Toxicity and Immunosuppressive Effects of Diazinon in Grass Carp, Ctenopharyngodon Idella (Cuvier and Valenciennes)

Grass carp is one of the valuable warm water fish species that is currently being cultured in polyculture system in Iran. Despite of large scale grass carp farming in the East Asian countries, only minimum data is available concerning the fish immune system and the effect of organophosphate chemicals on the fish immune response. Diazinon is one of the major organophosphate pesticides currently used in Northern and Southern part of Iran. Unfortunately, these areas are also the main regions for grass carp culture and there are regular reports of the disease outbreaks particularly in the provinces of Gilan and Khozestan. In addition, previous studies has indicated that Aeromonad septicemias was one of main factor in the high mortality occurrences in the grass carp, in particular whenever the fish immune system seems to be suppressed by some toxicants. The specific objectives of this study were: (i) determination of 96-h LC5() diazinon in grass carp; (ii) purification and partial characterization of grass carp IgM; (iii) assessment of some humoral and cellular immunoresponses of non- immunized and immunized grass carp, following exposure to diazinon. In this study, LC50 of diazinon at 96 hour in grass carp was determined to be 15.13 mg/L. The examination of hematological and tissue enzyme parameters indicated that diazinon at sublethal levels had caused an effect similar to anemia. In addition, a significant decrease of lymphocytes values and significant increase of PMNs values were observed. There were also significant and insignificant changes in some blood parameters such as monocytes and myelocytes counts, MCH, MCHC, AST, ALP, ALT and LDH values at different days of post exposure of diazinon. Such fluctuations indicated that fish hematopoietic tissues were in stress and were in constant struggles to maintain normal condition. The pathological effects of diazinon on the liver, kidney, spleen, gills, and nostrils of grass carp examined under light and electron microscope, showed that diazinon caused severe damage to the cell structure such as congestion of blood vessels, haemorrhage, cellular infiltration, pyknosis of cells nuclei, vacuolar degeneration and general necrosis in the tissues of kidney, spleen and liver. There were also degenerative changes of interstitial tissue, detachment of tubular basement membrane in kidney. In the gills, hyperplasia and fusion of secondary lamellae, separation and sloughing of epithelium from the underlying basement membrane were also observed. In the lysozyme study it was indicated that grass carp reacted to diazinon by raising the level of lysozyme in tissues of spleen and kidney and also in serum of Aeromonas-immunized fish exposed to diazinon and control positive (immunized only), as compared to control negative (non-immunized and not exposed to diazinon). However, the level of lysozyme in immunized fish exposed to diazinon is lower than control positive that indicated the depressive effect of toxicant on fish immune system. Analysis on data of leucocytes chemiluminescent response indicated that cellular factors of fish immunity following immunization with A. hydrophila and also under influence of diazinon, responded by changing its functional activities, as evidenced by a high chemiluminescent response in both of immunized fish exposed to diazinon and control positive groups as compared to the control negative group. However, the level of chemiluminescent response in immunized and exposed group was insignificantly lower than control positive group that indicated the negative effect of diazinon on fish immune system. In summary, an of the above findings proved the immunosuppressive effect of diazinon on nonspecific immune system of grass carp. The level of IgM in serum of normal grass carp was found to range from 3 to 4 mglml. Estimation of molecular weight of grass carp IgM was performed using three different methods. Affinity chromatography method gave the approximate values of about 480 and 640 KDa in SDS-PAGE, while gel chromatography and ion-exchange chromatography methods showed an identical molecular weight with an approximate value of 490 KDa. In antibody study, the titers of immunized fish were significantly higher than immunized fish exposed to diazinon. In addition, a strong positive correlation was also demonstrated between the results of ELlSA and agglutination titers. These observed results confirmed the immunosuppressive effect of diazinon on specific immune system of grass carp.

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