**7. Hematological parameters**

GPx activity in gills, muscle, liver and brain of treated fishes with parathion were also reported,

The SOD enzymes are enzymes that catalyse the dismutation of superoxide into hydrogen peroxide and oxygen whereas CAT catalyzes the decomposition of hydrogen peroxide to water and oxygen. The increased SOD and CAT activities in hepatocytes of fishes exposed to diazinonmight be biochemical responses to over production of superoxide radicals and H2O2in hepatocytes, respectively (Banaee et al., 2012). It has been shown that the CAT activity may be related to H2O2 production in a xenobiotic detoxification process (Achuba and Osakwe, 2003; Monterio et al., 2006). A previous study by Monterio et al., (2006) reported similar changes in the hepatic CAT activity of freshwater fish, *Brycon cephalus* exposed to methy parathion. Following 2-cholrophenol exposure, alterations in SOD and CAT activities in *Carassius auratus* were reported (Luo et al., 2006). Hai et al, (1997), and Box et al, (2007) showed that organophosphate pesticide and exposure to environmental pollutants caused a significant reduction in CAT activities in different tissues of *Ictalurus nebulosus* and *Mytilu sgalloprovin‐ cialis*, respectively. Whereas, Isik and Celik, (2008) reported in rainbow trout exposed to diazinon and methyl parathion a decrease in SOD activities in liver, gills and muscle tissues

Banaee et al., (2012) found that the levels of total antioxidant capacity in hepatocytes of fishes exposed to both concentrations of diazinon were significantly decreased. Similar results were observed in carps exposed to sub-lethal concentrations of cyfluthrin (Sepici-Dinçel et al., 2009). The overproduction of free radicals during pesticide detoxification may be associated with the decrease in the hepatic total antioxidant capacity (Monterio et al., 2006). Impairment in the synthesis of enzymatic and non-enzymatic antioxidant may be the most important factor in reducing levels of cellular total antioxidant. Therefore, the decline in the hepatic total

Glutathione S-transferases (GSTs), a family of cytosolic multifunctional enzymes, are detoxifying enzymes that are present in different tissues of fish. They catalyze the conjugation of glutathione with a variety of reactive electrophilic compounds, thereby neutralizing their active electrophilic sites and subsequently making the parent compound more water soluble. For example, the toxicity of diazinon can be decreased by the action of carboxylesterase enzyme which catalyses the hydrolytic degeneration of diazinon and by the action of glutathione *S*transferase which catalyses the formation of excrete-able conjugate (Keizer et al., 1995). In addition to catalytic functions, the GSTs can also bind covalently/non-covalently to a wide

Thioltransferase catalyzes the reversible thiol-disulfide interchange reactions. The enzyme has a major role in maintaining intracellular thiol in the reduced state and functions in this capacity by coupling to glutathione and glutathione reductase. Thioltransferase also has a role in the cellular regulation by catalyzing the reversible modification of proteins by thiol-disulfide

Xanthine oxidase (XOD) is an essential enzyme that converts hypoxanthine to xanthine, subsequent to uric acid. This enzyme contains FAD, molybdenum and Iron are exclusively

antioxidant levels make the fish cells more vulnerable to oxidative stress damage.

number of hydrophobic compounds, such as insecticides.

interchange (Bernstein et al., 1982).

by Monterio et al. (2006).

110 Insecticides - Development of Safer and More Effective Technologies

separately.

The main hematological parameters in fish including red blood cell counts (RBC), hematocrit (Ht), hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) may be influenced by in‐ trinsic and externalfactors. Toxicology studies show that the disruptive action of different in‐ secticides on the erythropoietic tissue such as kidney and spleen may decrease erythrocyte number and hemoglobin content as an anemic sign, and even lead to death of fish. A low red cell or hemoglobin count indicates anemia, or severe bleeding. Low hemoglobin usually means the animal has anemia. Anemia results from conditions that decrease the number or size of red cells, such as excessive bleeding, a dietary deficiency, destruction of cells because of a transfusion reaction or mechanical heart valve, or abnormality formed hemoglobin (Hisa and Connie, 1998). Decreases in the number or size of red cells also decrease the amount of space they occupy, resulting in a lower hematocrit. A low hematocit, combined with abnormal blood tests, confirms the diagnosis. Decreased erythrocyte count and haemoglobin content in fresh‐ water fish *Channa punctatus*, (Anees, 1978) and *Cyprinus carpio* (Svoboda,et al., 2001; Banaee et al., 2008) after acute and sub-lethal exposure to diazinon.

Another type of hematological response to the effect of organophosphrous compounds was a significant increment of mean corpuscular volume (MCV) associated with increase of hematocrit value and drop of MCHC. MCV is the index most often used. It measures the average volume of red blood cell by dividing the hematocrit by RBC. The MCV categorizes red blood cells by size. Under a microscope, stained red blood cells with a high MCV appear larger than cells with a normal or low MCV. Mean corpuscular hemoglobin (MCH) measures the average amount of hemoglobin within a red cell. A similar measurement, mean corpuscular hemoglobin concentration (MCHC), expresses the average concentration of hemoglobin in the red blood cells. In contrary, values of MCV, MCH and MCHC registered in during exposure to diazinon based pesticide in 60 and 120 µg/L concentrations to common carp were comparable with the control group (Banaee et al., 2008). Alteration in values of MCV, MCH and MCHC in *Cyprinus carpio* was reported (Svoboda et al., 2001).

