Determination of LD50 and Embryotoxicity of Pure and Commercial Formulations of Fipronil, an Organic Insecticide, by means of Hen’s Egg Test

Abstract

Fipronil, an organic insecticide, is a member of relatively new and small class of pesticides, the phenyl pyrazoles or fiproles group of chemicals. It is a highly effective against a variety of insect pests and its use worldwide is increasing. The aim of this study was to determine the effects of both pure and commercial formulations of fipronil on the development of chicken embryos. For this purpose, solutions of pure fipronil dissolved in acetone and commercial fipronil diluted with distilled water (20 µl/egg) were injected into the air space of fertilized hen’s eggs at the beginning of the incubation. The eggs were opened on the day 15 of the incubation and the following parameters of each group were examined: Dead and abnormal embryo numbers, malformation types, both live and relative embryo weights and Crown-Rump Lengths (CRL). Mortality values of 31.25, 62.50, 125 and 250 µg/egg pure fipronil groups were 26.32%, 33.33%, 40.00% and 66.67% and mortality values of 50, 125, 250 and 500 µg/egg commercial fipronil groups were 25.00%, 31.58%, 44.40% and 61.11%, respectively. Regression analysis estimated the LD50 (the 50% lethal dose) for pure fipronil and commercial fipronil to be 161 µg/egg and 383 µg/egg, respectively. The differences in abnormal embryo rates were not statistically significant among the control and the fipronil groups (p>0.05). However, the live and relative embryo weights and embryo CRL values of the fipronil groups were significantly lower than that of the control group (p<0,001). Based on the results, it was concluded that both pure and commercial fipronil have significant embryotoxic effects but no teratogenic effects on chicken embryos.

Keywords

• Fipronil
• LD50
• chick embryo
• embryotoxicity
• teratogenicity

Organik İnsektisit Fipronilin Saf ve Ticari Formülasyonlarının Tavuk Yumurtası Testiyle LD50 Tayini ve Embriyotoksik Etkilerinin Belirlenmesi

Abstract

Organik bir insektisit olan fipronil, pestisitlerin fenil pirazoller veya fiproller olarak bilinen nispeten diğerlerine göre daha yeni ve küçük bir grubuna dahildir. Zararlı böceklere karşı son derece etkili olup, tüm dünyada kullanımı artmaktadır. Bu çalışmada fipronilin saf ve ticari formülasyonlarının tavuk embriyolarının gelişimi üzerindeki etkilerinin belirlenmesi amaçlandı. Bu amaçla asetonda çözdürülmüş saf fipronil ve steril bidistile saf suda sulandırılmış ticari fipronil solüsyonları (20 µl/yumurta) kuluçka başlangıcında döllü tavuk yumurtalarının hava boşluğuna enjekte edildi. Yumurtalar kuluçkanın 15. gününde açılarak grupların ölü ve anormal embriyo sayıları,malformasyon tipleri, canlı ve rölatif embriyo ağırlıkları ile embriyoların tepe-kıç boyları (Crown-Rump Length, CRL) belirlendi. 31.25, 62.50, 125 ve 250 µg/yumurta saf fipronil gruplarında mortalite sırasıyla %26.32, %33.33, %40.00 ve %66.67 olarak, 50, 125, 250 ve 500 µg/yumurta ticari fipronil gruplarında ise mortalite sırasıyla %25.00, %31.58, %44.40 ve %61.11 olarak gerçekleşti. Regresyon analizi yöntemiyle saf fipronilin LD50 (Letal Doz %50) değeri 161 µg/yumurta, ticari fipronilin LD50 değeri 383 µg/yumurta olarak tayin edildi. Saf ve ticari fipronil grupları ile kontrol grupları arasında anormal embriyo oranı bakımından farklar istatistiksel öneme sahip değildi (p>0.05). Bununla birlikte saf ve ticari fipronil gruplarında canlı ve rölatif embriyo ağırlıkları ile embriyoların CRL değerlerinin kontrol grubuna kıyasla önemli oranda azalma gösterdiği belirlendi (p

