from the experimental gas
I

C A R B O N DIOXIDE (%)

Fig. 6.-The mortality of immature stages of C. gramria due to exposure to carbon dioxide. Oxygen a s i n air. Mortality based on emergence compared with concurrent controls.

mixture and subsequent incubation gave information on the grcss effects only as the age composition of the immature stages was mixed. For example, the grain in the first replicate could contain no stages older than 3 days, whereas, if the weevils had been laying eggs throughout the

42

S. W. BAILEY

experiment, the last might contain individuals whose ages varied from 0 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, even a t the end of the incubation period, a t concentrations that are not lethal to the adults suggests either that the immature stages were killed early i11 life or that the adults failed to oviposit. Owing to the difficulty of identifying egg plugs and the possibility that some oviposition may have taken place during the first few hours of an experiment, the results of egg plug counts and dissection of the grain can only indicate the probable causes. Table 3 shows the effects of carbon dioxide and oxygen on oviposition and is based on the 28th day replicate.

Fig. 7.-The mortality of immature stages of C. granaria due to exposure to reduced oxygen concentrations. Carbon dioxide a s in air. Mortality based on emergence compared with concurrent controls.

Table 3 indicates that the rate of egg laying was much reduced but not entirely suspended in gas concentrations that did not permit development of immature stages. Dissection of the grain showed that in most cases the eggs had hatched and the larvae died in the first instar. This suggested that under some conditions eggs laid between the day of removal and 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 all replicates) emerged not earlier than 5 weeks after their removal from

AIR-TIGHT STORAGE O F GRAIN. I

43

the gas mixture. All these, therefore, had been eggs or very young larvae when removed.
TABLE3
EFFECT O F OXYGEN A N D CARBON DIOXIDE O K OVIPOSITION BEHAVIOUR O F C. GRANARIA

Carbon Dioxide*
(%'C)

No. of
Egg 'lugs

No. of F1 Generation Emerging

1718 77 2 0 0

Oxygen'

No. of
Egg 'lugs

t%)

No. of F1 Generation Emerging

1718 158 103 0 0

0 19.5 30.2 34.2 35.9 37.8 41.7

$ $
47 19 0 0 0

20.9 10.9 5.0 3.1 2.1

t t

2 0

0 0

* P e r cent. oxygen a s in air.

Jy P e r cent. carbon dioxide a s i n air. $ E g g plug counts not possible owing to holed condition of grain.

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 concentrations causing partial mortality. A suitable quantity of conditioned

E F F E C T O F OXYGEN A N D CARBON DIOXIDE O N I M M A T U R E STAGES O F C. GRANARIA

The numbers i n the body of the table represent the F1 generation adults emerging a f t e r incubation Age Groups (days)
0-7 7-14 14-21 21-28 28-35 31.1 Per cent. Carbon Dioxide* 7 Days' Exposure
6 42 56 0 1 1

- 4.4 Per cent. Oxygen? 14 Days' Exposure 3 42 36 0 8 7 Days' Exposure

TABLE 4

14 Days' Exposure 2 35 42 1 6

Controls 28 44 41 1 10

8 34 52 0 8

* P e r cent. oxygen a s in air. .F P e r cent. carbon dioxide a s in air.

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

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S. W. BAILEY

was removed after 7 days, the other replicates and the controls after 14 days. All were incubated in room air a t 25OC and 70 per cent. R.H. and the number of F, adults recorded. The results are shown in Table 4. For each age group, excluding 21-28 days in which the low numbers were believed to be due to a breakdown in the temperature control of the incubator during the preparation of the grain, a X"est was made of the variability between treatments in the proportion of grains from which
TABLE5
RESPIRATORY QUOTIEKT* OF C. GRANARIA AT

250C

AND

70

PER CENT. R.H.

Replicate No.

