**6. Oxidative stress**

During the detoxification process, ROS are produced (Üner et al., 2006; Isik and Celik, 2008) and they can indiscriminately attack and damage cellular macromolecules -lipids, proteins and DNA- in living cells resulting in serious disturbances in physiological cell processes (Sureda et al., 2006; Tejada et al., 2007). Li et al. (2010c, d; 2011b) believed that cellular antioxidant responses could be used as potential biomarkers for monitoring residual xenobiotic present in aquatic environment. For example, Salvo et al. (2012) found that endosulfan at the sub-lethal concentration in subchronic exposure caused significant changes in liver somatic indices as well as induction of the phase I biotransformation system and oxidative stress in juvenile common carp (*Cyprinus carpio*). Similar results was observed in gar (*Atractosteus tropicus*), Tilapia (*Oreochromis niloticus*), tropical reef fish (*Acanthochromis polyacanthus*) exposed to ethorophos (Mena Torres et al., 2012), lamba-cyhalothrin (Piner and Uner, 2012), Chlorpyrifos, respectively (Botte et al., 2011; Xing et al., 2011; Oruc, 2012).

**5. Physiological dysfunction in various biological systems of fish by**

exposed to insecticides were the main symptoms evidenced in the toxicology studies.

Özkul, 2010), dimethoate (Auta et al., 2002), respectively.

**6. Oxidative stress**

Behavioral alterations and the change of body's color pattern of fish or darkness of skin and mucosa increase to the skin and gill surface, as well as bleeding around the eyeball and the base of pectoral fins and also, the volume increase of the liver and the gall bladder in fish

Behavioral changes are the most sensitive indicators of potential toxic effects. Most insecticides affect the behavioral patterns of fish by interfering with the nervous systems and sensory receptors and consequently it can lead to disorders in the fish response to environmental stimuli. The effect of certain insecticides on the activity of acetylcholinestrase may also lead to a decreased mobility in fish (Banaee, 2012). Several studies have demonstrated that insecticides are metabolized in liver to toxic derivate via cytochrome P450 mono oxygenases (Fujii and Asaka, 1982; Hamm et al., 2001; Schlenk, 2005) and finally, these metabolites were hydrolyzed in microsomes (Keizer et al., 1993; Keizer et al., 1995) and excreted from the body. Nevertheless, rainbow trout was very sensitive to organophosphate insecticides toxicity due to a lack of esterase activity and a very sensitive acetylcholinesterase activity to OPs inhibition (Keizer et al., 1995). The phosphorus group of organophosphate insecticides attacks the hydroxyl group of the serine amino acid at the active site of acetylcholinesterase inhibiting the enzyme (Üner et al., 2006; Banaee et al., 2011). Inhibition of AChE in fish was accompanied by an increase in acetylcholine levels (Üner et al., 2006; Banaee, 2012) that can be dangerous since it will impact feeding capability, swimming activity, identification, and spatial orientation of the species (Banaee et al., 2008; Banaee et al., 2011). Thus, AChE inhibition is considered to be a specific biomarker of exposure to organophosphorus and carbamate insecticides like diazinon, chlorpyrifos, propoxur, isoprocarb, (Üner et al., 2006; Cong et al., 2008; 2009; Wang et al., 2009; Banaee et al., 2011;). Similar results have been observed for pyrethroids insecticide toxicity (Koprucu et al., 2006). Disorder in γ-aminobutyrate (GABA) system in brain of rainbow trout exposed to sub-lethal lindane was reported by Aldegunde et al., (1999). GABA receptors inhibit the transmission of nerve impulses; thus disturbances in this receptor would also lead to an over stimulation of the nerves. Researchers have reported the same alterations in *Oryzias latipes*, *Cyprinus carpio*, *Labeo rohita*, *Oncorhynchus tshawytscha*, *O.latipes*, *Cirrhinus mrigala*, *Oreochromis niloticus*, *Clarias gariepinus* treated with chlorpyrifos (Rice et al., 1997; Halappa & David, 2009), malathion (Patil & David, 2008), diazinon (Scholz et al., 2000), endosulfan (Gormley & Teather, 2003), Fenvalerate (Mushigeri & David, 2005), fenitrothion (Benli &

During the detoxification process, ROS are produced (Üner et al., 2006; Isik and Celik, 2008) and they can indiscriminately attack and damage cellular macromolecules -lipids, proteins

