EFFECTS OF SOME PESTICIDES ON THE EMBRYONIC STAGES OF CHICK
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1 EFFECTS OF SOME PESTICIDES ON THE EMBRYONIC STAGES OF CHICK Subrata Datta Department of Zoology, East Calcutta Girls College, Lake town Kolkata (INDIA) Received August 17, 2007 Accepted January 4, 2007 ABSTRACT It has been reported that different insecticides and pesticides which are commonly used in the field, not only cause harm to the adult animals but also in their embryonic stages. Of the different pesticides, we have used Furadan-3G (Carbofuran insecticide) and Malathion (Organo phosphorus/ water soluble) to know their effects on the embryonic stages of the chick. Rhode Island Red and white leg horn-these two varieties of poultry chickens were used in this study. Sterile distilled water was used for control set and the whole experiment was done in aseptic (sterile) condition. Immediately after fertilization, the fertilized eggs were incubated in different hours in an artificial incubator. For this, first, the Furadan was injected on to the airspaces of the freshly fertilized eggs of the chickens in differents doses at ug (microgram) level and immediately after injection, the injected portion was sealed by a mixture of molten paraffin and lac. Similarly, the Malathion was used. Immediately after treatment, the eggs were incubated in an incubator. L D 50(lethal dose) was 50 ug /ml. After 24 hours, 48 hrs. and 72 hrs. of incubation the treated eggs were ruptured and the embryos of different hours were preserved in 70% alcohol in respect to the control sets in each case. After dehydration and eosin staining and mounting, the permanent slides containing embryos were examined. The Furadan and Malathion treated embryos showed Terata (abnormal morphology) formation with head trunk fusion. When the experiment was carried out for 21 days, most of the treated embryos died whereas only 5-7% of the embryos developed into adult with abnormal growth as terata (Head trunk fusion and deformed legs) in both cases (Furadan and Malathion) and in both varieties (Rhode Island Red and White leg horns). For the histological study, Furadan and Malathion treated 24 hrs., 48 hrs. and 72 hrs. chick embryos were sectioned (Microtomy). The treated set showed abnormal morphology, particularly in the limb bud region (About 72 hrs.) while the control showed normal histology. Biochemical analysis of the Treated embryos showed deviation in the standard curves plotted against hours and also variation in protein and nucleic acid analysis when their O.D. value was calculated through colorimeter but the control did not show such variation. From the above study, it can be concluded that terata formation is definitely due to the effect of the Furadan and Malathion pesticides.. Key Words : Pesticides, Carbofuran (Furadan-3G), Malathion, Chick embryos, Terata, Aseptic condition INTRODUCTION It has been reported by several workers that not only the pollutants but also some pesticides are also responsible for causing 340
2 Journal of Environmental Researh And Development Vol. 2 No. 3, January-March 2008 human health hazards including poultry birds, cattles, dogs and other animals 2. Some of the pesticides used by the farmers to control the pests may cause remarkable abnormalities during developmental stages of some of the animals including the adult morphology 3. May be the effect of these chemical pesticides is severe or mild, but it should not be ignored. Poultry birds, particularly the chickens, ducks or other birds have been effected 4,5. OBEJECTIVES Therefore the objectives of this study is to know the preliminary effect of some of the chemical pesticides, especially Furadan-3G (Carbofuran Insecticide) and Malathion (Organo phosphorus insecticide, Gangopadhyay, 2000) on the poultry-chickembryos of both of the varieties Rhode Island Red and white Leg-horn poultry chickens at the morphological, histological, biochemical and if possible at genetic levels during their embryonic development from an immediately fertilized-egg, since, human beings when consume such eggs or their flesh are also effected indirectly. MATERIAL AND METHODS (a) Fertilized poultry eggs and application of insecticides : Furadan and Malathion were dissolved in sterile water. In both the cases, Lethal dose (LD)- 50 was determined which was 50 ug / ml. The eggs (immediately after their fertilization) of Rhode- Island-Red and white leg horn were collected from a local poultry farm. The fertilized eggs of both of these varieties were washed by spirit and 50 ug/ml. of these insecticides were injected separately onto the airspaces of these freshly fertilized eggs by using sterile disposable syringe and the injected portion of the eggs were sealed by a mixture of molten paraffin and lac(waxlac). The whole experiment was done in a completely aseptic-condition (sterile) to avoid inection. 