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PROJECT WORK Toxicity evaluation of Pyridone derivatives on Sitophilus oryzae Linn (Coleoptera: Curculionidae)

By:                                              		   under guidance of             Jayshree V. Shinde                         		   (Dr. A. E. Desai)

2011-2012

“Toxicity evaluation of Pyridone derivaties on Sitophilus oryzae Linn (Coleoptera: Curculionidae)” A DISSERTATION SUBMITTED TO THE UNIVERSITY OF PUNE IN PARTIAL FULFILLMENT OF THE DEGRRE OF MASTER OF SCIENCE IN ZOOLOGY BY MRS.SHINDE JAYSHREE VITTHAL

UNDER THE GUIDANCE OF DR.A.E.DESAI (M.Sc. PH.D.)

P.G.DEPARTMENT OF ZOOLOGY K.T.H.M. COLLEGE NASHIK-02 2011-2012

K. R.T.ARTS, B.H.COMMERCE AND SCIENCE COLLEGE, NASHIK. Certificate University of Pune This is to certify that the incorporate in this dissertation entitled “Toxicity Evaluation of Pyridone derivaties on Sitophilous Oryzae Linn (COLEOPTERA:CURCULIONIDAE)” was satisfactorily carried out by Mrs. SHINDE JAYSHREE VITTHAL  of M.Sc. II bonafide student of this college. She has completed this project under my supervision and guidance during academic year 2011-2012. This project work submitted by her is original and the scientific information obtained from other sources has been duly acknowledged.

Project guide                     Examiner                      Head, Dept. of Zoology (Dr. A. E. Desai)			 (Dr. S. M. Nikam)

Acknowledgement

It is the moment of great pleasure for me in venturing out to present the project. I would like to express my deep sense of gratitude towards my project guide, Dr. A.E.Desai for his advice encouragement and discussion throughout the project work which was very valuable.

I am equally thankful to our respected Head of Department (Zoology), Dr.S.M.Nikam for providing laboratory facility during the project work.

I would also like to  extend  my  gratitude towards our respected principal Dr.V.B.Gaikawad for providing laboratory   facilities for research paper.

I can’t forget the help extended by our department .I kind like to thanks to our senior Mrs.Sonali Devore and also my classmates  and laboratory staffs for their kind help & co-operation.

P.G. Department of Zoology K. T. H. M. College, Nasik. Mrs. Shinde Jayshree Vitthal M.Sc. Zoology

INDEX

Sr. No Title 1	        Introduction 2	        Material and method 3	        Observation & Calculation 4	        Results & Discussion 5	        References

