**8. Blood biochemical parameters**

Measurement of blood biochemical parameters are used as important diagnostic tool for the detection of abnormalities in the liver and other tissues (Banaee et al., 2011). Liver serves to protect the body from potentially harmful ingredients absorbed from the intestinal tract, gills, and skin as well as toxic by-products of metabolism by various mechanisms that previously described.

The significant differences in glucose concentrations in plasma between the control and treatment fish, following the action of different insecticides, which may be considered to be the manifestation of stress (Banaee et al., 2011). In agreement with our results, Ceron, et al., (1997) report significant glucose increase in common eel (*Anguilla anguilla*) following a 96 h action of sub-lethal concentrations of diazinon. Bhatia et al., (1972) and Weiss et al., (1984) reported a pronounced increase in blood sugar level which was going parallel to the inhibition of the cholinesterase and the appearance of manifestation of cholinergic stimulation as a result of parathion intoxication.

Glucose increase is a general response of fish to acute pollutant effects, including organophos‐ phates insecticides (Banaee, 2012). Plasma glucose was elevated in treated silver catfish *Rhamdia quelen* after all periods of clomazone exposure (Lazzari, et al., 2006).

Albumin and globulin make up most of the protein within the body and are measured in the total protein of the plasma. Total protein, albumin and globulin tests are used to monitor the 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 pollutants and insecticides (Vijayan et al., 1997; Velisek et al., 2009).

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)

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.,

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.

Measurement of blood biochemical parameters are used as important diagnostic tool for the detection of abnormalities in the liver and other tissues (Banaee et al., 2011). Liver serves to protect the body from potentially harmful ingredients absorbed from the intestinal tract, gills, and skin as well as toxic by-products of metabolism by various mechanisms that previously

The significant differences in glucose concentrations in plasma between the control and treatment fish, following the action of different insecticides, which may be considered to be the manifestation of stress (Banaee et al., 2011). In agreement with our results, Ceron, et al., (1997) report significant glucose increase in common eel (*Anguilla anguilla*) following a 96 h action of sub-lethal concentrations of diazinon. Bhatia et al., (1972) and Weiss et al., (1984) reported a pronounced increase in blood sugar level which was going parallel to the inhibition of the cholinesterase and the appearance of manifestation of cholinergic stimulation as a result

Glucose increase is a general response of fish to acute pollutant effects, including organophos‐ phates insecticides (Banaee, 2012). Plasma glucose was elevated in treated silver catfish

Albumin and globulin make up most of the protein within the body and are measured in the total protein of the plasma. Total protein, albumin and globulin tests are used to monitor the

*Rhamdia quelen* after all periods of clomazone exposure (Lazzari, et al., 2006).

can be an indicator of immune system deficiency.

112 Insecticides - Development of Safer and More Effective Technologies

**8. Blood biochemical parameters**

described.

of parathion intoxication.

(1990) in common carp after an acute effect of trichlorfon.

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

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 carp to diazinon.

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

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 14th and 28th day. However, elevated levels of this enzyme in plasma indicate a transient damage to either muscle fibers or other tissues. Previous results reported that continuous exposure to acute pesticide (bifenthrin) concentrations resulted in significantly increased levels of CK in common carp at 96 h (Velisek et al., 2008).

zyme activity, chemiluminuscence (CL) response and immunocompetent cells popula‐ tion size, IgM levels, value of white blood cells (WBC) and respiratory burst activity, head kidney phagocytes and peripheral blood leucocytes, etc., have been reported by scholars (Betoulle et al., 2000; Khoshbavar-Rostami et al., 2006; Banaee et al., 2008; Cues‐ ta et al., 2008; Girón-Pérez et al., 2009; Shelley et al., 2009; Ahmadi et al., 2011; Jin et al., 2011, Wang et al., 2011). The exposure to sub-lethal concentrations of insecticides is what probably makes fish vulnerable to infectious diseases because of their immune-de‐ pressiveeffects (Zelikoff et al., 2000). For example, the susceptibility of juvenile chinoock salmon (*O.tshawytscha*) to infectious hematopoietic necrosis virus was significantly in‐ creased in fish exposed to sub-lethal concentrations of esfenvalerate (Clifford et al., 2005). Similar results were reported in goldfish and common carp that were exposed to carbaryl and lindane respectively (Shea, 1983; Shea & Berry, 1984; Cossarini-dunier&Hat‐