Keywords

• Fipronil
• LD50
• tavuk embriyosu
• embriyotoksisite
• teratojenite

References

1. 1. Tingle, C.C.D., Rother, J.A., Dewhurst, C.F., Lauer, S. and King, W.J. Fipronil: Environmental fate, ecotoxicology, and human health concerns. Rev. Environ. Contam. Toxicol. 176: 1-66 (2003)
2. 2. Tomlin, C.D.S. The pesticide manual 2000. British Crop Protection Council, Twelfth Edition (2000)
3. 3. Aydınoğlu, H., Dursun, H.Y. ve Bayraktar, L. Bitki Koruma Ürünleri. T.C. Tarım ve Köy İşleri Bakanlığı Koruma ve Kontrol Genel Müdürlüğü Yayınları, Ankara (2002)
4. 4. Ishaaya, I. Biochemical processes related to insecticide action: An overview. In: Biochemical Sites of Insecticide Action and Resistance. Ishaaya, I. (ed.) Springer-Verlag Berlin, pp. 1-16 (2001)
5. 5. Casida, J.E. and Quistad, G.B. Why insecticides are more toxic to insects than people: The unique toxicology of insects. J. Pestic. Sci. 29: 81-86 (2004)
6. 6. Hovda, L.R. and Hooser, S.B. Toxicology of newer pesticides for use in dogs and cats. Vet. Clin. Small Anim. 32: 455-467 (2002)
7. 7. USEPA. Fipronil pesticide fact sheet. EPA 737-F-96-005. U.S. Environmental Protection Agency. Washington, USA (1996)
8. 8. ACP. Evaluation of fipronil use as a public hygiene insecticide. Advisory Committee on Pesticides. Food and Environment Protection Act 1985, part III. Control of Pesticides Regulations 1986. No. 187. Issue No. 52. Pesticides Safety Directorate, MAFF, York, UK (1999)

