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Abstract Rainbow trout (Onchorhyncus mykiss) and Nile tilapia (Oreochromis niloticus) were exposed to low levels of copper sulfate (CUS04), (0.01, 0.1, 0.5 and 1.0 ppm), malathion (ML) (0.0001, 0.001, 0.01 and 0.1ppm) and paraquat (PO) (0.0001, 0.001, 0.01 and 0.1 ppm) for 96 hours. The comet assay was used in rainbow trout models, along with humoral immune response, histopathology and blood enzymes to determine the toxic effects of these pesticides. Histopathology and blood enzymes only used to determine the toxic effects of these pesticides on the two freshwater fishes, Onchorhyncus mykiss and Oreochromis niloticus. Examination of liver and gill tissues of Onchorhyncus mykiss following CUS04’ ML and PO treatment revealed necrosis, vacuolation of hepatocytes, expanding hepatic sinusoids, hyperplasia and fusion of gill lamellae, fibrosis and separation of epithelial layers. In liver and gill tissues, these symptoms increase in severity with higher pesticide doses.Liver of Oreochromis niloticus exposed to 0.01, 0.1, 0.5 and 1.0 ppm of CUS04 for 96 hours showed pyknotic cells going through necrosis that causes damage in many areas in the organ. By increasing the exposure, fish liver revealed hemorrhaging as infiltration of erythrocytes, expanding of the hepatic sinusoids, vacuoles as lipidosis, where most of hepatocytes turned to fat cells. At the highest exposure CUS04’ liver showed extensive expanding of hepatic sinusoids and formation of aneurium as a cluster of lymphocytes underneath the liver capsule. Also, melano-macrophage cells appeared in the fish exposed to 1.0 ppm CUS04. Liver of Oreochromis niloticus showed the same changes when exposed to increased concentrations ML and PQ.Gills of Oreochromis niloticus exposed to increasedconcentrations of CUS04 revealed hyperplasia of interlamellar epithelium that results in total fusion of secondary lamellae, hemorrhage and infiltration of erythrocytes, necrosis and destruction of secondary lamellae and infiltration with macrophages. The same symptoms were seen in gill when exposed to ML. With ML exposure the hyperplased layer form aneuria at the free end of the secondary lamellae. All aneuria consists of aggregation or cluster of lymphocytes. This layer of gill also infiltered with macrophages and erythrocytes. Also, necrosis and vacuolation can be seen. With PO exposure fish gills revealed the same changes as in CUS04 and ML exposure. Additionally, the hyperplased interlamellar epithelia form pulps at the free ends. Those pulps look like bridges for the secondary lamellae. Also, lifting of interlamellar epithelia can be seen in the organ.Onchorhyncus mykiss exposed to CUS04 displayed hyperplasia of hepatocytes occurred with an increase in cell numbers from 42 cells I 400x field for the control to 58 cells I 400x field at 0.5 ppm. Fish exposed to ML displayed increased hepatocytes number from 42 cell I 400x field for the control to 51 cell I 400x field at 0.1 ppm. While fish exposed to PO . showed hyperplasia of hepatocytes occurred with an increase in cell numbers (from 42 cell I 400x field for the control to 65 cellI 400x field at 0.1 ppm PO).On the contrary, Oreochromis niloticus exposed to CUS04displayed hypoplasia of hepatocytes occurred with a decreasein cell numbers from 322 cells I 400x field for the control to 128 cells I 400x field at 0.5 ppm. Fish exposed to ML displayed decreased hepatocytes number from 322 cellI 400x field for the control to 97 cellI 400x field at 0.001 ppm. While fish exposed to PO showed decrease in cell number from 322 cellI 400x field for the control to 81 cell/400x field at 0.01 ppm PO. The connective tissue, represented in tunica adventetia of the hepatic portal vein increased, in average width from 2 urn in control to 6.7 urn following CUS04 exposure at 1.0 ppm. While it ranged from 2.1 urn in control to 6.3 urn at 0.01 ppm with ML exposure and from 2.0 urn in control to 3.3 urn at 0.01 ppm with PO exposure.Lactate dehydrogenase (LDH), aspartate aminotransferase (AST) or GOT, alanine aminotransferase (ALT) or GPT and alkaline phosphatase (AlP) from