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115

Lysozymes are a family of enzymes with antibacterial activity characterized by the ability to damage the cell wall of bacteria. Ahmadi et al. (2011) found that exposure to diazinon induced a dose-dependent decrease in rainbow trout plasma lysozyme activity. Peroxidases are a large family of enzymes which play important role as natural antibacterial agent in animal immune system, e.g., myeloperoxidase (Clark and Klebanoff 1975). Ahmadi et al. (2011) indicated that

Globulin is made up of subunit of α1, α2, β, and γ globulins, which are considered as the source of almost all the immunologically active proteins in the blood (Jha et al., 2007; Ahmadi et al., 2012).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). Significant decrease in globulins levels in plasma of fish exposed to diazinon could be due to a disruption in protein biosynthesis (Ahmadi et al., 2011). In the other word, diazinoninduced tissue destruction and hepatocyte apoptosis might be the most important agent responsible of reducing the synthesis of total protein, albumin, and immunoglobulin by the

Complement includes over 20 different plasma proteins that are produced by a variety of cells including, hepatocytes, macrophages, and gut epithelial cells. Some complement proteins bind to immunoglobulins or to membrane components of cells. The complement system is an essential and effective part of the innate immune system. It can rapidly distinguish and opsonize bacteria for phagocytosis by specialized phagocytes or destroy them directly by membrane disorder (Rooijakkers and van Strijp 2007; Ahmadi et al., 2012). Ahmadi et al. (2011) believed that significant depression of complement levels clearly indicated the comprehensive effects of diazinon on immune system of rainbow trout. Thus, insecticides may alter the function of the immune system and result in immune-depression, uncontrolled cell proliferation, and alterations of the host defense mechanisms including innate immunity and

diazinon treatment of rainbow trout significantly affected the peroxidase activity.

tenberger, 1988).

liver (Banaee et al., 2011)

acquire immunity against pathogens.

Creatinine is a breakdown product of CK in muscle. The creatinine test has been usually used to diagnose impaired kidney function and to detect renal damage (Toffaletti and McDonnell, 2008; Banaee et al., 2011) However, the lack of changes in creatinine levels are in accordance with the previous results obtained by Gilbert et al (1989) and Banaee et al. (2011) which reported that plasma creatinine was usually not an accurate biomarker to distinguish dysfunction in kidney tissue. The creatinine test has been usually used to diagnose impaired kidney function and to detect renal damage (Toffaletti and McDonnell, 2008).

The alkaline phosphatase (ALP) plays a significant role in phosphate hydrolysis and in membrane transport as well as is a good bio-indicator of stress in biological systems. The importance of measuring alkaline phosphatase is to check the liver dysfunction (Banaee et al., 2011) and the cellular membrane health. Alkaline phosphatase (ALP) is an enzyme found in different body tissues; in liver, is produced by the cells lining the small bile ducts (Agrahari et al., 2007). Increased activity of ALP in blood plasma on 14th day may be related to hepatic tissue damage and dysfunction due to pesticide toxicity; the elevation in ALP activity on may be due to an increase in transphosphorylation activity (Sharma, 1990). Agrahari et al. (2007) found increased levels of ALP in plasma of *Channa punctatus* exposed to monocrotophos. These changes in ALP activities in plasma were earlier reported in tilapia (Rao, 2006) and in common carp (Banaee et al., 2008). The return of ALP activity to basal values could indicate a fish adaptation to the diazinon treatment at sub-lethal concentrations.

Acid phosphatase plays an important role in carbohydrate metabolism. This enzyme can be found inside the membrane of lysosomes. So, any damage to the membrane of lysosomes can cause the release of this enzyme into muscle and increase its levels.