INTRODUCTION

INTRODUCTION

Insect have existed on each for at least 20 million year while man came on scene only half year ago. It is obvious that  must  have  faced the  problem of insect pest quite early  in the  history  of   their  existence. Insects are  injurious to living plants and animal including man .The third great phase of insect injury rises to stored product .The organism which damaged the stored grains and its product are called as grains pests. e. g. Rodents, nematodes, fungi, weeds, birds, bacteria and viruses etc. Stored food including grains ,cereals, cereals products, dried food, nuts spices, drugs and many other liable to infested by a large number. of insects most of which are cosmopolitan and wide spread all over world .According to Metcalf and  Flint  (1973) the pest of stored products are the most expensive of all insects, because    they  feed  upon  product  that  have been  grown  harvested,  some time manufactured are of prime importance as pest of stored food. Grains constitute the most important staple foodstuff for the ever growing population in the tropics. As in field crops, a wide range of insects   attack stored products with the commonest among   them   being    beetles  and moths (Obeng-Ofori et Aal.,1997). Insect pest damage to stored grain results to measure economic losses to farmers throughout the world. In most tropical countries, post harvest losses of cereals and pulses due to attack by insect pests have been estimated at 20-30% (dick, 1988) According to their feeding habitat    these  pest  are classified into two categories like primary and secondary stored g rains pests. Primary  pest attack the whole grains and larvae feed and develop with kernels (seeds) so called as internal feeder,while secondary pest are external feeder because  they  feed     on grain dust and broken kernels. Thus, both type of pest ultimately damage the grains .Primary Pests includes granary weevil ,maze weevil, laser grains bore and anquimois grain moth while secondary pest includes flat and rusty grain beetle ,red flour beetle saw toothed beetle and Indian meal moth. Stored grains pest should be controlled for increasing availability of food since  ancient way are in used to control grain pest. Some examples  biological ways (Plant extract/ botanical insecticides). Use of   turmeric  powder, rock salts,   sandal dhoup,  neem  leaves and     other   some natural   ingredients  which  are  using  since and  ancients  period, this method are successful in repelling the insects but they cannot kill the insects. Sun drying is also one of   the traditional  mechanical ways used to control  this insect,  but heating of grain may accelerate    the development of the insect. An air   tight containers is also   one of  the good methods to avoid the attack of pests,   but sometimes it may provide suitable environment for fungal and bacteria growth. Since,1950’s synthesis insecticides have been used to control stored grain products,insect (Subbramoryam and Haqstrum,1995) such as chloropicrin, methyl bromine,phosphine fumes etc.It is severe problem of today since pest have developed resistance against these chemicals. So today it is very necessary to discover an alternative as well as effective pesticide against stored grain pest. Now in view to control stored grain pests various mode of action of plant products are used. Viz: Toxicant / Insecticides, Attractants, Repellants, Solvent for insecticides (Cotton shell flour), Dust carriers (soyabeen and walnut shell flour). Various botanical insecticides and plant products that have been used as insects repellant in our country form ancient times. Dried and powdered leaves of Neem against woolmoth, Calatropis against white ants etc. Some examples of plants extracts / botanical insecticides as : 1.	Nicotine: This is the important alkaloid of plant Tobacco possess an insecticidal property two plant species like Nicotina tobaccum and Nicotina rustica are only employed for nicotine extraction. It leaces no harmful residues. 2.	Pyreyhrum: This is obtained from the white flower of Crysanthemum cineraria folium. The active ingredients are esters. This acts as contact insecticides by affecting nervous system of insect like chewing lice of livestock and an aphid.

3.	Rotenone: This substances occur in 68 species of certain leguminous plants the more important species is Derris and lonchocorpus. It act as contact as wel as stomach poison which affects respiration by inhibition of oxygen utilization by  body cells resulting in death of insects. Dust are effective incase of ectoparasites. 4.	Qussia: This is obtained from a Surinam tree Quassia amara. The decoction action of Qussia chips. In water mixed with soap have been used to control aphids and sawflies. Much work has been done on growth inhibitory effects of plants extracts some terpenoid (sunflower) e.g. Kaurenaice and Trachybbauoic acid inhibit larval growth (Eliger, et. al; 1976) while others e.g. carvocrol embryonic development in insects (Ohigoshi, 1976). The plant products are known to destroy essential part of theendocreine system of certain insects and prevent their reproduction (supavarm et. al; 1974, saxena and saxena 1992). Pyrethroid introduce in Japan in 1983 against this resistant moth were highly effective, however, high resistance to these purethroids was observed within only few years (Makino and HHorikiri, 1985; hama 1986, 1987). Resistance organophosphorus insecticides could gradually develop even in susceptible strains by selecting with organophosphorus insecticides for several generation (Sasaki 1982; Cheng et al; 1985; Noppum et al; 1986; Hama, 1986b). it has been suggested that insecticides resistance can be suppressed by immigration of susceptible individuals (Gorghiou and Taylor, 1977). Synthetic chemical are expensive, erratic in supply due to foreign exchange constraints and cot benefit wise often not economical to use by resource poor farmers (Niber, 1994; Udo, 2005). The laboratory experiment showing insect growth disrupting effects of Azadirachta indica flower extract on Dysdercus cingulatus koeigiz by saxena and Harshad, (1992), Chawdagart et al; (1985) received literature pertaining to plants species possessing pest control properties including JH and insect growth disrupting like activities. An alkaloid piperin acetone extract of piper nigrum proved highly toxic against Sitophilus oryzae grain pest. The aim of this project work is the synthetic compounds of pyridine derivatives synthesized and prepared in organic research laboratory of K. T. H. M. college, Nasik has been evaluated for their toxicity on the stored grain pest, Sitophilus oryzae Linn as a part applied science and to protect stored grains in view of economic loss to house wives and farmers.

