Chapter II MATERIALS AND METHOD 34
2.0 MATERIALS AND METHOD Experimental procedure During the study of biodeterioration of paper manuscripts and their control the following materials and methods were used. Some observation was made on the gravity of the severe infestation made by the insect pest on paper manuscripts in various repositories like museums, archives, and libraries. The egg clusters, larvae and adults of Lepisma saccharina and Gastrallus indicus were collected from the infested books of the Maulana Azad Library and Book Bank of Zakir Husain College of Engineering and Technology, AMU, Aligarh. Some specimens of bookworm were cultured in the research laboratory of the Department by using an environment study chamber to conduct experiments for control measures. Lepisma saccharina posses very long life cycle and it takes years to develop into adult. Hence certain specimens were directly collected from the libraries to conduct experiments. Since both the nymph and adult are destructive in nature, all of the stages were directly used for experiments. The identification of the different species of insects was done from the Entomology Section, Department of Zoology, AMU, Aligarh. The culture of Gastrallus indicus was raised in small booklets made of old paper which measured about 6 x4 x1. Besides, this culture was raised in paper mache medium in petridishes of 14 cm in diameter. The 35
booklets and the petridishes were kept in trays and glass jars respectively, covered with fine muslin cloth by using elastic bands. The bionomics of different type of insects was studied in the laboratory under controlled conditions of 28 o -30 o C temperature and 60-70% R.H., by using an environmental study chamber. Thereafter experiments were initiated on the control measures of these insect pests. Certain experiments on fumigation treatment were carried out under normal room temperatures ranges between 28 o -34 o C, R.H. 60-65% and atmospheric pressure 70 mm Hg. A few old books of approximately same sizes and volumes having dead infestation were selected to be used for the experiments. Ten larvae of both species having almost same sizes were released in each book at different places by marking their location and noting the page numbers. At a time one book (along with ten larvae) was exposed to 15 gm of paradichlorobenzene in a closed chamber (15 x15 x15 ) and kept for a week. The first observation on mortality count was recorded after 3 days of exposure. Later subsequent observations were made after every 24 hours. Subsequently the same procedure was repeated using different quantities of the fumigant i.e. 20 gm, 25 gm and 30 gm and every time using a separate book with ten larvae each. Thus the toxic efficacy of different quantities was assessed by the total mortality count of the larvae during the entire periods of the experiments. While counting the number of mortalities the morbid larvae were also regarded as dead. The experiment was repeated five times with different quantity of each fumigant. The mortality data thus obtained was subjected to logit analysis and used for drawing sigmoidal regression curves to determine the LD 90 value of each quantity of toxicant as described by Khan (2001). 36
Experiments conducted on bookworm and silverfish for their food and foraging preferences, yet little of this appears to have been included into bait formulations. This is because the insect pests of cellulose and paper materials evolved to feed on the collection of books and manuscripts. From the perspective of the silverfish, the difference between natural food and bait formulations is very important. Some experiments were conducted to determine the efficacy of various synthetic insecticides used as the bait formulations like sodium fluoride, borax and boric acid against the silverfish and bookworm under laboratory conditions. The relative toxicity of these was determined by exposing the pests to these insecticides mixed with the preferred food of the insect pests. The present study examined the effects of three compositions of artificial bait formulations on the silverfish. The following experiments aimed to determine the ability to attract and kill the insects by the different combination of toxicants. The present author used various combination of different ratio of mixtures of boric acid, borax, sodium fluoride, paper mache and starch. The three chemical baits used for the treatment for the seven sets combination. These chemical combinations have least negative effects on the paper materials and most positive effect on the mortality counts as the treatment does not have any direct contact with the collection. There is no published data available on the bait formations on this type of collection. However diatomaceous earth, boric acid were generally used as pesticide baits for domestic purpose. During the experiment author used trapping bait stations with the combination of different baits. Author used thoroughly mixed dry baits and then distributed them in each jar with two types of preferred feeding materials. First one is the normal old paper and second contains the bait 37
combination. It was observed that most of the time silverfish preferred to feed on starchy bait formulation rather than an old handmade paper. Silverfish gave very good results comparatively to bookworm as it is a fast moving insect and showed apparently decrease in the population. All the information have been compiled regarding the natural products used as the pesticides and their efficacy in controlling the insect pests of paper was discussed in experiments of indigenous control methods. The author discussed many indigenous natural products which have been used in pest control for paper manuscript repositories. The present author also designed some experiments on the different essential oils as repellent particularly on insect pest of paper. Some of them posses insecticidal properties which gives good mortality rate. These essential oils are ecofriendly, biodegradable and less toxic to human beings. Silverfish and bookworm were collected from the infested books of Maulana Azad Library, AMU, Aligarh and Book Bank, Zakir Hussain College of Engineering and Technology, AMU, Aligarh during March to August and reared in glass containers containing cellulose and mixed feed (starch, cellulose and paper mache) at 25 o +3 o C in 70% R.H. Collection was done with the help of blunt brushes, sticking strips and trapping system and then immediately transferred to glass containers. The culture reared them in a biological incubator, to provide the feasible climatic conditions. Later on the collected insects were taken for the further treatments and experiments.the essential oil used for the experiment was extracted in the laboratory using the steam distillation plant. Extracted oils were analyzed by using FTIR spectra to know the active functional groups. Later on oils were used for the experiments. 38
