Section 6 - Inspection and detection methods for storage insect pests

Inspection procedures for grain handling facilities and methods for detecting stored grain insects

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By R. L Semple

I. INTRODUCTION

The inspection of storage facilities and the stored food commodities that they contain, is of paramount importance in preserving grain for human consumption. Stored grain is not an inert substance, but a respiring, living organic entity. It deteriorates during storage, either quantitatively by the amount of dry matter lost (DML), or qualitatively by discoloration, mould contamination, sprouting and caking, etc., promulgated by the activity of microorganisms, invertebrate (insects and arachnids) and vertebarate pests (rodents and birds). Further weight losses are incurred during the handling, transporting and processing of stored food commodities.

Accurate information on all these forms of loss can only be obtained by thorough and regular inspection and sampling procedures. This is imperative in formulating sound management decisions involving the adoption of any remedial action against these biodeteriorating agents or the disposal of grain with due cognizance to the condition of both the commodity or the storage facility. Regular inspection helps maintain a storage environment which is conducive to good grain quality by monitoring any significant buildup in pest populations, grain temperature, moisture migration, spillage and grain residues.

It further promotes or encourages general maintenance of the storage structure and surroundings in a sound condition thereby denying access to pests as far as practicable. It also removes potential sites for infestation developing in cracks and crevices within the storage fabric and the removal of disused or obsolete machinery, used and often infested bags and dunnage.

Connell (1975) stated that, "Inspection and sampling work involves the exercises of judgement based principally on experience and an intimate knowledge of the nature, properties and physical behavior of grain. Inspection, although considered of major importance, is more often than not underestimated or in fact neglected, because it is time consuming and precise methods cannot be adequately specified. General principles however are the same whether the object of the inspection for pests is for quarantine, grading or quality determination, or a scientific study; or whether it is performed in a grain warehouse, mill or processing plant, farm or ship. Information on the following is required:

• the presence of any pests
• if so, what species, population densities, location
• damage inflicted prior to the inspection
• potential for further development of these populations (their growth potential) or,
• whether the populations will eventually die out if conditions persist or they remain uncontrolled (seriousness)
• duration at safe storage of commodities or,
• the need for treatment or disposal. (urgency)

Subjective estimates must be critically avoided. While a numerical estimate of population numbers is seldom a practical approach, some quantitative estimate based on an arbitrary, internationally accepted and comparative scale must be pursued.

It will be expanded in a later section, but it is important to realize that after adopting a thorough inspection programme, the inability to find any insect infestation does not automatically preclude their absence, and especially in the humid tropics of Southeaast Asia, the presence of low level populations now (i.e. 1 insect per 100 kg), will result in damaging population densities developing within four months. Continuous monitoring is therefore the essence of preventing serious deterioration of grain in storage.

II. THE OBJECTIVES

The objective of any sampling and inspection programme is to formulate the basis for future planning and action. Armed with precise and complete records of the history of stocks on hand, their present condition and presence of pests, the initiation of a feasible and economic control programme can be undertaken. Secondly, it forms an important base for determining its monetary value and whether the condition and quality of grain will satisfy the requirements of any potential buyer. Decisions based on a sample of grain that does not statistically reflect the overall condition of the bulk will result in erroneous conclusions being drawn concerning its fitness for human consumption. This problem is further compounded by the multiplicity of acceptance in quality standards; the nil tolerance for live insects in international export wheat as well as white flour in Western countries where it is condemned both in the eyes of the consumer and legally. Conversely, quite heavily infested grain or grain products maybe considered the norm in some developing countries depending on the economic status of the community.

2.1 Problems in systems assessment

It is considered a prerequisite that the inspector has a working knowledge of the insect species that are commonly occurring in the produce with which he is dealing. Recognition of the taxonomic and morphological differences. becomes increasingly important in establishing the status on endemic pests as well as identifying the introduction of any exotic species. Any species that is subject to quarantine regulations must be accurately identified by an appropriate authority. It is also advantageous for the inspector to have some knowledge on the biology, ecology and behaviour of various species which he may encounter, the differences between primary, secondary, field and associated pests, as well as beneficial parasites and predators.

Other practical difficulties have been elucidated by Howe (1966), and can be summarized as:

• inaccessibility due to bad stacking or to inadequtely designed buildings and machinery
• sheer bulk of produce and size of premises
• variety of ecological niches and habitats
• insect behaviour: detection of latent or hidden infestation remaining inside the stack or bulk
• non-random or uneven distribution or insect populations

Problems associated with inspection techniques all focus on the desirability of investigating locales where insects are likely to abound, or to search for signs of the presence of insects rather than the insects themselves, particularly by recording rises in temperature (hotshots) with the use of thermocouples or thermistors inserted into bulk or built-in to bagged stacks.

III. INSPECTION AND SAMPLING TECHNIQUES

Normal visual inspection of storage facilities and stored grain is subjective in nature, and therefore any results can only be recorded in a descriptive way. So long as the methodology remains uniform, direct comparison and therefore useful appraisals of different situations can be used. For more accurate and quantitative measurements it becomes necessary to sample the grain whereby the results can be interpreted by physical or chemical methods.

3.1 Oualitative inspection methods

Inspection is carried out with the objective of assessing the degree of insect infestation, although all forms of deterioration should be subsequently identified. Freeman (1948) first developed the need for standardization of estimates of infestation into defined categories for all species and all products, and in some cases his estimate have been modified for the tropics where the degrees of infestation are somewhat higher than in Britain (Hall, 1953).

Recording the results of a visual inspection have further been simplified by a useful shorthand notation as described by Ashman (1966; 1970) and has been widely adopted by inspectors. (Table 1).

For example,

Living adults (A) Living larvae (L) Living pupae(P) Dead adults (a) Dead larvae (1) Dead pupae (p)

General inspection may involve checking grain held at storage facilities for any obvious infestaion without drawing grain samples or it may involve looking for sources of residual infestation within the fabric of the storage structure or the immediate surrounds.