MATERIALS AND METHODS

MATERIALS AND METHODS

A)	Collection and culture of Sitophilus oryzae Linn: The stored grain insect Sitophilus oryzae Linn were collected from warehouses of Food Corporation of India centre, Manmad. The insect were brought to laboratory and culture in propylene plastic containers disinfected wheat grain at temp 26±2, RH:65-70% and photoperiod (L:D)- 12 : 12 hrs. The insects are preserved and some fresh specimens were used for toxicity testing against the pyridone derivatives . B)	Synthesis of chemical compounds : Following chemicals were synthesized in the organic research laboratory of chemistry department, K.T.H.M. college, Nasik. 1. COMPOUND NO 1 (PN-104)

2. COMPOUND NO 2 (PN-101) 3. COMPOUND NO 3( PN-105) 4. COMPOUND NO 4( PN-106)

Experimental bioassay: Culture of Sitophilus oryzae Linn. Were maintained under laboratory conditions at temp 26±2, RH: 65-70% and photoperiod L: D 12:12 on wheat grains. These insects were used for toxicity testing. Synthetic chemicals were synthesized and obtained from organic research laboratory of chemistry department of K.T.H.M. College, Nasik. Stock solution was prepared for all chemical compound by using DMF as solvent and further Concentration of each chemical compound were made as 5ppm, 10ppm, 15ppm and 20ppm in distilled water. Adult Sitophilus oryzae Linn. Were selected for toxicity testing. Petriplates were taken and labeled a control, 5ppm, 10ppm, 15ppm and 20ppm. the filter paper were put in each petriplates according to their inner diameter size. The disinfected and dried it wheat grain were kept in each petriplates. 5-6 drops of 5ppm, 10ppm, 15ppm and 20ppm were smeared with wheat grain and where kept respective petriplates, 10-individuals of Sitophilus oryzae were released in each plate. The control set was arranged with DMF. Mortality were observed at the interval of 6 hrs for 24 hrs.              Observations was recorded for mortality and LC50, Heterogenecity, variance and fiducial limits were calculated by Finney’s method (1971)

OBSERAVATIONS

a).	Sitophilus oryzae Linn Kingdom: 	Animalia Phylum: 	Arthropoda Class : 	Insecta Order: 	Coleoptera Family: 	Curculionide Genus: 	Sitophilus Species: 	oryzae Linn                    Common Name:  Rice weevil

Pest status:

This is very; serious pest of stored grain. It is cosmopolitan in distribution.

NATURE OF DAMAGE S. oryzae feed on whole grains of rice, wheat, Jawar, Bajara, maize,  Barley etc. which may lead to heal grain. Larvae and  adults  feeds  on  an   endosperm  of  seed  and  followed  out  up to the thin porous husk remains     only   which    results into     reduction in weight and quality of germination may take place, producing a weak seedling which is vulnerable to attack by moulds, bacteria and other insects. Adults which are winged are even known to fly from  the Godown    to the field. In the vicinity where they being to infest the grain in the field. Taining with white, dusty excreta of this insect contaminate the grains                       and  thus make it unpalatable.

LIFE CYCLE The adults are small, reddish brown about 4mm in length   with a large rostrum and thorax. Rice  weevil  prefers  warmer   climates  and so as is more prevalent in the southern states. Rice weevils are internal  feeders which means larvae develops inside whole grain kernels. Mating often occurs within 24 hrs of adult emergence from grain kernels. Using her long, slender mouth parts, the female rice weevils bare a small hole into a grain kernel and then lays an egg in the hole. After sealing the hole with a gelatinous secretion. She proceed to the next kernel to  repeat   the process. Over the life span at 4 to 5 months, the female will lay from 300-400 eggs. Although as general rule about 50% of the  eggs does  not hatch. The eggs  hatches  into short, stout ‘C’ shaped Larvae i.e. creamy, white apodous. The larva feed by chewing away at the inside of kernel and will eventually hallow out the inside of kernel. In warm conditions the period from egg to pupa can last as few as 26 days but usually it  takes   longer   time. After pupation the adult beetle remains inside the kernel maturing and hardening. The adults  when  chew its way out, leaving a small round hole is the grain. These open   round hole are a sign of a weevil  infestation. Rice weevils  are  prolific  breeders     and can  build   up huge population  in   stored  grain  to the point where the grain has little value as a food product.