50 ul of sterile distilled water was injected for the control set aseptically (under a laminar flow) and the experiment was repeated. (b) Incubation : A wooden-glass incubator having optimum temperature (37 0 c) and moisture was used. Immediately after treatment, both the treated and control sets were kept on the incubator with proper marking. (c) Preservation, Staining and Preparation of whole mount of the chick embryos : After 24 hrs, 48 hrs. and 72 hrs. of incubation, the Insecticide treated eggs along with control were taken out from the incubator and ruptured on a tray and the embryos of different hours were preserved in 70% alcohol, dehydrated and eosin stained embryos were mounted on the slides as the whole mount as per standard protocol. (d) Histological assay : 24 hrs., 48 hrs. and 72 hrs. treated and control-embryos were first preserved in 70% alcohol and dehydrated through a series of graded alcohol and finally embedded in molten paraffin (57 ) blocks and sectioned by a microtome (5-6 um) and stained by routine haematoxlin-eosin procedure. (e) Biochemical analysis : Colorimetric estimation and biochemical analysis of RNA, DNA, protein etc. from the whole chick embryos of different hours of treated and control set was done and plotted against a standard curve according to standard protocol 6. RESULTS AND DISCUSSION Terata - formation is not uncommon among the birds, especially among the poultry chickens. Teratogenesis may be one of the keyhazards among the vertebrates. Furadan-3G and Malathion treated embryos exhibited abnormal morphology (Terata) with head-trunk fusion and almost without any neck in 24 hrs., 48 hrs. and 72 hrs. of chick embryos (Fig. 1,2,3,4,5,6,7 and 8) and deformed legs after 21 days of incubation in comparision with the completely normal morphology of the control sets. The experiments were repeated. When 341
3 the experiment was carried out for 21 days, most of the treated embryos died whereas only 5 to 7 % of them developed into adults with abnormal growth as terata (Head-trunk fusion and deformed legs) in both varieties but the control developed into normal chicken (Figures not shown). Histologically, the treated set showed abnormal morphology, particularly in the limb bud region especially in about 72 hrs. of incubation while the control set showed normal histology. Biochemical analysis(total protein, RNA, DNA, etc.) of the entire treated embryos showed deviation in the standard curves when plotted against hours and also variation in protein and nucleic acid etc. but the control did not show such variation. Fig. 1 : Control showing normal chick embryo of about 24-hrs. of incubation. Note embryo with membranes. Fig. 2 : Terata of the Furadan-3G-treated chick embryo of about 24hrs. (Left) & Malathion-treated (Right). 342
4 Journal of Environmental Researh And Development Vol. 2 No. 3, January-March 2008 Fig. 3 : Normal (control) chick embryo of about 48 hrs. of incubation. Fig. 4 : Furadan-3G (Carbofuran) treated chick embryos of about 48 hrs. of incubation. 343
5 Fig. 5 : Malathion treated chick embryo of about 48 hrs. of incubation showing Terata. Fig. 6 : Normal (Control) chick embryo of about 72 hrs. of incubation. 344
6 Journal of Environmental Researh And Development Vol. 2 No. 3, January-March 2008 Fig. 7 : Terata (Head Trunk fusion) of about 72 hrs. of Furadan treated - chick embryos (Right Maximum fusion). Fig. 8 : Terata of about 72 hrs. of Malathion treated chick embryo. 345
7 CONCLUSION From the above study, it can be concluded that Terata -formation atleast in the chick embryos is definitely due to the toxic effect of both of the pesticides- Furadan 3G (Carbofuran) and Malathion which can even cause hazards and abnormalities in : (a) morphological (b) histological and (c) biochemical levels in the developmental stages of the chick embryos. The study at genetical level is in progress. Thus, the chicken consuming the Pesticide-treated grains consume pesticides atleast in lower doses (at microgram level) which in-turn is consumed by the human beings through chick eggs or flesh and thereby causing human health hazards. Therefore it can be safely concluded that farmers should be prevented from using chemical pesticides for killing the pests, rather it can be much better if Biological control device is adopted by constructing the genetically manipulated strains against the pests for betterment of society and to keep our environment free from pesticide and pollutanthazards. ACKNOWLEDGEMENT The author is grateful to Lord Shiva and thankful to our principal (Dr. Mrs.Sukla Hazra) and Saswata Dutta for constant encouragement and to U.G.C for financial support. The author also pay his thanks to Mr. Bidyut Chatterjee for typing the entire manuscript. REFERENCES 1. Balinsky B.I, An introduction to embryology. 5 th edition, Thomson Publication, EWP, , (2004). 2. Gangopadhyay P.K, Application oriented Chemistry, 1 st Publication, Books Syndicate Pvt. Ltd., India, 45, (2000). 3. Kormondy J.E, Concepts of ecology, 4 th edition, Prentice Hall of India , (1996). 4. Laemmli U.K, Cleavage of structural proteins during the assembly of the head of the bacteriophage T4, Nature (London) 227, , (1970). 5. Patten B.M, Early embryology of the chick. T.M.H edition, Tata Mc Graw-Hill Publishing Co.Ltd., India, 96, (1976). 6. Shukla J.P, Malathion induced biochemical alterations in the Liver of fingerlings of Channa punctatus, Actahydrochim. Hydrobiol., 6, , (1988). Save Water 346
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