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In the past several insect pests had entered India and are causing economically perceptible damage to native crops. The Government of India, in line with the International Standards on Phytosanitary Measures (ISPM), notified ‘Plant Quarantine Order, 2003’ to reduce the risk of introduction of exotic insect pests in India. The storage insect pests of quarantine concern for India have been listed in Table 9.1.

As the name indicates, micro-organisms are very minute, cellular, living organisms which cannot be seen with the naked eyes (however, large colonies of fungi can be seen). Micro-organisms affecting stored grain can be divided into the following groups in order of their significance:

1. Fungi
2. Actinomycetes
3. Yeast
4. Bacteria
5. Protozoa (not found in sound food grains)

Fungi, bacteria and actinomycetes are grouped under the Thallophyta of the plant kingdom. The most important factors for their normal development are: (i) Nature of Food, (ii) Moisture, (iii) Oxygen and (iv) Temperature.

Economic Importance of Rodent Pests

• They consume and damage foods in the fields and in stores. In addition, they spoil it in store by urine and droppings reducing the sale values.
• Due to their gnawing and burrowing habit they destroy many articles (packing, clothes and furniture) and structures (floors, buildings, bridges, etc). By gnawing the electrical cables they can cause fires.
• They are responsible for transmitting diseases dangerous to human and domestic animals.

Among many pests of agricultural crops and stored grain products, rodents are the major pests which do the great damage. Rodents including rats, mice and squirrels are gnawing animals having chisel like teeth. They have highly developed sense of smell, taste and hearing. They are omnivorous and feed on grains, vegetables, fruits, meat and other products in the houses or in the fields. Rats daily consume food equal to about 10 per cent of their body weight and the figure may go up to 30 per cent in case of mice. Rats are among the most destructive pests of paddy in the field causing about 5-10 percent damage. It is estimated that these animals destroy 30 per cent of the total world’s cereal production. In India, 10 million tonnes of food grains are lost every year. It is often said that if rodent ravages could be controlled, a huge amount of food grains can be saved.

Of the 84 species of order Rodentia in India, 50 belong to family Muridae. The important ones belong to two sub-families, viz., Murinae and Gerbillinae. Murinae has 18 species of genus Rattus, 2 of Bandicota, 7 of Mus, 2 of Apodemus and a few each under eight other genera. The sub-family Gerbillinae has 2 species of Gerbillus, one each of Tatera and Meriones. Family Sciuridae has 10 species of flying squirrels and 13 species of tree squirrels in India.

It is interesting to note that only 25 of about 1200 species of birds found in India have been reported to inflict damage to crops, vegetables and fruits. The exact ecological causes for population increase of individual species have yet to be studied. It would appear that, in general, the granivorous birds became abundant with the increase in area under grain crops and the subsequent storage of grain in godowns and bulk storage. The details of important bird pests are given below:

There are several species in this cosmopolitan family that are of importance chiefly as pests of stored foods, flour, grain, seeds and bulbs, feeding on these products or the fungi that develop on them. These mites are mostly very minute, almost colourless, with the body bearing a few long setae, and with the legs terminating in one claw and usually a sucker. When very abundant the mites and their cast skins form a buff-colored dust and give rise to a characteristic odour. The living mites move slowly away from a source of light. An interesting characteristic of these mites is a nymphal resting stage (hypopus) during which they take no food but attach themselves to other mites, insects and humans by means of suckers and are thereby transported to other suitable places for development, where they drop off, moult, feed, and develop into adult stage. The hypopus may be resistant to fumigants. However, the cheese mites, Tyrophagus casei do not have a hypopial stage. Prolonged storage of products subject to infestation without cleaning of pantries and shelves where such materials are placed. These mites can subsist on organic debris in cracks, crevices and corners and are readily spread to other areas.