Table no 1: 2,5dihydro-7,8dimethoxy-4(3-methoxyphenyl)-2-oxo-1H-in deno (1,2-b)pyridine-3carbonitrile. Sr.no. Dose in ppm	No.of insects released	Mortality for 6hrs           	12hrs	18hrs	24hrs 1	5	10	1	2	3	6 2	10	10	2	3	3	7 3	15	10	2	3	4	8 4                	20	10	3	3	4	9

Table no 2: 2,5dihydro-7,8dimethoxy-4(3,4-dimethoxyphenyl)-2-oxo-1H-in deno (1,2-b)pyridine-3carbonitrile. Sr.no. Dose in ppm	No.of insects released	Mortality for 6hrs           	12hrs	18hrs	24hrs 1	5	10	2	2	2	5 2	10	10	2	3	3	6 3	15	10	3	3	3	8 4                	20	10	2	3	4	9

Table no 3: 2,5dihydro-7,8dimethoxy-4(3,4,5-trimethoxyphenyl)-2-oxo-1H-in deno (1,2-b)pyridine-3carbonitrile. Sr.no. Dose in ppm	No.of insects released	Mortality for 6hrs           	12hrs	18hrs	24hrs 1	5	10	1	2	3	6 2	10	10	2	3	3	8 3	15	10	2	3	4	9 4                	20	10	3	3	3	9

Table no 4: 2,5dihydro-7,8dimethoxy-4(thiophen-2-yl)oxo-1H-in deno (1,2-b)pyridine-3carbonitrile. Sr.no. Dose in ppm	No.of insects released	Mortality for 6hrs           	12hrs	18hrs	24hrs 1	5	10	2	2	3	6 2	10	10	2	3	3	7 3	15	10	2	3	4	9 4                	20	10	3	2	3	9

CALCULATIONS: The calculation were done as per following method: 1.	In column 1 the does concentration mentioned in ppm

2.	The no. of insect exposed are mentioned in column no III. and it is designated as N

3.	To calculate % mortality, Abborts (1925) formula was employed. This formula gives the % of the original no of insect died by treatment. P= om- cmX 100 100-cm Where, P= corrected mortality Om=observed mortality cm = control mortality it was observed that there was no mortality in control gr. Of insect therefore the mortality data obtained in experimental insect for each dose was calculated by Finner formula (1971) P= rXN/100 Where, P=% mortality r = mortality observed N = no of insect exposed.

4.	Logarithm is taken of each concentration and is recorded in column I and

5.	The empirical probit value was read for % mortality from table 1 (transformation of % of probit) from Finner’ s book and recorded in column V and designated as Y.

6.	The value of weighing coefficient is read from IX 2 (weighing coefficient and probit value to be used for final adjustment) and recorded in column VI and designated as W

7.	Weight is recorded in column VII which is calculated by formula W= nw Where, n= no of insect exposed W = weighing coefficient

8.	Column VII is multiplied by y and recorded in column VIII and read as nwx

9.	Column nw is multiplied by y and recorded in column IX and read as nwy

10.	Column X is designated as nwx2 in which nwx is multiplied by X

11.	Column nwy is multiplied by y and recorded in column VI and read as nwy2

12.	Column nw is multiplied by y and X and recorded in XIII read a nwxy

13.	Column XIII contain the working probit found out by calculation.

14.	Summation is taken from column VII to XII and note down at the bottom of table

15.	The Lc50 value is calculated by formula Lc50 = X+5-y /V Where X = ∑ nwx / ∑nw Y = ∑ nwy/ ∑nw B= ∑xy/∑xx Where ∑xy =∑nwxy + (∑nwy) (∑nwx)∑nw ∑xx= ∑nwx- (∑nwx)2/∑nw

16.	The regression equation is written as y= a+bx i.e. a = y-bx

17. Heterogenecity, X2 (n-1)= {∑nwy2-y(∑nwy)}-b {(∑nwxy)-x(∑nwy)} Table value of x2 read from the statistical table at (n-1) degree of freedom, if the calculated value p is less than table value, the hypothesis is accepted. And if p is greater than the table value is not accepted Here n= no of experiments.