**insecticides**

**5.1. Behavioral response**

108 Insecticides - Development of Safer and More Effective Technologies

The antioxidant enzymes that provide the first line of cellular defense to ROS include superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR), glutathione *S-*transferease (GST) and xanthine oxidase (XOD), etc. However, an imbalance between the activities of cellular antioxidant enzymes and ROS production results in oxidative stress and cellular damage. If the antioxidant system is not able to eliminate or neutralize the excess of ROS, there is an increased risk of oxidative damage (Üneret al., 2006; Oruç and Usta, 2007; Isik and Celik, 2008). It is well established that waterborne pollutants induces oxidative stress and cellular damage in affected aquatic organisms (Sureda et al., 2006; Box et al., 2007).

GR plays a vital role in recycling oxidized glutathione (GSSG) to reduced glutathione (GSH) (Jos et al., 2005; Box et al., 2007; Sureda et al., 2009). GR plays an important role in diazinon detoxification because diazinon can be directly conjugated with GSH facilitating the excretion from the animal body (Banaee et al., 2012). GSH also participates in neutralizing free radicals (Jos et al., 2005; Sureda et al., 2009). This GSH consumption leads to an increase in GR activity in order to recycle GSH. The increase in GR activity observed after seven days of exposure to sub-lethal concentrations of diazinon was followed by a declining trend, which is clearly manifested after 28 days of diazinon contact (Banaee et al., 2012). These results agree with a previous study carried out on fishes that had been exposed to environmental pollutants (Franco et al., 2008). Banaee et al., (2012) found that the decreased activity of GR at the 28th day after an initial antioxidant response may be indicative of a disorder in cell metabolism. GR activity is severely dependent on cellular NADPH levels (Peña-Llopis et al., 2003). It has been reported that the contact with pesticides decreased the synthesis and accelerated the breakdown of GR mediated by a disorder in NADPH synthesis and decreased activity of glucose-6-phosphate dehydrogenase (G6PDH) enzyme (Ozmen et al., 2004; Li et al., 2010c).

Since an increase of GPx activity is necessary to eliminate the excess of H2O2 and lipid hydroperoxides produced in hepatocytes of fishes exposed to. The increased activity of GPx accelerates the utilization of GSHto GSSG. This increased GSSG, indicative of a more oxidized state, may explain the decreased levels of total antioxidant capacity in liver cells of fish after exposure to pesticide. A decrease in GPx activity to basal values is probably related to decreasing cellular GSH levels on the days 14 and 28, although it cannot be discarded a direct effect of diazinon on the biosynthesis of the enzyme. Similar alterations in GPx activity were observed in different tissues of *C. carpio* exposed to diazinon (Oruç and Usta, 2007). Decreased GPx activity in gills, muscle, liver and brain of treated fishes with parathion were also reported, by Monterio et al. (2006).

found in liver, intestine and little amount in other tissues of animals (Sathyanarayana, 2005) also stated XOD played a vital role in transformation of toxic ammonia into nontoxic uric acid. Xanthine oxidase produces hydrogen peroxide which is very dangerous to the animal, and then it converts into HO and O2. Further, the uric acid may act as an antioxidant and free radical scavenger protects the cells from oxidative damage (Sheehan et al., 2001; Guskovet al., 2002). Naveed and Janaiah (2011) reported that the reduction in XOD activity in liver of fish, *Channa punctatus* exposed to triazophos leads to increase in cellular damage and may be due to non-

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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

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

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

availability of Iron to the fish during toxic period.

al., 2008) after acute and sub-lethal exposure to diazinon.

and MCHC in *Cyprinus carpio* was reported (Svoboda et al., 2001).

**7. Hematological parameters**

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 separately.

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

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 number of hydrophobic compounds, such as insecticides.

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 interchange (Bernstein et al., 1982).

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 found in liver, intestine and little amount in other tissues of animals (Sathyanarayana, 2005) also stated XOD played a vital role in transformation of toxic ammonia into nontoxic uric acid. Xanthine oxidase produces hydrogen peroxide which is very dangerous to the animal, and then it converts into HO and O2. Further, the uric acid may act as an antioxidant and free radical scavenger protects the cells from oxidative damage (Sheehan et al., 2001; Guskovet al., 2002). Naveed and Janaiah (2011) reported that the reduction in XOD activity in liver of fish, *Channa punctatus* exposed to triazophos leads to increase in cellular damage and may be due to nonavailability of Iron to the fish during toxic period.