Conditions Approx. 150 mixed age adults only; no grain After: 5 days 8 days 0- to 21-day-old immature stages in grain After: 1 3 days 20 days 23 days 28 days 0- to 21-day-old immature stages in grain and associated adults After: 24 h r 28 h r 2 days 1 3 days 23 days quotient

Carbon Dioxide (%)

Oxygen (%)

Respiratory Quotient

1 2

16.57 16.72

0.37 0.18

0.81 0.81

1 2 3 4

13.73 5.03 13.42 14.24

0.114 12.72 0.19 0.11

0.68 0.62 0.65 0.69

1 2 3
4 5

12.1 15.9 15.9 15.9 16.1

02).

4.5 0.57 0.17 0.11 0.13

0.74 0.78 0.79 0.77 0.78

* Respiratory

= 72

CO2/ (20.88 - %

adults emerged. Only for the 0-7 day old age group were the differences significant. The difference between the 7 and 14 days' exposure to the experimental concentrations of carbon dioxide and oxygen did not reach significance, and the overall significance is due wholly or almost wholly to the difference between the applied treatment and the control. For older age groups the results do not indicate significant differences between treatments although the 14-day exposure tended to give lower emergence than the 7-day.

( c ) The Respiratory Quotient of C . granaria

The combined concentrations of oxygen and carbon dioxide that occur in infested hermetically stored grain is governed by the respiratory quotient (R.Q.) of the insects, that of the grain making only a small

AIR-TIGHT STORAGE OF GRAIN. I

45

contribution (Lindgren 19% ; Oxley 1945). Three series of experiments were therefore set up to determine the R.Q. of the adult insects, of the immature stages, and of a mixed population. In each series the material was placed in 100-ml flasks containing room air, which were then sealed. After a suitable time had elapsed the gaseous contents of the flasks were analysed. In the second and third series the flasks were completely filled with the grain, thus containing about 40 ml of room air (Page and Lubatti 1937 ; Jones 1943). A number of replicates were set up in order to avoid any effects which might have been caused by the reduction in atmospheric pressure due to the extraction of successive gas samples from a single flask. I t was to be expected that the indicated R.Q. would be a little lower in the presence of grain as some of the carbon dioxide produced by the insects might have been absorbed by it (Howe and Oxley 1952). The results are shown in Table 5 . In order to determine the contribution due to the respiration of the grain a further 100-ml flask was filled with grain only. This produced 1.71 per cent. of carbon dioxide in 35 days. The age structure of an infestation in stored grain is inevitably complex. Here it is sufficient to take a respiratory quotient of 0.8 as a basis for the estimation of carbon dioxide and oxygen concentrations that might be found in practice. Dendy and Elkington (1918) using an entirely different method, determined the R.Q. of adult C. granaria and obtained values ranging from 0.78 to 0.86 with a mean of 0.815. Figure 8 shows the combined concentrations of the two gases that occur on the basis of an R.Q. of 0.8.

IV. DISCUSSION The maximum variations that can occur in the components of the intergranular air in wheat infested witn C. granaria under the physical conditions of these experiments are shown in Figure 8. This allows a maximum concentration of less than 17 per cent. carbon dioxide, even assuming that the insects are able to make use of all the oxygen initially present. The experimental results on the tolerance of C. granaria to carbon dioxide show that this concentration does not kill any of the adults or prevent oviposition and that a large proportion of the immature stages survive and complete their development. On the other hand the oxygen tension drops to a very low value, and it seems clear that in practice it is the depletion of oxygen and not the build-up of carbon dioxide that is the important factor. When the oxygen tension is low the carbon dioxide present has little additional effect. The great importance of reduced oxygen tension is a t variance with the generally kield belief that the effectiveness of hermetic storage is due to the accumulation of carbon dioxide. I t explains the failure of attempts to fumigate grain by the simple addition of carbon dioxide, generally in

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S. W. BAILEY

the form of dry ice (Johnston and Sorenson 1952; Shellenberger and Fenton 1952). Concentrations as high as 40 per cent. for 3 weeks, and possibly longer a t lower temperatures than 25OC, would be necessary and these would be very difficult to maintain in normal concrete silos or sheeted stowages owing to leakage and diffusion. Attempts to reduce the practical difficulties by maintaining a higher concentration for a shorter period would be ineffective, as some experiments run to check this point gave survivals of up to 20 per cent. of adult C. yrwnaria exposed to approximately 100 per cent. carbon dioxide for 5 days.