A number of insects infest seeds in storage. These insects infest seeds to fulfill their feed and shelter requirements, which result into qualitative as well as quantitative loss of seeds. Indian climate is very favourable for their multiplication and growth throughout the year. Storage pests move along with commercial products from one area to another and even to different countries in the world. Some adult insects are strong fliers and may spread from one to another place of storage or from source of infestation. Generally, all insects infesting seeds in storage belongs to insect order Coleoptera and Lepidoptera (Table 4.1). Some minor species belong to Pscoptera. These pests cause losses to man by destroying large quantities of grain, corn, legumes (beans, lentils and peas), milled cereal products, flour, bran, macaroni and other paste products, dried fruits, dried vegetables, cheese, nuts, candy and other food materials. They contaminate and thus eliminate for human consumption, far more food than they eat and either cause direct loss or transmit diseases to man and his domestic animals. These insects can be categorized as major or minor pests according to the loss of seeds caused by them. Similarly, on the basis of their feeding behavior, they can be grouped as follows:

The damage to food grains in the storage may be caused due to insect attack, mould, fungi, bacteria, rodents, heat and moisture. The value of grain for milling purposes and human food is directly correlated with the amount of insect contamination present. The immature stages of rice and granary weevils, lesser grain borer, Angoumois grain moth and occasionally the flat grain beetle, cadelle, rust red flour beetle and saw toothed grain beetle live inside the kernels where they are hidden from view until they emerge.

Grain from casual inspection appears to be in good condition but may contain enough internal infestation which render it unfit for human food or unfit for purchase for future sale as milling stock. In general, millers have found that wheat with more than 0.5 per cent of infested kernels is unfit for milling. The need for a rapid and accurate method of estimating insect contamination in grain offered for sale has become more and more important to both grain dealers and millers.

Methods for Estimation of Damage

In order to assess the damage caused to food grains due to any one or combination of various factors; physical, chemical and micro-analytical methods are employed according to the necessity and information required. Various methods have been employed by field workers as well as by laboratory technicians in estimating damaged grains in food grains with particular reference to detection of insect infestation which alone cause maximum damage. These methods have been described below.

Insect infestation occurs in stored grains and grain products to variable extent depending upon the storage conditions in developing countries. The losses of nutritional compounds are proportional to the extent of insect infestation, nature of insect feeding and distribution of nutritional chemicals in different seed components.

1. Sources of Insect Infestation

There are many sources that leads to infestation in storage, viz., left over seed from bins or stores or spilled seeds under the stacks, cleanings, sweepings of storage structure, processing plants, old infested stores, old gunny bags, insect infested transport vehicles, storing insect infested seeds with fresh stock or vice-versa, entry of insects from neighbouring stores and carry over of field infestation

A series of efforts have been made to initiate the work on post-harvest grain technology in India. H.M. Lefroy, the 1^st Imperial Entomologist appointed by the British Government in India in 1905 had given a tentative list of storage insects in 1906. This list of storage insects was further supplemented by T.B. Fletcher in 1914. The problems of grain storage were discussed at Pusa, Bihar at the Entomological meetings held during the years 1917, 1919 and 1923.

The Department of Food was established in 1942 by the Government of India. The executive wing of the Food Department was responsible for the purchase, movement, transport and distribution of food grains. Central Food Technology Research Institute (CFTRI) at Mysore was established in 1950 with a mandate to train personnel and evolve technology relating to food processing and preservation. The Central Warehousing Corporation was set up on 2^nd March, 1957 under Agricultural Produce (Warehousing and Development) Act 1956. On 1^st January, 1965 Food Corporation of India was established.

In 1958, the Department of Food started a centre at Hapur known as Grain Storage Research and Training Centre with the main objective to train the personnel of State Government engaged in food procurement, storage and distribution. This centre was upgraded during 1968-1973 with the technical and financial assistance of UNDP and this centre was redesignated as Indian Grain Storage Institute (IGSI). Two field stations at Ludhiana and Bapatla (later shifted to Hyderabad in March 1979) were established in 1968-69. Subsequently, three stations of IGSI at Jabalpur (October, 1980), Udaipur (February, 1981) and Jorhat (May, 1981) were also established. The overall objective was to minimize losses of food grains by development of modernized grain storage practices for the farmers, traders, cooperatives and large storage of food grains in the country and also to evolve the methodology to be adopted by the Save Grain Compaign which has its headquarters in the Department of Food.