9. 9. Hamon, N.M., Gamboa, H. and Garcia, J.E.M. Fipronil: A major advance for the control of boll weevil in Columbia. In: Herzog, G.A. Hardee, D.A. (chairs), Ottens, R.J., Ireland, C.S., Nelms, J.V. (eds.). Proceedings, Beltwide Cotton Conferences USA, Vol. 2, Jan. 9- 12 1996, Nashville, T.N. Cotton Insect Research and Control Conference. N.C.C., Memphis, TN, pp. 990-994 (1996)
10. 10. Hamburger, V. and Hamilton, H.L. A series of normal stages in the development of chick embryo. J. Morphol. 88: 49-92 (1951)
11. 11. Sümbüloğlu, K. ve Sümbüloğlu, V. Biyoistatistik. Hatipoğlu Yayınevi, 3. Baskı, Ankara (1990)
12. 12. Fischer, L.D. and Van Belle, G. Biostatistics: A Methodology for The Health Sciences. John Wiley&Sons, New York (1993)
13. 13. Conover, W.J. Practical Nonparametric Statistics. Second Edition, John Wiley&Sons, New York (1980)
14. 14. Jelinek, R. The chick embryotoxicity screening test (CHEST). In: Methods in prenatal toxicology. Neubert, D., Merker, H.J. and Kwasigroch, T.E. (eds.), Georg Thieme, Stutgart, pp. 381-386 (1977)
15. 15. Kemper, F.H. and Luepke, N.P. Toxicity testing by the hen’s egg test (HET). Fd. Chem. Toxic. 24 (6/7): 647-648 (1986)
16. 16. Nishigori, H., Mizumura, M. and Iwatsuru, M. The hen’s fertile egg screening test (HEST): A comparison between the acute toxicity for chick embryos and rodents of 20 drugs. Cell Biol. Toxicol. 8 (4): 255-265 (1992)
17. 17. Wolf, T. and Luepke, N.P. Formation of micronuclei in incubated hen’s eggs as a measure of genotoxicity. Mutat. Res.-Gen. Tox. En. 394: 163-175 (1997)
18. 18. Jelinek, R., Peterka, M. and Rychter, Z. Chick embryotoxicity ecreening test-130 substances tested. Indian J. Exp. Biol. 23: 588-595 (1985)
19. 19. Vesely, D. and Vesela, D. The use of chick embryo for prediction of some embryotoxic effects of mycotoxins in mammals. Vet. Med. Praha. 36 (3): 175-181 (1991)
20. 20. Özcan, M. Hidrokinon’un gelişim toksisitesinin döllenmiş tavuk embriyosunda analiz ve değerlendirilmesi. G. Ü. Sağlık Bilimleri Enstitüsü, Yüksek Lisans Tezi, Ankara (1992)
21. 21. Jelinek, R. and Marhan, O. Validation of chick embryotoxicity screening test (CHEST). A comparative study. Funct. Dev. Morphol. 4 (4): 317-323 (1994)
22. 22. Davies, W.J. and Freeman, S.J. Chick embryotoxicity screening test (CHEST I and II). In: Methods in Molecular Biology. In vitro toxicity testing protocols. O’Hare, S. and Atterwill, C.K. (eds.), Humana Press Inc., Totowa, NJ, 43: 307-310 (1995)
23. 23. Bryan, T.E., Gildersleeve, R.P. and Wiard, R.P. Exposure of Japanese quail embryos to o,p´-DDT has long-term effects on reproductive behaviors, hematology, and feather morphology. Teratology 39: 525-535 (1989)
24. 24. Seifert, J. Teratogenesis of polychlorocycloalkane insecticides in chicken embryos resulting from their interactions at the convulsant recognition sites of the GABA (Pro) Receptor Complex. Bull. Env. Cont. Toxicol. 42: 707-715 (1989)
25. 25. Powell, D.C., Aulerich, R.J., Meadows, J.C., Tillitt, D.E., Giesy, J.P., Stromberg, K.L. and Bursian, S.J. Effects of 3,3`4,4`,5-pentachlorobiphenly (PCB 126) and 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) injected into the yolks of chicken (Gallus domesticus) eggs prior to inncubation. Arch. Environ. Contam. Toxicol. 31 (3): 404- 409 (1996)
26. 26. Powell, D.C., Aulerich, R.J., Meadows, J.C., Tillitt, D.E., Stromborg, K.L., Kubiak, T.J., Giesy, J.P. and Bursian, S.J. Organochlorine contaminants in double-crested cormorants from Green Bay, Wisconsin: II. Effects of an extract derived from cormorant eggs on the chicken embryo. Arch. Environ. Contam. Toxicol. 32: 316-322 (1997)
27. 27. Paul, B.S. and Vadlamudi, V.P. Teratogenic studies of fenitrothion on white leghorn chick embryos. Bull. Env. Cont. Toxicol. 15 (2): 223-229 (1976)
28. 28. Schom, C.B. and Abbott, U.K. Temporal, morphological, and genetic responses of avian embryos to azodrin, an organophosphate insecticide. Teratology 15 (1): 81-87 (1977)
29. 29. Meiniel, R., Lavergne, J. and Autissier-Navarro, C. Teratogenic effect of dicrotophos on the embryonic chick tibia; histological and cytological studies. Toxicol. Eur. Res. 2 (3): 133-140 (1979)
30. 30. Rashev, Z. and Vasilev, V.K. Teratogenic effect of the pesticide preparation metathion. Vet. Med. Nauki 19 (1): 79-89 (1982)
31. 31. Misawa, M., Doull, J. and Uyeki, E.M. Teratogenic effects of cholinergic insecticides in chick embryos. III. Development of cartilage and bone. J. Toxic. Env. Heal. 10 (4-5): 551-563 (1982)