3.1.1 General inspection of grain in store.

The following developed by the Ministry of Agriculture, Fisheries and Food Inspectorate, Britain, have international sanction and are therefore recommended for any general commodity inspection.

3.1.2 Inspection of storage facilities and handling equipement.

The following categories apply to general inspections of the structural condition of the warehouse, silo or mill, or any handling and convey ance equipment that may offer suitable localities for endemic or residual insect infestations.

Table 1: Notation used for recording the results of a visual inspection or general inspect tion without taking samples and commodity inspection after drawing samples (After Ashnan. 1970)

In describing the species of insects present, it becomes necessary to employ the scientific generic and specific names, since the common names associated with insects sometimes vary between different countries. If the inspector is not absolutely positive of completely identifying species that are found, the uncertainty should be specified in the report, with every effort made to have the specimens in question identified by a specialist taxonomist.

The location where the species of insects were collected during the inspection should also be established in the report. They may not necessarily have complete distribution within the warehouse unless very heavy infestations are involved and this information will become an important consideration when embarking on any follow-up control procedures. Individual inspectors may develop their own shorthand notations or use floor plan sketches to supplement their report.

For example, the findings of an inspection maybe recorded as follows:

The main activities associated with a worth while inspection programme are making the effort for a prolonged search, observation and accurate recording of results. There exists no satisfactory alternative to be becoming actively involved either by getting into the grain, on to the stock, into the premises around handling equipment and physically searching for live insects, or the obvious signs for their presence. Because of the time-consuming nature of an inspection, the initial phase should

concentrate on observing places where insects if present are likely to congregate. However, there are certain practices that maybe employed to make the job a little easier, most of which aim at stimulating the insects to come to you rather than the reverse.

3.1.3 Additional aids for visual inspection. During an inspection, a few bags should be opened at random and the folds of sacking and bag corners examined; Trogoderma granarium larvae and Tribolium castaneum adults are frequently found this way. Some bags should be at least lifted and set aside and the exposed surfaces of neighbouring bags quickly examined for adults and more carefully for larvae and pupae; such as for Ephestia cautella.

For the detection of light infestation, a more detailed examination is required. The following techniques may prove rewarding.

(i) agitation of bags:

This is effective for low population densities of sitophilus granaries, S. orgzae and S. zeamais which will often walk out of sacks after they have been sufficiently disturbed. A long stick maybe drawn over surfaces of vertical stacks or they can be hit to activate small numbers of adult moths which are therefore more readily observed.

(ii) the feel of grain in bulk:

Walking across the surface of bulk grain with bare feet may prove an excellent guide to its general condition. If it feels cool and free flowing chances are there is no cause for immediate concern. However, if a hotspot exists, this can be exemplified by solid caked patches indicative of high dust content and moisture migration with subsequent rises in temperature.

(iii) traps:

Various traps have been designed to exploit the activities of many species of insects. Tube traps, consisting of a smooth inner surface and rough external surface (approximately 7.5 cm long x 2.5 cm diameter) can be inserted into bags to catch species such as Tribolium castaneum which are unable to escape by climbing the smooth inner surfaces. It is a useful cumulative trap, but is not effective in trapping Sitophilus sp. or Oryzaepilus surinamensis (plus others) and may become ineffective where the webbing activity of moth larvae is apparent.

An extension of this simple design for trapping insects at varying depths in bulk grain has been developed in Canada (Loschiavo and Atkinson, 1967).

Various forms of home-made, fly paper-type, sticky traps are commonly used to give an indication of the presence of flying insect pests at an early stage of infestation. Attractant traps, such as light taps (incandescent, flourescent or black light) as well as suction traps, can be helpful in large warehouses where suitable power is available to give reliable early indications of the presence of moths and beetles that fly readily. Traps that employ a sex attractant or pheromone as a lure offers a potential approach not only for estimating the degree of infestation but ultimately as control measure. Various traps have been adequately described by Bailey (1975).

(iv) artificial crevices:

Sections of corrugated cardboard (4 cm wide x 20 cm long) can be placed between bags to attract pupating moth larvae. It can be examined after 24 hours for insects, but it is more applicable if the bulk is only lightly infested in which case examination may be done after 4 to 5 weeks.

Plank traps, consisting of two strips of wood 15 cm wide and 60 cm long, hinged together but held 3 to 4 mm apart, is use ful for Trogoderma granarium larvae and Tribolium castaneum adults. These are inserted in bags and left for several days before withdrawal and examination.

(v) dead insects:

When residual protectants have been applied as a surface treatment and dead insects continue to accumulate, the conclusion might be reached that the treatment has been fully effective. Usually it indicates a source of live insects in the area, or from infested bags, some of which bemay obscured whithin the stack. If these insects are removed and the location marked for future reference, any further accumulation of dead insects indicates the need for further action.

(vi) repellents:

Insecticidal formulations that possess a strong repellency action such as pyrethrin or synergised pyrethroids, can be particularly useful in exposing hidden insects in cracks and crevices. A light application will often stimulate insects to crawl onto exposed surfaces before they finally succumb to the insecticide.

(vii) grain temperature and moisture content:

As mentioned earlier, it is offer more rewarding to investigate for the signs of insect presence rather than looking for live insects. Localized rises in grain temperature or moisture within a bagged stack or grain bulk are most important indicators of insect activity.

Dr. R. W. Howe has further discussed how the knowledge of insect pests and the physical behaviour and properties of stored grain should be used to help the inspector. He states, "Most storage insects are inconspicuous and secretive, and as a consequence are difficult to find. Nearly all storage insects are more easily found in dark premises because they are more active in the dark than in the light. They also lay eggs more readily in the dark.