In this study author used bioassay to evaluate the degree to which the essential oil repelled silverfish and bookworm. During these experiments insects kept in fumigation chamber to evaluate the mortality percentage and the insecticidal activity of essential oils of different plants. Different quantity of essential oils was used for the cent percent mortality calculation. As the increased quantity of volatile essential oils the active components also got an increment, which results in increased mortality rate. Fumigation treatment shows excellent results for those insect pests, who use to live in tunnels or holes such as bookworms and termites.to calculate the repellency rate, repellence bioassay was performed. For this, various amount of essential oils applied to filter papers that were placed into acrylic chamber contains 40 insects. All the glass containers were kept in biological incubation chamber for 10 hours. The incubation chamber maintained at 26 o C and 70% R.H. The mortality of the insects was recorded periodically for up to 10 hours then 16 hours and finally after 3 days. Out of number of pesticidal tendency owing essential oils, author found the best results with cedarwood and nutmeg oil. The author analyzed the composition of all used essential oils to find out the functional groups. Rate of repellency was calculated by using an acrylic box with three chamber. Some insects were released to middle chamber filter paper impregnated with essential oil whereas impregnated filter paper with essential oil both in the adjacent chambers kept plane filter paper. Then the observations were made according to the motility of insects and reading was taken daily. The oils such as cedarwood and nutmeg oil which causes high mortality in pest populations can be used as fumigant. 39
Materials Several equipments including environment study chamber, fumigation chamber, digital balance, ph meter were used during the experiments. Materials used for the research and experiments are describe as follows with particulars listed in the Appendix A. Environment study chamber The required optimum humidity and temperature maintained for the insect culture in the environmental chamber. Rearing of insects was done at particular temperature and humidity for conducting experiments. Collected silverfish was also kept as stock in the chamber till used for the experiment. Specifications of the thermometer are listed in the Appendix A. Fumigation chamber It is an improvised air tight wooden chamber of measurement 15 x15 x15. The facility of optimum temperature and humidity could also maintain inside the chamber. The fumigation by experiment using paradichlorobenzene was conducted in the chambers. Vacuum thermostat Thermostat was used for drying of plant specimens to extract the essential oils,which was available in the research laboratory of the Department of Museology, AMU, Aligarh. 40
Steam distillation plant for oil extraction This apparatus consists of two large containers. The first container has an inlet at the bottom, in which steamed water, heated at low pressure, is sent in. This container is loaded with the aromatic raw materials i.e. the part of the plant to be distilled. The steam rises, gently simmering the contents. It is the most common method of extracting essential oils. The heat of the steam forces the intercellular pockets that hold the essential oils to open and release them. The temperature of the steam should not be very high as it can damage the botanical material but should be high enough to open the pockets which hold the essential oil.tiny droplets of essential oil evaporate and attach to the steam. The steam which then contains the essential oil, is passed through a cooling system to condense the steam, which forms a liquid from which the essential oil and water is then separated by decantation. The oil forms a layer on the water surface as it does not dissolves in water and hence is separated easily. Digital thermohygrometer The temperature and relative humidity of the lab, environment study chamber and fumigation chamber was measured with digital thermohygrometer. Specifications of the thermometer are listed in the Appendix A. Digital balance Weighing of the chemicals and feeding materials were conducted by electronic balance for the quantity of the materials for the experiments. It was available in the research laboratory of the Department of 41
Museology, AMU, Aligarh. Specifications of the thermometer are listed in the Appendix A Blunt brushes It is used for the mechanical collection and handpicking of insects pests from infested books. Glassware Rearing jars, test tubes, measuring test tubes, beakers, measuring flasks and other glassware used with specifications are listed in Appendix E. Paper mache preparation Paper mache was prepared by grinding old wet paper to convert into a paste and then dried in sun light. Chemicals for fumigation Paradichlorobenzene was used for the fumigation treatment to the Gastrallus indicus and Lepisma saccharina. (Experiment No. 3.1) Chemicals used for bait formulations Sodium fluoride, borax and boric acid were used for baiting of insect pests in Experiment No. 3.2. The specifications are listed in Appendix B 42
Filter papers Impregnation of different essential oils was done with the help of filter paper. Acrylic box An acrylic box was designed by the author to test the repellency percentage for different essential oils. The volume of test chambers A, B and C, were the same size of (15x10x10 cm 3 ). 43
Figure 2.1 Fungal deterioration of paper manuscripts Figure 2.2 Tunnels on paper manuscripts made by bookworm 44
Figure 2.3 Pinholes and tunnels caused by bookworm Figure 2.4 Damage due to pinholes by bookworm 45
Figure 2.5 Severe deterioration of manuscripts by bookworm 46
Figure 2.6 Severe damage of manuscripts due to silverfish and bookworm 47
Figure 2.7 Faecal materials deposited inside the tunnels made by bookworm on paper manuscript 48
Figure 2.8 Paper manuscript damaged by bookworm 49
Figure 2.9 Paper manuscript damaged by Silverfish 50
Figure 2.10 Termite infestation on paper 51
Figure 2.11 Collection of Silverfish for experiments Figure 2.12 Rearing of bookworm in environmental study chamber 52
Figure 2.13 Larva of Gastrallus indicus (50 times enlarged under microscope) Figure 2.14 Dorsal view of a Juvenile Bookworm Beetle (50 times enlarged) 53