CARBON DIOXIDE

(%)

Fig. 8.-The concentrations of carbon dioxide and oxygen occurring in the intergranular air among grain infested with C. granaria. Based on a respiratory quotient of 0.8.

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

AIR-TIGHT STORAGE O F GRAIN. I

47

air. In the experiments on immature stages the gases in the intergranular air only were analysed and the immature stages may have been exposed to 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 are the ones required. I t has been shown that a maximum concentration of approximately 17 per cent. carbon dioxide can be expected; nevertheless higher concentrations have been reported. Analyses of the intergranular air in the Argentine pits have given values as high as 27 per cent., representing an R.Q. of 1.3, and the reasons for this require investigation. Possible explanations include the presence of other species of insects, and these may have higher respiratory quotients though it is unlikely that any will exceed 1.0. The physical conditions of temperature and moisture content may also play a part. A more probable reason is the existence of some anaerobic respiration by microflora infesting small damp areas in the bulk of the grain or against the floor or walls of the pits. A further explanation is suggested by the results obtained from early attempts to determine the R.Q. of C. yranaria. In these early experiments infested grain was placed in conical flasks which were then carefully sealed with corks and wax. Results were very variable, R.Q.'s ranging up to just above unity. It was not until flasks with ground glass stoppers were used that consistent results (Table 5) were obtained, and i t seems likely that diffusion between the contents of the flasks and room air occurred in the early experiments. Owing to the different physical properties of oxygen and carbon dioxide, diffusion past the waxed corks might produce a high apparent R.Q. The rate of diffusion is directly proportional to the pressure and inversely proportional to the square root of the density of the gas, and under these conditions oxygen will diffuse into the flask more rapidly than carbon dioxide will diffuse out. This additional oxygen will be used by the insects to produce more carbon dioxide. Equilibrium will be reached when the effect of the increased carbon dioxide pressure within the flask equals the effect due to the different densities. Calculation shows that equilibrium 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 tension shown in many experiments, similar values being reported by Dendy and Elkington (1920). If, as has been shown, death of all stages occurs when the oxygen has dropped to 2 per cent. some other mechanism must be responsible for the further reduction in oxygen tension. The value of 2 per cent. was determined by exposing the insects to a constant flow of gas in which the concentration of oxygen was maintained a t the predetermined figure, death occurring after several days. In the flasks, however, any respiration occurring during the period that the insects were dying would be a t the expense of the remaining oxygen. As death is not a rapid response and as the oxygen tension dropped comparatively rapidly, it is