18.	Variance is calculated by formula Variance= 1/b2{1/∑nw+(m-x)2/∑nwx2-(∑nwx)2 /∑nw} Here, m= Lc50 value

19.	The standard error (s.E≥)value is calculated by formula S.E.= √V

20. The Fiducial limit is calculated from variance by formula Lower limit m1= m-1.96(√V) Upper limit m2=m+1.96(√V)

Results & Discussion

RESULT

Acute toxicity test were carried under laboratory condition for 24 hrs duration for chemical compound, the LC50 value for chemical compound were calculated by the method described by Finney (1971) and simplified by Busvine (1971)as in table No 3. LC50 value of these compounds against Sitophilus oryzae Linn were worked out as, 1.5515+0.9982(2,5dihydro-7,8dimethoxy-4(3-methoxyphenyl)-2-oxo-1H-in deno (1,2-b)pyridine-3carbonitrile.), 3357+0.7354,( 2,5dihydro-7,8dimethoxy-4(3,4-dimethoxyphenyl)-2-oxo-1H-in deno (1,2-b)pyridine-3carbonitrile.),1.5885+0.9567(2,5dihydro-7,8dimethoxy-4(3,4,5-trimethoxyphenyl)-2-oxo-1H-in deno (1,2-b)pyridine-3carbonitrile.), 1.5134+0.8954,(2,5dihydro-7,8dimethoxy-4(thiophen-2-yl)oxo-1H-in deno (1,2-b)pyridine-3carbonitrile.) Thus, as the concentration of chemical compound is gradually increased. The mortality of insect increases concentration. The mortality is highrest in concentration 20ppm and 15ppm shows moderate mortality while at 5ppm and 10ppm concentration the compound showed less toxicity Thus comparatively in 20ppm the effect of chemical compound is very prominent than other

DISCUSSION There is significant increase in mortality from low to high dose of chemical compound. Farmers mostly rely on the  contact    insecticides for the control of stored grain pests of raw pulses but such treatments may results in the presence of residues in the   stored grain    products       (Pourmirza and Tajbakhsh, 2008). Therefore the number. of suitable contact insecticides that can be used in the control of stored grain pests are limited, (white and Leech, 1995. Arthur, 1999; Golam et, al.2011 reported 100%  mortality of   S. oryzae and P. interpunctella when  exposed    to   nitrogen and phosphin mixtures in PVC bins. Azadirachtin exhibited   good  efficacy   against  Musca  nobulo L,  Culex fatigans,  Tribolium castaneum  and   Anthrenus flavipes   (Paul et al.1963)  Rhyzooertha  dominica, Sitoroga cerealla ( Savitri and Rao, 1976) Tribolium  (Tabassum, at, al; 1994).	S. oryzea, R. dominica, T. granarium and T. castaneum show high mortality when exposed to biogas in PVC bins (Bennett, 2003). Khaire et, al. (1992) reported the effect of ten vegetable oils against C. chensis as grain protectant and coconut oil is best protectant as natural product against C. macultus (Swella and Mushobozy, 2007). Li, et al. (2009)observed that T. castaneum, P. interpunctella, S. oryzae and C. maculates have shown high mortality when treated with red spring wheat. From the above researchers it is concluded that a new approach in insect control could be the use of less hazardous chemical compounds which are compatible with the environment. In the present study among the pyridone derivatives are most effective and revealed high mortality PN101,LC50=1.5515+0.9982,PN104, LC50=1.3357+0.7354,PN105, LC50=1.5134+0.8954, PN106, LC50=1.5885+0.956+7)and safe for application.

REFERENCES