In sub-tropical agro-climatic conditions of India, a variety of insect-pest, weeds and diseases hamper the economic growth of field crops. For effective control of these pests, a number of pesticides including insecticides, fungicides, herbicides and nematicides are to be applied. Estimation technique for single pesticideonly can not be followed for detection of residues of all categories of pesticides. Hence, this constantly expanding use of pesticides on food crops accentuates the need for rapid, precise and sensitive method for determination of pesticide residues of all the major groups of pesticides. In such situation, multi-residue analytical technique can be efficiently followed for detection and estimation of multiresidues of intra and inter class xenobiotics.

The purpose of multiresidue analysesistodeterminetheresidues ofas manypesticides as possible withina short period of time even if the recoveries of somecompounds are low. In multiresidue methods the recoveries up to 70% are accepted.The recoveriesless than70%have tobe mentionedspecifically. Multiresidue analysis is relativelymore sensitiveand requires extreme vigilance on the part of the analyst during processingof samples. Additionally, instrument, GLC,need to beequipped withcapillary columnsforhigh resolutionand sophisticated detectorslikeECD, NPDand MSD for detectingmulti-residues of the pesticides. Multiresidue analytical methods/techniques havebeen developed for determination of general pesticide residues for some environmental componentsand differentfood commodities.Criteria formulti-pesticides screening as per California Department of Food and Agriculture (CDFA) are:

Crops treated with pesticides invariably contain small amount of these chemicals and the hazard depends on the amount of pesticide residues that remain on the crop and their toxicity. The amount of residue that may remain on crop or food commodity and environmental commodities depend on the nature of pesticides, good agricultural practice, environmental conditions and adoption of safe waiting periods and other perfect methods of storage. As residues are inevitable by product of pesticide use, hence pesticide residue in food commodities and their entry in to food chain has become major cause of concern all over the world. The plant science industry has a responsibility to assist farmers to produce crops that are acceptable to the food chain and consumers alike.

It is clear that pesticide residues in farm produce should be kept as low as practicable, and eliminated whenever possible through Good Agricultural Practices (GAP). However, the use of pesticides can be regulated to ensure minimum residues on food which can be considered safe to human consumption and environment as a whole.

To regulate pesticide residues to safe levels, a concept was introduced by Joint FAO/WHO Expert Committee on Food Additives (1955) and Codex Alimentarius Commissionwas established in 1964 to implement the Joint FAO/ WHO Food Standard Programmes. The Commission has laid down principles for aiming at the maximum residue limits (Tolerance limits) of pesticide on food commodities. The recommendations are based on:

In the present era, residue analyst is exposed to array of analytical choice and as per International regualtions, presence of pesticide residues in environmental components and food commodities are becoming more stringent.Generally, multiresidue methods are based on GC equipped with selective detection either electron capture detector, nitrogen phosphorous detector, flame ionization detector etc. One most widely applied technique in pesticide residue analysis is HPLC, equipped with different detectors. Another up-datemethod for confirmation is gas chromatography-mass spectrometry(GCMS)and HPLC-MS applyingparticlebeam or thermo sprayionisationtechnique. Recently one more analytical technique which has gained ground in the field of pesticide residue analysis, is high performance thin layer chromatography (HPTLC).The criteria of good analytical technique like simple, rapid, flexible, versatile, inexpensive, easily documented and validated has been the inherent advantage of HPTLC. Strictly speaking, HPTLC is not different in principleor application from conventional TLC, but due to improvements in materials, its effectiveness has also enhanced.The technique has a potential to operate upto femtogramlevel. HPTLC is a sophisticated and automated form of TLC. It is a technique which can generate about 5000 or more theoretical plates. The high performance in TLC is achieved by optimized plate coating materials, by improved sample application techniques, and by developing newmethods for the development of mobile phase. Thus HPTLC is an instrumental approach at different stages of TLC. A flow chart for HPLC principle is shown in Figure 8.1. The principle of separation is based on adsorption and this leads to easyseparationof isomers, includingchiralcompounds.Main difference between HPTLC and TLC is given in Table 8.1