48

S. W. BAILEY

reasonable to assume that the insects would not die as soon as the oxygen fell to 2 per cent.; instead they would be motionless and anaesthetized but would continue to respire a t the expense of the remaining oxygen. Even after death occurs the respiration of the tissues may continue as long as the cytochrome system remains intact. Bacterial activity on the dead tissues is another explanation (Davidson 1941). The high tolerance shown by C. g r a m r i a to carbon dioxide build-up and oxygen deficiencies cannot in any way be regarded as an adaptation to its normal environment, as it has been shown by Oxley and Howe (1944) that the intergranular air among infested grain stored in orthodox containers rarely contains more than 1 per cent. of carbon dioxide, owing to leakage or diffusion. Howe (1943) does describe an infestation in a small open-top metal bin where carbon dioxide reached 20 per cent. a t one point, but as he points out this is exceptional. Powning (1947), analysing the intergranular air in huge bulks of Australian wheat heavily infested with Rhixopertha 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 taken for a n infested bulk of grain that has been hermetically sealed to become completely sterilized, and (ii) the amount of damage that is likely to be 1 done by the insects between the times of sealing and sterilization. 0 1 the first of these will depend the effectiveness of air-tight storage as a method to be used for short-term as well as long-term storage of grain, and its possible use as an alternative to fumigation before grain is used or exported. The time (i) will be dependent on the degree of infestation present, the temperature, and possibly the moisture content of the grain. Howe and Oxley (1944) describe a method of determining the degree of infestation of a sample of grain, the measure being expressed as the percentage of carbon dioxide present in the intergranular air in a flask that has been completely filled with grain, sealed, and incubated a t 25OC for 24 hr. This method is now widely used and is clearly very appropriate to this study. Figure 9 shows the time taken for the oxygen in the intergranular air in flasks completely filled with 2 lb of grain infested with 120 adult C. g r a m r i a to drop to 2 per cent., the grain having an initial carbon dioxide value of 5.8 per cent. Extrapolating from this value, lightly infested grain with a carbon dioxide value of 0.5 per cent., equivalent to about six weevils per lb of grain, will take 39 days for the oxygen to drop to 2 per cent. To this is added a further 17 days (Fig. 3 ) , giving a total of 56 days. Heavily infested grain with an initial carbon dioxide value of 5 per cent. would require a total of 21 days. These estimates of time can be regarded as maxima, as in practice the oxygen will continue to drop to 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 estimates are based on the physical conditions of these experiments, namely, 25OC and 14 per cent. moisture content. Different conditions will cause varia-

AIR-TIGHT STORAGE O F GRAIN. I

49

tions in the time factor, the effect of temperature being particularly important. Lindgren (1935) determined the rate of respiration of C. gramria a t various temperatures, but his findings must be used with caution here a s the limits of tolerance of the insects to oxygen depletion may also vary with temperature and moisture. A further possible cause of small variations in the response of the insects, applying to all sets of physical conditions, may be the rate a t which oxygen depletion takes place.

1 0

20

30

40

50

60

70

80

90

100

TIME (HR)

Fig. 9.-Reduction in oxygen concentrations due to a n infestation of C. granaria. producing a carbon dioxide value of 5.8 per cent. Mean of three experiments.

Lindgren believed that the respiration rate of the adult insects was depressed by carbon dioxide concentrations in excess of 9 per cent. Figure 9 indicates a progressive reduction, but in fact very considerable variation was found. I n some experiments respiration continued a t a constant rate until oxygen had dropped as low as 3.5 per cent., equivalent to 14 per cent. carbon dioxide ; in others the rate decreased from the start of the experiment. Howe and Oxley (1952) report a similar situation and found that the rate was depressed by concentrations of carbon dioxide between 4 and 14 per cent. The estimates of the time taken for lethal conditions to be brought about also serve to illustrate the degree of advantage to be gained by completely filling the silo. As, in any given set of conditions, the time is directly proportional to the amount of oxygen present, the existence of a

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S. W. BAILEY

10 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 insects before they die is difficult owing to a number of factors, but a useful estimate can be made, again based on the amount of oxygen present a t the time of sealing. One ton (2240 lb) of grain occupies approximately 47 cu. ft. Of this 40 per cent. or 18.8 cu. ft. is intergranular air containing 3.9 cu. ft. of oxygen. This is equivalent to 158 g of oxygen and, assuming for the sake of simplicity that starch (R.Q. = 1 ) is being utilized by the insects, this oxygen is sufficient to oxidize 133 g of food. Thus for every ton of grain stored just under 5 oz will be consumed by the insects. This amount is negligible, representing 0.013 per cent., and is independent of either the number of insects present or the physical conditions. However, it is dependent on the amount of free space above the grain, the absolute amount 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 g per half-ton of grain. The effect of a 50 per cent. air space will therefore be to increase the loss from 4.7 to 16.4 oz/ton. This is still negligible, and it is clear that in practice the amount of loss will be controlled entirely by the degree of perfection of the sealing.