The proportion of insects at or near the surface of produce varies with the insect species and the produce concerned. This is related to the size of the insect and its developmental instars and to the grain size of the product. Packing of stacks, diurnal rhythms, a tendency to stay near boundaries when brought to them by random movement, upward movement stimulated by disturbance, and outward movement stimulated by heating-all tend to bring insects near the surface of bagged stacks where the inspector has some chance of finding them.

Some of the reactions of insects to stimuli also help the inspector. Most prefer the dark; some are thigmotactic and collect in cracks between bags or under rubbish; most seek out wetter spots and many drink; yet others react to temperature gradients. Therefore, the inspector should examine dark places, the conical tufts of sprouting grain under leaks in the roof, the wet surfaces of bags and areas of produce known or thought to be wetter than the rest, and the tops of stacks especially those under metals roofs if Trogoderma is likely to be present."

3.2 Quantitative sampling inspections

The aim of drawing random samples of the commodity is to determine the mean value and the variability of the level of infestation or contamination (% discolored kernels) in any given situation.

Ashman (1970) devised a tentative "sequel sampling" procedure, involving spear samples of the commodity taken at random and then examined by sieving (NB: does not account for latent infestation of the immature stages hidden within individual kernels). The procedure involves collecting a number of spear samples from several bags (dependent on the total number of bags in the consignment, the number of which should not be less than the square root of the total number) until a 1 kg sample is obtained, and examined for insects by sieving. Resampling occurs if low numbers are found and may involve a further three consecutive sampling occasions consisting of 3, 9 or 22 kilograms.

Degrees of infestation and need for appropriate action was as follows:

N.B. Population of Trogoderma granarium Everts requires control measures at any degree of infestation.

3.2.1 Spear sampling. There are many inherent problems with such a classification and could therefore be open to a variety of interpretations.

• Different insects preferentially attack different commodities and consequently inflict greater damage (i.o. pest status is variable)
• When insects are present in low numbers and are unevenly distributed, sampling spears are likely to give an inadequate assessment of the infestation, either grossly over or underestimated.
• The sampling size required to give an accurate assessment of the infestation using sampling spears is often laborious or time-consuming if the consignment is 10 bags then not less than 10 bags should be sampled at random: (1000 bags require 32 bags to be sampled).
• The sample taken is rarely a random sample, due to the difficulty of sampling from the central portion of the stack.
• Samples taken from individual bags is rarely a random sample or truly representative of the condition within the bag. At least six more samples should be removed from each bag to make up the primary sample, a practice that is rarely adhered to (approximately 1 kg sample).
• Sampling spears damage sacking and consequently create potential for more spillage.

Problems related to spear sampling are further exemplified in Figures 1 and 2 as depicted by Golob (1977). Insects do not distribute themselves randomly or uniformly in any container of grain. They are most often found in pockets associated with dust, broken grain, and foreign materials towards the bottom or in areas of localized heating or wetting. It is very difficult to sample with a grain trieur or spear close to the peripheral margins of the bags, especially at the top and bottom. Therefore, a large population crawling on the bottom could be completely missed. Alternatively, small pockets of insects within the bulk maybe, by chance, included in a spear sample. For example, 6 sitophilus oryzae in a 500 g sample is not equivalent to 1200 individuals in a 100 kg. bag because of nonuniform distribution, and in fact the bag may contain less than 10 individuals altogether.

Typical spears for sampling bagged grains are represented in Figure 3. If used with due recognition of their limitations, they offer a cheap, simple and quick method in obtaining grain from bagged produce. To obtain more representative samples, a sectional probe should be utilized. These are available in sizes suitable for bags, or in larger sizes for probing deeper piles in bins, sheds, transport vehicles, etc. For larger samples in bulk grain, suction samplers consisting of a small hollow spear, hollow extension rods and suitable end attachments for the connection of an industrial type vacuum cleaner are adequate for drawing samples from up to 10 m below the surface. A more sophisticated version consisting of double construction interlocking aluminum extension tubes connected by flexible hose to an electrically driven vacuum pump is available.

Samples are drawn into a collecting compartment, with the advantage that grain friction can be minimized by the vacuum and the probe inserted to any grain depth. The major disadvantage is its relative cost.

3.2.2 Other direct sampling methods involve either snaking of bags, coning and quartering and sieving.

(i) bag snaking:

A number of bags maybe emptied by pulling the open bag backwards over the floor surface, allowing a small stream of grain to flow out gradually. Most visible insects will be concentrated in the latter portion and will be readily observed at the sides of the band.

(ii) coning and quartering:

It is a simple and cheap method of obtaining highly representative samples (approximately only 10% sampling error and therefore more accurate than spear samples) but suffers from time and capacity constraints. The procedure involves tipping bags into the floor forming a cone, constantly mixing materials from the periphery to the apex of the cone, then spreading it evenly and dividing into 1/4, 1/8, 1/16, etc. subsamples depending on the volume required.

(iii) sieving:

"Hand held sieves" are particularly useful in assessing the dust content and live insects from small samples. Different-sized mesh openings can be used for different particle size, or a combination of appropriate sizes can be used for mixed commodities varying in particle size.

"Sack sieves" have also been developed to sample an entire sack; the time taken can be between 5-15 minutes. The recovery of insects is dependent on insect species, time of sieving, slope of the oscillating sieve mesh and mesh size, but tests have shown better than 90% recovery of insects and is independent of population density.

Hugh and Simmonds (1978) have also developed a vibratory screen detector applicable to free ranging adult forms. Insects pass through the vibrating screen along with fine materials which accompanies them. Commercially available models with a throughput of 1 kg. min-1 is available from Eriez Magnetics Pty. Ltd., Sydney, Australia, and this equipment has the ability for scaling up if larger throughputs (10 kgs. min-1) are required. Similar flow rates have been developed by Sweco, Inc., Los Angeles, California.

Fig. 1. Typical spears for sampling bagged grain. A= Closed spear for sampling large particples, such as maize or coffee; B= closed spear for sampling small particles, such as wheat or rice C= open spear.