The phenomenon and behavior of supercritical fluid (SCF) has been the subject of research right from 1800's and suggestion of supercritical fluid chromatography (SFC) came in to exixtence in 1958. Supercritical fluid chromatography (SFC) was first developed in 1960, and that time, it was considered as a science fiction chromatography and a revolutionary separation technique. But its reputation has slowly ebbed over the years and it is now moving forward by leaps and bounds as a stable analytical method with many advantages over the existing chromatographic methods. It is an unit operation that exploits the high dissolving power of fluids at temperature and pressurre above their critical values for extracting analytes from sample matrices.The first commercial packed column of SFC was made available in 1981 and the first commercial capillary column SFC instrument was introduced in 1985. Several refrences are useful for understanding the super critical fluid extraction and super critical fluid chromatography

High performance liquid chromatography (HPLC) was developed during late 1960s and early 1970s. Today it is a widely accepted separation technique for both sample analysis and purification in a variety of areas. HPLC, basically a highly improved form of column chromatography and popularly known as liquid chromatography, like other forms of chromatography, whether paper, thin layer or gas chromatography, needs two essential components, the stationary and the mobile phase. It has the unique ability to separate and quantitate residues of polar, non volatile and heat labile chemical compounds. HPLC uses high pressure pumps up to 400 atmospheres that makes it much faster, short and narrow columns packed with micro particulate phase and a detector. To affect a separation, mixture to be analysed is dissolved in a solvent (ideally this solvent is identical to mobile phase) and then forced to flow through analytical column along with mobile phase. In the column the constituents of mixture get resolved and are detected with a suitable detector by a proper choice of column, mobile phase and detector. HPLC is a technique having high degree of versatility. There are several excellent texts published over the last decade which may be consulted for general information on the instrumentation in liquid chromatography

Chromatography was first employed by Ramsay (1905) to separate mixtures of gases and vapours. The first scientist to recognize chromatography as an efficient method of separation was the Russian botanist Tswett (1906), who used a simple form of liquid-solid chromatography to separate a number of plant pigments. The colored bands he produced on the adsorbent bed evoked the term chromatography for this type of separation (color writing).Althoughcolor haslittletodo with modern chromatography, the name has persisted and, despite its irrelevance, is still used for all separation techniques that employs the essential requisites for a chromatographic separation,viz. a mobile phase and a stationary phase.

Gas-liquid chromatography (GC) was invented by James and Martin (1952) and is a separation technique in which the mobile phase is a gas (usually helium or nitrogen) and the stationary phase is a liquid. In the original columns used by James and Martin, the liquid stationary phase was adsorbed on the surface of an inert support such as Celite (a diatomateous earth) or calcined Celite (a form of brick dust). The support was usually deactivated before use by acid treatment and subsequent reaction with hexamethyldisilazane. The technique was extensively used for the separation of a wide range of volatile substances. Thus, chromatography has been defined as a separation process that is achieved by distributing the components of a mixture between two phases, a stationary phase and a mobile phase. Those components held preferentially in the stationary phase are retained longer in the system than those that are distributed selectively in the mobile phase. As a consequence, solutes are eluted from the system as local concentrations in the mobile phase in the order of their increasing distribution coefficients with respect to the stationary phase.By separating the sample into individual components, it is easier to identify (qualitate) and measure the amount (quantitate) of the various sample components.