The white blood cell (WBC) count determines the total number of white cells (leukocytes) in bloodsample. Fewer in number than the red cells, WBC are the body's primary means of fighting infection. There are five main types of white cells (lymphocytes, monocytes, neutrophil, eosinophiland basophiles), each of which plays a different role in responding to presence of foreign organisms in the body. The number of white blood cells may increase or decrease significantly in certain diseases. Banaee et al., (2008) observed significant decrease of leukocyte count of common carp in during exposure to sub-lethal concentration of diazinon. A low white blood cell count may mean dysfunction in hematological tissues (spleen and kidney) or certain infectious diseases. Lower than normal levels of lymphocytes (lymphopenia) can be an indicator of immune system deficiency.

course of diseases in immune disorders, liver dysfunction and impaired kidney activity (Banaee et al., 2011). According to the test results, levels of total protein, albumin and globulin were decreased in fish exposed to diazinon. Decreased total protein levels may be due to starvation, malnutrition and chronic liver diseases (Banaee et al., 2011). Other authors also found that the levels of total protein and albumin are decreased in the fish exposed to different

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Decreased globulin levels have been reported in immune deficiency. In fact, the effect of diazinon on the immune system of tilapia (*Oreochromis niloticus*) and beluga sturgeon (*Huso huso*) as immunomodulator has been studied by other authors (Khoshbavar-Rostami et al., 2006; Gokcimen et al., 2007). Diazinon-induced tissue destruction and hepatocyte apoptosis might be the most important agent responsible of reducing the synthesis of total protein,

Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are found in the liver, heart, skeletal muscle, kidney, pancreas, spleen, erythrocyte, brain and gills (Banaee et al., 2011). When diseases or injuries affect these tissues, the cells are destroyed and these enzymes are released into plasma. Keizer et al (1995) showed that diazinon in fish liver can be metabolized to diazoxon and other metabolites including pyrimidinol by cytochrome P450 monoxygenase and during this process, reactive oxygen species (ROS) are generated. The increase in intracellular levels of ROS may lead to lipid peroxidation resulting in an increased permeability of liver cell membrane. As a result, liver enzymes including AST and ALT are released into plasma. Similarly, ROS produced from the metabolism of diazinon may damage other tissues such as gills, muscle, heart, kidney and spleen causing the leakage of enzymes into plasma. In this sense, if the cellular injury is chronic AST and ALT levels will remain elevated (Banaee et al., 2011). Increased activities of AST and ALT were observed in plasma of *Channa punctatus*(Agrahari et al., 2007) exposed to organophosphorus insecticides. Banaee et al., (2008) have reported increased levels of AST and ALT followed by the exposure of common

Lactate dehydrogenase (LDH) is an enzyme found in almost all body tissues, such as heart, kidneys, liver, skeletal muscle, brain, erythrocyte and gills (Banaee et al., 2011). LDH measurement is used to detect tissue disorders and as an aid in the diagnosis of tissue damage (Banaee et al., 2011). Although a significant decrease in LDH content in plasma was observed in fish exposed to diazinon on 7th day, LDH activity was not significantly reduced on 14th and 28th days. Our data on 7 days after initial experiment are in accordance with several reports that revealed decreased LDH activity in tissues under various pesticide toxicity conditions in different fishes such as *O. mossambicus* (Saha and Kaviraj, 2009) and *C. punctatus* (Agrahari et al., 2007). Hernández et al. (2006) reported some insecticides, such as organophosphates are able to cause inhibition of LDH activity. However, the recovery evidenced on 14th and 28th days in LDH activity could indicate that diazinon concentration used in the present study was

Creatine kinase (CK) is an enzyme found in muscle, heart, gills and brain (Banaee et al., 2011). Although, fish exposed to higher dose of diazinon exhibited higher CK activities in plasma on 7th day, plasma CK concentrations resumed to its normal levels or even a reduced activity on

pollutants and insecticides (Vijayan et al., 1997; Velisek et al., 2009).

albumin, and immunoglobulin by the liver (Gokcimen et al., 2007).

not enough to produce chronic LDH alterations (Banaee et al., 2011).

carp to diazinon.

Poisonous substances treatments can also deplete the body's supply of lymphocytes, as can exposure to diazinon. Lymphopenia as a consequence of methy-parathion based pesticide was reported by Nath and Banerjee (1996) in *Heteropneustes fossilis* and also by Siwicki et al., (1990) in common carp after an acute effect of trichlorfon.

Decreased in lymphocyte and monocyte percentage in smear were showed in *Cyprinus carpio* (Svoboda et al., 2001). In contrary, Banaee et al., (2008) observed significant increment of neutrophil percentage in smear of common carp in during exposure to 60 and 120 µg/L concentration of diazinon. The most common and important cause of neutrophilia is infection, and most infections cause neutrophilia. The degree of elevation often indicates the severity of the infection. Tissue damage from other causes raises the neutrophile for similar reasons. Poisonings, and severe disease, like kidney failure all cause neutrophilia (Holland et al., 1997). Ghosh and Banerjee (1993) reported lymphopenia and increased in both neutrophil and eosinophil in *Heteropneustes fossilis*, after an effect of dimethoate in 96h LC50 concentration.