Fig. 1. Typical spears for sampling bagged grain. A= Closed spear for sampling large particples, such as maize or coffee; B= closed spear for sampling small particles, such as wheat or rice C= open spear. - continue

For greater accuracy and representation, it is advantageous to take larger primary samples and then take subsamples to form a suitable working sample. Various methods (such as coning and quartering mentioned earlier) can be employed, or by using specific apparatus designed for the purpose. These consist of the gravity mechanical types such as the Boerner conical divider, a simplified alternate channel box divider or the motorized centrifugal types such as the Gamet divider. Simplified dividing trays are also available.

TSPC developed the produce flow sampler (PFS) for taking samples from entire bags originally designed for sampling incoming loads from road transport. The time it takes is approximately 20 seconds for a 100 kg. bag, all flowable commodities can be sampled, and because samples are completely random, it is far more accurate than spear sampling.

IV. DETECTING HIDDEN INFESTATIONS

Most of the damage and weight loss caused by insects on grain are inflicted by the primary grain feeders. They are capable of penetrating sound whole kernels of grain and their life cycle is completed entirely within the kernel in which the egg is laid or entered by the first instar larva.

The absence of any live adults of storage insects in grain samples separated by the previous methods, does not necessarily mean the absence of an infestation and consequently many methods have been devised to identify individual kernels that have become the home of the immature stages of the major insect pests.

4.1 Concentration of infested grains in a sample Any detection method is greatly enhanced if the infested material can be adequately separated from sound grain and hence reduce the number of grains and the amount of dust and broken kernels that have to be examined. It becomes desirable to concentrate the insect-damaged kernels as a preliminary step. For this purpose, flotation in an air stream or liquid maybe employed.

4.1.1 Flotation separation in liquids. The feeding activity of insect larvae progressively reduces the density of the grain, and by immersing the grain sample in a liquid of suitable specific gravity, the infested grains should float and the sound ones sink. At specific gravities between 1.050 and 1.190, the floating layer contains only infested kernels and aproximately 50 to 70% of all infested kernels were separated (White, 1956). Absolute separation is therefore unlikely, but the presence of hidden infestations can be estimated quite accurately; while a general indication of the severity of infestation (the degree) will also be obtained.

4.1.2 Flotation separation in the air. A vertical column with a fan which produced a stream of air sufficient to float the grain sample was used by Milner (1953). By progressively increasing the intensity of blowing, it was noted that virtually all insect-damaged kernels were removed in the first two fractions, from which no emergence had occurred. The detection of insect-damaged grain (i.e. those containing exit holes) can then be a relatively quick and efficient operation, and may speed up the exit-hole inspection procedure in commercial samples by a factor of ten or better.

4.1.3 Projection separation in air. By projecting the grains of a sample through the air at an initially constant velocity, they should separate according to their relative density (Bailey, 1975). Air drag and gravity then further separate the grain according to kernel size, shape and the surface texture which also determine the bulk density or test weight of any particular grain example. Infested kernels tend to fall short of sound kernels of the same size and shape.

This separation has enormous practical implication. Wheat that has been designated as heavily infested can be separated into different bins reclaiming as much as 50% of the grain for food purposes rather than condemning the entire consignment for feed purposes.

4.2 Methods available for detection of insect infestation

4.2.1 Physical methods:

(i) Acoustic detection (Adams et al., 1953; Bailey and McCabe, 1965). This method provides an immediate answer if active immature forms are present, and has an advantage of not destroying the sample. A microphone, low-noise amplifier, and loud speaker or a cathode ray oscilloscope display tube are required, and to limit extraneous laboratory background interference, the sample and microphone needs to be insulated or alternatively, the grain can be directly linked to a piezo-electric crystal.

Vibrations are noted at several characteristic frequencies, e.g., 200 cycles.sec-1 are associated with movement and dispersal, while frequencies round 1200-1500 cycles.sec -1 are associated with feeding.

This method is potentially valuable for detection of insect activity within silos and other storage facilities, and possibly transport vehicles, provided extraneous noises can be successfully eliminated. The system can be modified for adequate sensitivity under field conditions.

Other limitations of acoustic detection are that quiescent stages (pupae) and eggs cannot be identified. However, a commercial unit has been developed by SASAD, Budapest, Hungary called the Insectofon (R).

Acoustic microscopy. involving the use of high frequency ultrasound (ca. 100 mHz) which had different velocities in tissues of different densities could be developed for insect detection in grain (Bruce and Street, 1978).

(ii) Grain Radiography (Milner et al., 1950 b). This method is appropriate for detecting hidden insects at most stages of development and has been developed and used extensively under commercial applications in the United States.

The use of X-rays, discovered in 1895 by Roentgen, was generally restricted to the examination of high density materials. Claussen and Shehan (1942) developed a process for making beryllium as a window material on X-ray tubes, and enabled the use of radiography with low density materials such as grain.

Several researchers at Kansas State University (Katz et al., 1950; Milner et al., 1950 b, 1952) pioneered radiography in its application to agriculture, and developed a method for detecting hidden infestation that was marketed by the General Electric company known as the "grain inspection unit". It suffered from being time-consuming (approximately 15 minutes) and was not suitable for routine spection in of grain. Stermer (1972) developed a completely automated X-ray system to inspect grain on kernel-tokernel basis that was more reliable and practical for use. His study also resulted in the development of a procedure using fine-grain "mammography" film which obtained a contrast of about 75%, after only 2.5 minutes exposure. The film is examined by low-power binocular microscope (6x to 30x) with transmitted light, and obtained an efficiency of close to 100% with fully grown larvae and pupae and 80 to 90% with early instar and eggs.

The automated X-ray analyzer requires four major phases.

The automated X-ray analyser suffers from being an expensive piece of laboratory equipment, does not distinguish between live and dead insects and treating with K2CO3 as a constrasting agent was a lengthy process.