Chromatography is an important analytical technique for the separation, isolation and identification of the constituents of a mixture. It is a physical method of separation in which the components to be separated are distributed between two phases; one of these phases consisting a stationary bed of large surface area, the other being a fluid that percolates through or along the stationary bed. All forms of chromatography do have the common features given in this definition. All of them involve the physical movements of the sample across a flat surface (tube like) or through a tube. The stationary phases in all of these possess a large surface area. In all the techniques, the movement of the sample is due to the motion of the fluid/mobile phase. The principle underlying all these methods is the selective retardation of the movement of the components by the stationary phase. These methods result in the production of bands of concentrated components. The basis of naming various chromatographic methods are different:

After having performed their function, pesticides in the parent form or in some converted form reach the soil, the ultimate sink for these moieties which are collectively termed as residues. According to FAO 1986, pesticide residue is defined as any substance or mixture of substances in food, agricultural commodities or animal feed resulting from the use of pesticides and includes any specified derivatives such as degradation products, metabolism, reaction products and impurities that are considered to be of toxicological significance. From the soil they move into other segments of the environment. The pesticide residues are absorbed by plants and enter the food chain, where they get concentrated in animal fat. Some residues are carried away by wind in the vapor form, which enables them to be transported to long distances from the source of application and then get collected by dust and rain before they are deposited on the ground again. Pesticide residues also move to aquatic environments through runoff, leaching, and atmosphere disposition.

The knowledge on the fate of pesticides, after application in the environment is useful to research workers associated with their safe and effective use. It is important to know whether a xenobioticwillpersist orwill be inactivated. Frequently persistence is desirable for sustained control. However, for many cases, rapid detoxificationof the compound may be required to avoid cumulative effects. Most of the pesticides are not very soluble in water andtheir oxidation orhydrolysis (or both)leads to formation of polar groups. Such reactions are called primary metabolic reactions, often, but not always. Theproducts ofthese primary reactions undergo secondary changes whereby they are conjugated with endogenous substances to form molecules that are more readily excreted. In higher animals, these changes facilitate the transportation of metabolites of pesticides in blood, which in turn leads to their eventual excretion.

Part A

1. Pesticides

A pesticideis defined as anysubstance intended for preventing, destroying, attracting, repelling or controlling pests including unwanted species of plant, or animals duringthe production, storage,transportation, distributionand processing of the food, agricultural commodities or animal's feed or which may be administered to the animals for the control of ectoparasites. Pesticides include insecticides, herbicides, fungicides, acaricides, nematicides, etc.

According to food and agricultural organization (FAO) 1986, the term pesticidehas been defined as“any substance or mixture of substances intended for preventing or controlling any pest and includes anysubstance or mixture of substances intended for use as plant growth regulator (PGR), defoliant or desiccant. The term excludes fertilizers and antibiotics or other chemicals administered to animals to stimulate their growth or to modify their reproductive behaviour”. Pesticides are the largest group of poisonous substances that are widely broadcast today.

Insect and non-insect pests cause extensive damage to standing crops, stored grains and stored products resulting in huge economic loss. Besides these, several pests cause annoyance, as well as attack livestock such as, cattle, poultry and pet animals. Some others damage household articles such as, clothing, upholstery, carpets, paper, books, leather articles, wooden and bamboo furniture and all sorts of wooden structures. These obnoxious creatures besides consuming any kind of food materials, spoil them by contaminating and polluting them with their faecal matter and urine and impart a repulsive, bad odor and make them unfit for consumption by humans and livestock. The damages caused by them pave the way for easy access and infection by some of the saprophytic microorganisms, which hasten the deterioration process. A few of these microorganisms produce mycotoxins, which are poisonous and may even be fatal to the consumers.

Among the household pests, cockroaches, crickets, silver fish, ants, white ants, carpet and powder post beetles and clothes moths are quite important. These insects not only cause annoyance but different types of flies, mosquitoes, bugs, fleas and lice either suck and feed on the blood from humans, cattle, poultry etc. but are also responsible for disseminating several disease causing microorganisms.