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

VI. REFERENCES
ARGENTINE REPUBLIC, MINISTERIODE AGRICULTURA Y GANADERIA (1949) .-Conservation de granos y almacenamiento en silos subterraneos. (Buenos Aires.) BAILEY, W. (1954) .-An apparatus f o r producing a number of different gas mixtures S. f o r a long period of time. J. Sci. Instnun. 31: 93. COLE, J. (1906).-The bionomics of grain weevils. J. Econ. Biol. 1: 63. F. DAVIDSON,J. (1941).-Wheat storage problems i n South Australia. Bull. Dep. Agric. S. Aust. No. 366. DENDY, (1918).-Report on the effect of air-tight storage upon grain insects. P a r t A. I. Rep. Grain Pests Comni. Roy. Soc. No. 1. DENDY, and ELKINGTON, (1918).-Report on the effect of air-tight storage A., H. D. upon grain insects. P a r t 11. Rep. Grain Pests Comm. Roy. Soc. No. 3. DENDY, and ELKINGTON, (1920).-Report on the effect of air-tight storage A., H. D. upon grain insects. P a r t 111. Rep. Grain Pests Comm. Roy. Soc. No. 6. FRANKENFELD, (1948).-Staining methods for detecting weevil infestation i n J. C. grain. Rep. U.S. Bur. Ent. No. ET-256. HOWE, R. W. (1943).-An investigation of the changes in a bin of stored wheat infested by insects. Bull. Ent. Res. 34: 145. HOWE,R. W., and OXLEY, A. (1944).-The use of carbon dioxide production a s a T. measure of infestation by grain insects. Bull. Ent. Res. 35: 11. HOWE,R. W., and OXLEY, A. (1952).-Detection T. of insects by their carbon dioxide production. (H.M.S.O.: London.)

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Rep. Tex. Agric. Exp. Sta. H. G., and SOREESON, W. (1952).-Prog. J. No. 1488. JONES, J. D. (1943).-Intergranular spaces in some stored foods. Food 12: 325. D. respiration of insects in relation to the heating and LINDGREN, A. (1935).-The fumigation of grain. Tech. Bull. Minn. Agric. Exp. Sta. NO. 109. J. of selective MILNER, M., BARNEY,D. L., and SHELLENBERGER, A. (1950).-Use fluorescent stains to detect insect egg plugs on grain kernels. Science 112: 791. OXLEY, A. (1945).-The T. spontaneous heating of stored cereals. Sci. J. R. Coll. Sci. 15: 71. OXLEY, T. A., a n d HOWE,R. W. (1944).-Factors influencing the course of a n insect infestation in bulk wheat. A n n . Appl. Biol. 31: 76. PAGE, B. P., and LLBATTI,0. F. (1937) .-Determination of fumigants. VIII. SamplA. ing from small spaces. J. Soc. Chem. Ind. 56: 54T. POWNING, F. (1947).-The R. sub-surface atmosphere of wheat infested with R l ~ i x o p e r t h adominica F. J . Coun. Sci. Industr. Res. A u s t . 20: 475. PRUTHI, S., and SINGH,M. (1950) .-Pests of stored grain and their control. I?zdian H. J. Agric. Sci. 18 ( 4 ) (spec. no.). on grain weevils, Calandra (Col., CurculioniRICHARDS, W. (1947) .-Observations 0. Soc. Lond. 117: 1. dae). 1. General biology and oviposition. Proc. 2001. SHELLENBERGER,A., and FEETON,F. C. (1952) .-Underground grain storage. Nowesl J. Miller (Milling Prod. Sec.) 248 (2) : 3a. SLEIGH,S. W. (1937) .-Portable apparatus f o r precise gas analysis. J. Soc. Cltein. Ind. 56: 430T. SPAFFORD, J. (1939).-Storing W. loose grain in South Australia. Bull. Dep. Agric. S. Aust. No. 352. D. WINTERBOTTOM, C. (1922).-Weevil in wheat and storage of grain in bags. (Govt. Printer : Adelaide.)