Simplicity of operator has been enhanced in various films such as self processing (Polaroid (R)) and cassettes for the Hewlett-Packard Faxitron(R) X-ray unit. The problem of interpretation of the radiograph which makes the X-ray techniques so different has prompted the USDA Federal Grain Inspection Services to scrap it altogether as a quick inspection method (Bruce and Street, 1978).

The low-intensity X-ray imaging scope (Lixiscope) developed by NASA, is a fully portable lightweight unit powered by a penlight battery, and may find application for grain inspection.

(iii) Nuclear magnetic resonance (Street, 1971). The application is similar to the X-ray techique but suffers from the same limitations of time constraints, sampling efficiency and relative cost.

(iv) Carbon dioxide evolution. This method gives an accurate measurement of the total metabolic rate of the grain, and therefore cannot be specifically applied to insects. The method requires enclosing a quantity of grain in a gas tight bottle at 35°C for 24 hours, then drawing a sample of intergranular air and analyzing it for percent CO2 evolved. Dry uninfested grain is normally < 0.25%, between 0.3-0.5% suggests a light insect infestation (or a mc> 15%), and if the CO2 evolution is > 0.5% in 24 hours, the grain is definitely unsuitable for storage without any further treatment (Howe and Oxley, 1944).

A further development requires the detection of respired CO2 by infrared absorption spectroscopy which is extremely sensitive, and is applicable to all stages except possibly the egg. The procedure requires a chamber containing the grain sample to be purged with a carrier gas, and then sealed during which time the CO2 evolved by insect activity reacts a concentration sufficient for detection. This takes approximately one minute and is followed by flushing with a carrier gas for 2 minutes, and moves into the infrared detector by a pusle-flow movement. It is capable of picking up slight increases in CO2 above the natural atmospheric background concentration of 300 ppm and can detect one 4th instar larva of Sitophilus oryzae (L) in 350 ppm grams of wheat (Bruce and Street, 1974).

Two commercial prototypes based on their original concept were developed by Technico Instruments Inc., USA, which can process three samples simultaneously, and by Horiba Instrument Inc. of Japan which is a smaller portable unit. The Lawrence Livermore Laboratories of the USA developed a miniaturized atmospheric carbon dioxide detector system (MACDS) which is extremely sensitive and portable (Bruce and Street, 1978).

Other sophisticated detection systems that may find future application in the grains industry involve infrared radiation detection such as Far infrared (FIR) imaging, photoacoustic spectroscopy and thermal imaging with a pyroelectric vidicon.

All these detection systems are based on the principle that all physical bodies with a temperature greater than absolute zero have an infrared radiation spectrum which is a function of the body's absolute temperature and that living metabolizing tissues (such as insects) generally have a higher temperature than its surrounding environment, thus giving off different radiation characteristics.

(v) Breeding out. Grain suspected of being infested maybe incubated thus allowing insects to complete their life cycle. The biggest disadvantage is the time factor since even under the optimum conditions of temperature and moisture content (2630°C; 14-16% m.c.), at least 4-6 weeks will be required to breed out the full population of grain weevils and even longer incubation periods will be required for many other storage species.

(vi) Visual examination of exit holes is the simplest method. It has been standardized by the FDA of the United States that > 3 holes per 100 grains was cause for rejection. An experienced observer can also detect the presence of mature, late instar larvae and pupae of weevils in grain by changes in colour transmitted through the seedcoat. This only indicates the presence of an advanced infestation, but neither the degree nor the severity of the infestation.

(vii) Grain dissection will reveal any internal infestation, and gives a valuable indication of the stage of development of an infestation relevant for any impending control method. It is best done under a binocular microscope, and dissected with a sharp scalpel after the grains have been presoftened by soaking for 2 hours.

(viii) Cracking flotation has long been employed to determine the internal insect content of grains. Insect fragments are released after coarse grinding of the sample. Concentration of insect material is achieved from an alcoholic solution by flotationseparation with the addition of mineral solvents. This enables microscopic examination and identification, and provides objective data on the number of insects present as well as an indication about the stages of development within the test sample.

The technique is complicated and laborious, requires the facilities of a grain test laboratory and sufficient skill and experience on the part of the technicians.

(ix) Electrical capacitance and resistance methods have been investigated by Wirtz and Shellenberger (1962) where changes in both these properties occur when insests are persent within the grain.

Additionally, temperature testing as mentioned earlier is of extreme value in detecting the presence of insects, as well as the effectiveness of applied control measures (i.e. fumigation) as indicated by a gradual decline in grain temperature compared to the ambient.

4.2.2 Chemical Methods:

(i) Alkali treatment. This method is often referred to as the sodium hydroxide gelatinisation procedure which depends on rendering the seedcoat and endosperm translucent so that the more advanced stages of immature insects become visible. Tests by Keppel and Harris (1953) have shown that it has too many constraints to be adaptable as a quick and efficient method, although its reliability is unquestionable.

(ii) Egg plug staining. (Milner et al., 1950 a). A soluble flourescent dye (berberine sulphate) is used to stain the gelatinous plug secreted by female Sitophilus spp. to cover the egg cavity in the grain. Grains are soaked in a dilute solution of 20 ppm followed by rinsing and examining the kernels under ultra-violet light for the greenish-yellow plugs. The degree of internal infestation can be then estimated by the number of egg plugs observed. This method is not particularly accurate, it is time-consuming, gives no indication on the stage of insect development and is only useful for weevil infestation.

(iii) Uric acid test (Pixton, 1961). Measurement of the uric acid content of infested grain will give an indication of the past insect infestations which may have been concealed during processing. The method was found too insensitive to detect present infestations, and was only useful when population densities were high, in which case they were visisly obvious anyway.