32. 32. Wyttenbach, C.R. and Hwang, J.D. Relationship between insecticide-induced short and wry neck and cervical defects visible histologically shortly after treatment of chick embryos. J. Exp. Zool. 229: 437-446 (1984)
33. 33. Varnagy, L. and Deli, E. Comparative teratological study of insecticide wofatox 50 EC (%50 methyl-parathion) on chicken and pheasant fetuses. Anat. Anz. 158: 1-3 (1985)
34. 34. Garrison, J.C. and Wyttenbach, C.R. Notochordal development as influenced by the insecticide dicrotophos (Bidrin). J. Exp. Zool. 234: 243-250 (1985)
35. 35. Rao, J.V., Swamy, A.N., Yamin, S., Rao, S.H. and Rahman, M.F. Teratism induced in the developing chick by RPR-V, an organophosphate. Fd. Chem. Toxic. 30 (11): 945-951 (1992)
36. 36. Kumar, K.B. and Devi, K.S. Teratogenic effects of methyl parathion in developing chick embryos. Vet. Hum. Toxicol. 34 (5): 408-410 (1992)
37. 37. Lenselink, D.R., Midtling, J.E. and Kolesari, G.L. Teratogenesis associated with oxydemeton-methyl in the stage 12 chick embryo. Teratology 48: 207-211 (1993)
38. 38. Varga, T., Cravedi, J-P., Füzesi, I. and Varnagy, L. Residues of fenitrothion in chick embryos following exposure of fertile eggs to this organophosphorus insecticide. Revue Med. Vet. 153: 275-278 (2002)
39. 39. Sahu, C.R. and Ghatak, S. Effects of dimecron on developing chick embryo: malformations and other histopathological changes. Anat. Histol. Embryol. 31: 15-20 (2002)
40. 40. Farage-Elawar, M. and Blaker, W.D. Chick embryo exposure to carbamates alters neurochemical parameters and behavior. J. Appl. Toxicol. 12 (6): 421-426 (1992)
41. 41. David, D. Influence of technical and commercial decamethrin, a new synthetic pyrethroid, on the gonadic germ population in quail embryos. Arch. d’Anat. d’Hist. d’Embryol. Norm. Exp. 65: 99-110 (1982)
42. 42. Dusek, Z., Holejsovska, I., Novotna, B. and Zemanova, Z. Embryotoxicity of 1,2- dibromoethane in chick embryos in ovo: early and late effects. Eur. J. Morphol. 39 (2): 105-112 (2001)
43. 43. Dusek, Z., Novotna, B., Vodickova, L., Naprstkova, I., Dostal, M. and Vilhelmova, M. Effects of 1,2-dibromoethane on haematopoiesis in the chick embryo. Xenobiotica 33 (4): 443-458 (2003)
44. 44. El-Sayyad, H.I., El-Gammal, S.A. and Kariem, S.A. Some aspects of growth deformities of chick embryos induced by flufenoxuron. J. Union. Arab. Biol. 5 (A): 313-329 (1996)
45. 45. Sur, E. Yumurtaya verilen Aflatoksin B1 (AFB1)’in tavukların lenfoid organlarının embriyonal gelişimi üzerindeki etkilerinin enzim histokimyasal yöntemlerle araştırılması. Doktora Tezi, S.Ü. Sağlık Bilimleri Enstitüsü, Konya (2001)
46. 46. Öznurlu, Y. Yumurtaya verilen Aflatoksin B1’in, etçi piliçlerin kemik dokularının embriyonik gelişimi üzerindeki etkilerinin belirlenmesi. Doktora Tezi, S.Ü. Sağlık Bilimleri Enstitüsü, Konya (2003)
47. 47. Prelusky, D.B., Hamilton, R.M.G., Foster, B.C., Trenholm, H.L. and Thompson, B.K. Optimization of chick embryotoxicity bioassay for testing toxicity potential of fungal metabolites. J. Assoc. Off Anal. Chem. 70 (6): 1049-1055 (1987)
48. 48. Hamilton, J.W., Denison, M.S. and Bloom, S.E. Development of basal and induced aryl hidrocarbon (benzo[α]pyrene) hydroxylase activity in the chicken embryo in ovo. Proc. Natl. Acad. Sci. USA 80: 3372-3376 (1983)
49. 49. Çelik, I., Oguz, H., Demet, O., Boydak, M., Dönmez, H.H., Sur, E. and Nizamlıoglu, F. Embryotoxicity assay of aflatoxin produced by Aspercillus parasiticus NRLL 2999. Br. Poult. Sci. 41 (4): 401-409 (2000)
50. 50. Mauldin, J.M. Quality control procedures for the hatchery. Poult. Sci. 93 (1): 1-24 (1993)
51. 51. Tuncer, A. Zaman aşımında ilaç aktivitesindeki değişikliklerin LD50 tayin yöntemiyle araştırılması. Yüksek Lisans Tezi, T.Ü. Sağlık Bilimleri Enstitüsü, Edirne (2002)
52. 52. Ameenuddin, S. and Sunde, M.L. Sensitivity of chick embryo to various solvents used in egg injection studies. Proc. Soc. Exp. Biol. Med. 175: 176-178 (1984)
53. 53. SBYKP. Kimya Sanayi Özel İhtisas Komisyonu Raporu, Tarım İlaçları Alt Komisyon Raporu. Sekizinci Beş Yıllık Kalkınma Planı. DPT: 2591-ÖİK: 603. Ankara (2001)
54. 54. Kucera, P. and Burnand, M.B. Routine teratogenicity test that uses chick embryos in vitro. Teratogen. Carcin. Mut. 7: 427-447 (1987)
55. 55. USEPA. Fipronil for use on rice (Regent, Icon) and pets (Frontline). HED risk assessment. United States Environmental Protection Agency. Washington, D.C., Office of Prevention, Pesticides and Toxic Substances (1998)