(iv) Chemical detection of insect phenols (Pottets and Shellenberger, 1952). A test based on spectrophotometry analysis for the concentration of a hydroxyphenol occuring in insect cuticle, which produces phenolidophenol dyes when chemically treated with 2, 6- dichloroquinone chlorimide was proposed. Bailey (1975) stated that the method showed particular merit based on Food and Drug Administration evaluations, but at that stage required more work to perfect the method.

(v) Ultra-violet light illumination. Ashman (1986) stated that insects or insect fragments when present in finely ground commodities will appear red when stained in crystal violet and illuminated with ultra-violet light. Similar to the uric acid test, it provides useful information of any previous insect infestations in the grain before processing.

(vi) Ninhydrin system for insect detection. Dennis and Decker (1962) described a machine for detecting pre-adult stages of insects in wheat utilizing the chemical ninhydrin. In practice, the grain and any internal insects are crushed between filter paper which has been impregnated with a 0.7% solution of ninhydrin in acetone. If the grains are infested, the amino acids contained in the body fluids of live insects are absorbed by the paper and chemically react with ninhydrin to produce a strong purple colouration. The machine developed was automated, with a sample flow rate of 300 kernels per minute, spot paper was previously heated to approximately 120°C. The test paper could be kept as a visual record and retained for more than two years. Unreacted treated paper loses some of its sensitivity with aging, resulting in a slight colour intensity reduction if the paper is used several months after treatment. This machine was limited because of its size (92 x 61 x 40 cm weighing 250 Ibs.), and a more versatile smaller prototype was developed at the Tropical Stored Products Centre (TSPC and reported by Ashman et al., (1970).

Compared to the X-ray technique for insect detection in a series of laboratory trials, the AshmanSimon Insect Infestation Detector proved superior in many respects, with the following deserving mention:

1. Cost of the X-ray technique was substantially higher.

2. Both techniques provide permanent records (impregnated paper can be photocopied and therefore retained indefinitely).

3. Determination of infestation was simpler and quicker.

4. The detector will detect 5-14% of eggs and early instar larvae; 40-60% of moderate sized larvae, 80-90% of late instar larvae in small cereal grains such as wheat, while the X-ray technique is incapable of detecting eggs and small larvae and is not significantly better in estimating mature larvae or pupae in grains.

5. Skilled technicians and expensive laboratory equipment are not required.

6. The machine is adapted to both manually cranked and/or electrical operation and is capable of dealing with a wide range of food grain of various particle size by altering the gap between the rollers (interchange of rollers with different diameters).

Some problems that may interfere with the ninhydrin test have been enumerated by Bailey (1975). Moisture content above 16% m.c. will give a faint general reaction, especially if contaminated by storage molds. Other forms of interference listed were 1) kernels damaged by cracking or checking; 2) kernels damaged externally by insect feeding or mechanical injury; 3) previously infested kernels where adult emergence has been completed; 4) kernels containing dead insects; 5) sooty or heat-damaged kernels; 6) kernels with a high free fatty acidity (FFA) value. These interfering reactions tend to produce distinct, sharply outlined spots.

When estimation hidden infestation of Sitophilus spp. in maize, individual kernels maybe infested by more than one larva, which produce an overlapping stain recorded as a single spot. The ninhydrin estimate in this case generally underestimated even though the grains have to be initially kibbled, but appears to be predictable and consequently a correction factor could be applied. The Ashman-Simon Insect Infestation Detector was originally designed for field applications to give a more subjective assessment of the infestation rather than a numerical estimate.

V. RECORDING OF RESULTS

Accurate and complete records give essential background information especially when compiled over a period of time. A standardized inspection record sheet should be used so that information is recorded in a systematic and comprehensive manner for convenient analyses. (Appendix lll).

Some of the points that need to be considered in compiling a report are:

weather conditions have an important influence on the development of a pest infestation and are less variable in tropical climates where temperature and relative humidity (which are functions of the EMC) approach the optimum requiement of many major insect pests.

• the structures used for storage of produce have an important effect on produce quality and subsequent build-up of populations. Accurate information of these structures, their condition, and potential sources for cross-infestation should be noted.
• the type of produce, its variety and grade, and observations on insect damage will often prove valuable.
• origin, destination and length in storage of commodities inspected have an important effect on future implementation of control and,
• previous applied control measures should foe documented as an assessment to their efficiency.
• a plan of the storage layout (bagged stacks and their dimensions) will also prove useful.

SUMMARY

The inspection of stored grain and storage facilities becomes necessary for the following reasons:

(i) To detect the presence or absence of insects and to determine stability of grain for export purposes, milling, etc.

(ii) To detect insects as they may affect grade and price.

(iii) To determine the suitability of grain for further storage.

(iv) Whether control measures will have to be implemented to secure its end usefulness for human consumption.

(v) If control measures have been applied previously, whether their implementation was successful or whether further controls are requried.

(vi) In research work, survey and inspection for the detection insects becomes necessary in the evaluation of biological, ecological and insecticide resistance studies of grain pests.

(vii) To continuously monitor for the possible introduction of pests subject to quarantine regulations (i.e. Khapra beetle).

Selected References

ADAMS, R. E. WOLFE, J. E., MILNER, M. and DHELLENBERGER, J. A. (1953). Aural detection of grain infested internally with insects. Science 118:163.

ASHMAN, F. (1966). Inspection methods for setecting insects in stored products. Trop. Stored prod. Inf. 12:481-494

ASHMAN, F. (1970). produce inspection and sampling technqiues. Food Storage Manual, World Food Programme T.S.P.L., Section 111, 671-645

ASHMAN, F., ELIAS, D.G. ELLISON, J.F. and SPRATLEY, R. (1970). Ashamn-simon Infestation Detector: An instrument for detecting insects within foo grains. Trop. Stored prod. Inf. 19:15-19

BAILEY, S. W. (1975). Detection and measurement of biological contamination ADAB Intern. Training Course in the Preservation of Stored Cereals, Selected Ref. papers l: 307-22

BAILEY, S.W. and McCABE, J. B. (1965). The detection of immature stages of insects within grains of wheat. J. Stored prod. Res. I: 201

BRUCE, W. A. and STREET, M. W. (1974). Infrared CO2 detection of hidden insect. J. Georgia Entomol. Soc. 9(4):260.

BRUCE, W. A. and STREET, M. W. (1978). The use of electromagnetic energy to detect hidden insect infestation. Proc. of Sec. Int. Work Conf. Stored prod. Spet. 10-16, 1978, p. 230-237.

CONNEL, M. (1975). Inspection procedures: grain stores and handling equipment. ADAB Int. Training Course. Selected Ref. papers l: 326335.

DENNIS, N.M. and DECKER, R.W. (1962). A method for detecting live internal infestation in wheat. J. Econ Entomol. 55:199.

FREEMAN, J. A. (1948). World foci of infestation and principal channels of dissemination to other points, with suggestions for detection and standards of inspection. U.N., F.A.O., agric. studies, 2: 15-34.

GOLOB, P. (1977). Techniques for sampling bagged produce. Postharvest Grain Loss Assessment Methods. Published by L.l.F.E. TPI. IN. FAO, GASCA, AACC (Harris, K.L. and Lindblad, C. J.) Appendix A:

HALL, D. W. (1953). Definitions for reporting the degrees of infestation in stored products in colonial territories. D.S.I.R, R.I.L. NEOS., 3p

HOWE, R.W. and OXLEY, T.A.. (1944). The use of carbon dioxide as a measure of infestation of grain by insects. Bull. Ent. Res. 35:11-22

HOWE, R. W. (1966). Insepction of produce and premises. Trop. Stored prod. Inf. 12:475-480.

HUGHS, R.T. and SIMMONDS, D. H. 91978). Automatic dockage testing and insect detection in wheat. Chem. Aust. 45 (10) 373.

KATZ, R., M.R. LEE and M. MILNER. (1950) X-ray inspection of wheat. Nondestrcution testing 9(2): 16-18.

LOSCHIAVO, S.R. and J. M. ATKINSON. (1967). A trap for the detection and recovery of insects in stored grain. Can. Ent. 99:1160.

MILNER, M., D.L. BARNEY and J.A. SHELLENBERGER. (1950a). use of selective flourescent stains to detect insect egg plugs in grain kernels. Science 112:933-935.

MILNER, M.,M.R. LEE and R. KATZ. (1952). Radiography applied to grain and seed. Food Tech. 6:44-45

STEAMER, R.A. (1972). Automated X-ray inspection of grain for insect infestation. Quality Detection in Foods (J. J. Gafney), Amer. Soc. Agric. Eng. 1976:110-114

WHITE, G. D. (1956). Studies on separation of weevil infested from non-infested wheat by floatation. USDA Agric. Mark. Report. 101.

Annex

1.1 Grain Purification:

The table above records the results of an experiment where the grain was projected from a moving belt into 16 separate fractions. The original sample was heavily infested (85%) and each fraction was characterized radiographically for internal infestation. Considerable concentration of the original infestation occurred in bins 1-7, which were closest to the point of projection, while grain samples from hoppers 12-16 yielded approximately half (48.9%) of the original sample weight with an infestation level of only 15% (7.4 x 100/48.9 = 15.13). This represents a 6x purification ratio which could then be successfully be treated and sold for human consumption.

1.2 Ninhydrin detector:

The detector, consisting of a hopper (H) leading to a pair of roughsurfaced steel rolls (D and C) through the 'nip' of which pases a continuous strip of specially treated paper (from A). The grain sample under test (G) is crushed by the action of the rolls on to the paper, and the body juices of any infestation present are expressed, forming an easily recognisable purple stain (B) on the paper. An arithmetical count of these marks then gives a clear indication of the level of infestation in the sample passed through the small machine. Two spring scrapers (E and F) keep the paper and exposed roll D free from crushed grain (J).

The detector measures 35.6 cm x 22.9 x 30.5 cm, weighs approximately 9.5 kg and is conveniently shaped with a built-in handle for local carrying by an operator.

Operating procedure is extremely simple and is as follows:

1. Select rolls appropriate to the size of the grain under test.

2. Lift off side cover, fit rolls, thread paper and replace cover.

3. Place a 50-9 sample of the grain in the top hopper.

4. Turn operating handle until sample has completely run through the machine; the crushed grain drops out of the base, of the machine.

5. Tear off the the extruded length of paper, and wait for marks to develop (the time lapse can be reduced to a few seconds by applying gentle heat to the paper).

6. Examine paper visually and record number of marks.

Fig. 2. Line drawing of the operation (after Ashman et al, 1970).

Cereals and pulses-Determination of hidden insect infestation- Part 2: Sampling

Ceréales et legumineuses-Détermination de l'nfestation cachée par les insectes-Partie 2: Echantillonnage

UDC 633.1 :635.65:632.7

Descriptors: agricultural products, cereal products, leguminous grains, determination, insects, contamination, sampling.

THIS DOCUMENT IS A DRAFT CIRCULATED FOR COMMENT AND APPROVAL. IT IS THEREFORE SUBJECT TO CHANGE AND MAY NOT BE REFERRED TO AS AN INTERNATIONAL STANDARD UNTIL ACCEPTED BY ISO COUNCIL.

IN ADDITION TO THEIR EVALUATION AS BEING ACCEPTABLE FOR INDUSTRIAL, TECHNOLOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT INTERNATIONAL STANDARDS MAY ON OCCASION HAVE TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL AS DOCUMENTS TO WHICH REFERENCE MAY BE MADE IN NATIONAL REGULATIONS.

(c) International Organization for Standardization, 1985

Cereals and pulses-Determination of hidden insect infestation Part 2: Sampling

0 Introduction

This Internatiroximately 9.5 kg and is conveniently shaped with a built-in handle for local carrying by an operator.

Operating procedure is extremely simple and is as follows:

1. Select rolls appropriate to the size of the grain under test.

2. Lift off side cover, fit rolls, thread paper and replace cover.

3. Place a 50-9 sample of the grain in the top hopper.

4. Turn operating handle until sample has completely run through the machine; the crushed grain drops out of the base, of the machine.

5. Tear off the the extruded length of paper, and wait for marks to develop (the time lapse can be reduced to a few seconds by applying gentle heat to the paper).

6. Examine paper visually and record number of marks.

Fig. 2. Line drawing of the operation (after Ashman et al, 1970).

Cereals and pulses-Determination of hidden insect infestation- Part 2: Sampling

Ceréales et legumineuses-Détermination de l'nfestation cachée par les insectes-Partie 2: Echantillonnage

UDC 633.1 :635.65:632.7

Descriptors: agricultural products, cereal products, leguminous grains, determination, insects, contamination, sampling.

THIS DOCUMENT IS A DRAFT CIRCULATED FOR COMMENT AND APPROVAL. IT IS THEREFORE SUBJECT TO CHANGE AND MAY NOT BE REFERRED TO AS AN INTERNATIONAL STANDARD UNTIL ACCEPTED BY ISO COUNCIL.

IN ADDITION TO THEIR EVALUATION AS BEING ACCEPTABLE FOR INDUSTRIAL, TECHNOLOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT INTERNATIONAL STANDARDS MAY ON OCCASION HAVE TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL AS DOCUMENTS TO WHICH REFERENCE MAY BE MADE IN NATIONAL REGULATIONS.

(c) International Organization for Standardization, 1985

Cereals and pulses-Determination of hidden insect infestation Part 2: Sampling

0 Introduction

This International Standard deals with methods for the determination of hidden insect infestation in cereals and pulses.

Part specifies methods of sampling. ISO 6639/3 and ISO 6639/4 describe the reference method and rapid methods for determining hidden insect infestation, respectively, while ISO 6639/1 describes the general principles of the methods.

1 Scope and field of application

This part of ISO 6639 specifies methods of sampling cereals and pulses, in bags or in bulk, for the determination of hidden insect infestation. The methods are applicable as a routine to grain) in any form of store or vehicle at any level of trade from producer to consumer.

2 References

ISO 950, Cereals-Sampling (as grain).
ISO 951, Pulses in bags-Sampling.
ISO 6639, Cereals and pulses-Determination of hidden insect infestation
ISO 950, Cereals-Sampling (as grain).
ISO 951, Pulses in bags-Sampling.
ISO 6639, Cereals and pulses-Determination of hidden insect infestation

Part 1: General principles.)
Part 3: Reference method.)
Part 4: Rapid methods.)

ISO 6644, Cereals and milled cereal products Automatic sampling by mechanical means.

3 Definitions

For the purpose of this part of ISO 6639, the following definitions apply.

3.1 consignment: The quantity of grain delivered at one time and covered by one set of shipping documents. It may be composed of one or more lots (see the notes to 3.2).

3.2 lot: A stated quantity, to be sampled using a particular sampling plan.

NOTES

1 There is no need to restrict the size of the lot when sampling for hidden insect infestation. A consignment of the same origin and history may be regarded as one lot may be split into several lots for sampling, whichever is the more convenient. It the consignment is received In several barges, railway waggons, lorries, stacks, etc., it is usually more convenient to treat each part as a separate lot for sampling purposes. Any parts of a consignment known to be of different origin and/or history are sampled as separate lots.

2 It should be noted that the definition of lot for the purposes of sampling for determination of hidden insect infestation differs from the definition of lot in International Standards relating to sampling of grain and pulses for the determination of other characteristics.

3.3 increment: A small quantity of grain taken from a single position in the lot.

3.4 bulk sample: The quantity of grain obtained by combining and mixing the increments taken from a specific lot.

3.5 laboratory sample: The quantity of grain removed from the bulk sample, or an increment (see 10.1), intended for examination.

4 General principles

NOTE - Usually there is little or no prior information on the size or distribution of any insect population that may be present in a lot to be sampled. In these circumstances, it is not possible to adopt a sampling scheme which is soundly based on statistical theory. Therefore, sampling schemes described in this part of ISO 6639 do not necessarily enable insect populations to be measured precisely, but have been designed to give a maximum of information in a practical manner.

4.1 Special care is necessary to ensure that all sampling apparatus is clean and dry before, during, and after the sampling of each lot. Sampling shall be carried out in such a manner as to prevent insects from elsewhere entering the samples, sampling apparatus and sample containers.

4.2 Laboratory samples shall be enclosed in sample bags (5.5) and shall be protected from direct exposure to sunlight, wetting or other extreme environmental conditions. Airtight containers shall not be used for samples as these may cause any insects present to be asphyxiated.

4.3 If related information about the grain, such as moisture content, is required, separate samples should be taken in accordance with ISO 9p>

Samples may be taken at any point from farmer to final destination.

NOTE - If samples are to be taken at different points in the distribution chain, it is useful to establish standardized sampling operations at all points and to collect the sampling data in order to form a more comprehensive picture.

Sampling is most easily carried out as the commodity is moved into and out of the storage structure or transit vehicle (railway waggons, lorries, containers, ships, lighters, etc.). If a commodity is stored for a long period in bulk or in bags, sampling becomes more difficult but more important. However, owing to the life cycle of the common species of insects responsible for grain infestation and to the need for any infestation to migrate to the areas where samples will be taken, it is, in general, not useful to take samples from commodities that have been stored for less than 3 weeks.

7 Pre-sampling inspection and identification of lots

7.1 The parties concerned shall agree as to what constitutes the lot or lots to be inspected and shall specify the species of insects (live or dead) to be reported on.

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