Onchorhyncus mykiss blood samples were also monitored as indicators of tissue damage. LDH levels increased from 9.0 mg/L in controls to 43.15 mg/L in fish exposed to 1.0 ppm CUS04. AST and ALT levels showed suppressed values (from 1193 Ilg/L in controls to 840 J..lQAST/L at 1.0 ppm and from 70 J..l9/Lin controls to 64.5 J..lgALTIL at 0.1 ppm CUS04)’ Both ALT and AST were elevated in fish exposed to 0.5 ppm. This is the first study that demonstrates low levels of CUS04 are toxic to rainbow trout. LDH and AST levels in trout showed suppressed values at the lower concentration of ML (from 8.95 mg/L for control to 5.2 mg LDH/L at 0.01 ppm and from 1.1 mg/L in contro~ fish to 0.4 mg AST/L at 0.01 ppm) then, sharply lncreased at 0.1 ppm (from 8.95 mg/L for control to 25.7 mg LDH/LI at 0.1 ppm and from 1.1 mg/L in control fish to 2.4 mg AST/LI at 0.1 ppm). ALT levels decreased from 70 ~g/L in contro fish to 34.5 ~m/L at 0.1 ppm. LDH levels increased from 8.951g/L in controls to 26.16 mg/L in fish exposed to 0.1 ppm PO. A T and ALT levels showed suppressed values (from 1193 ~g/L i controls to 661 & 864 ~g AST/L at 0.001 & 0.1 ppm and ppm).n Oreochromis niloticus exposed to CUS04. LDH, AST, from 0 ~g/L in controls to 61 & 46 ~g ALTIL at 0.001 & 0.1 ALT nd AlP were detected in fish serum after 96 hours expos reo Fish serum showed proportional increased levels of ! LDH, !from 245 ~g/L in controls to 333 ~g/L at 1.0 ppm of CUSd4’ Serum AST of treated fish exhibited a slight decrease to 66 ~gVL at 0.01 ppm but its level raised up to 220 ~g/L at 1.0 ppm ! of CUS04. Serum ALT levels appeared inversely irelati1nshiP with increased concentration of CUS04. Levels of serum AlP of treated fish showed lower values from 22 ~g/L in controls to 5.0 !l9/L at 1.0 ppm. The AlP levels in treated fish increased proportionally with CUS04 concentrations from 15 !l9/L in controls to 21 !l9/L at 0.5 ppm.With PO exposure, LDH levels showed increased valuesin treated fish from 245 !l9/L in controls to 317 Ilg/L at 0.1 ppm.Fish serum that exposed to PO revealed increased levels ofAST versus control. AST levels increased from 150 !l9/L in controls to 200 !lg AST/L at 0.1 ppm PO. Serum ALT of treated fish showed no big change. Serum AlP increased from 15 !l9/L in controls to 24 Ilg/L at 0.1 ppm when fish exposed to increased concentration of PO.Fish serum exposed to increased four concentrations ofML, showed proportional increasing of LDH values from 245Ilg/L in controls to 309 Ilg/L at 0.1 ppm. AST revealed decreased levels down to half amount in control, at 0.001 ppm of ML. By increasing the concentrations of ML to 0.1 ppm, ALT levels showed higher values (190 !l9/L). Serum ALT levels revealed increased levels from 22 !l9/L in controls to 44 Ilg/L at 0.1 ppm of ML. In treated fish serum, AlP levels showed small increase from 15 Ilg/L in controls to 19 !l9/L at 1.0 ppm of ML. The comet assay, a sensitive technique for evaluating DNA damage in individual cells, showed that CUS04, ML and PO are genotoxic (inflicts DNA damage) at these low levels. Liver, gill and blood cells displayed increased DNA damage as a tail moment with increased concentrations of CUS04, ML and PO. With exposure of CUS04’ DNA damage recorded increase amount from 2.0 in control to 10.6 (tail moment) at 1.0 ppm in gill and from 1.7 to 7.0 in liver cells and from 2.4 to 15.5 in blood. When fish exposed to increased concentrations of ML, DNA damage recorded increase amount from 2.0 in control to 6.4 (tail moment) at 0.1 ppm in gill and from 1.7 to 4.1 in liver cells and from 2.4 to 5.9 in blood. With exposure of PO, DNA damage recorded increase amount from 2.0 in control to 8.7 (tail moment) at 0.1 ppm in gill and from 1.7 to 6.6 in liver cells and from 2.4 to 9.5 in blood. Based on these numbers of DNA damage, it’s found that CUS04 was the most harmful to the three tissues. Also, we can find that blood was the most stressed and revealed high amount of DNA damage in fish exposed to CUS04 and PO, while gill tissue was in ML. Fish were exposed to two concentrations, 0.01 and 1.0 ppm, of CUS04 for 96 hours revealed slight decreased of IgM titer levels versus control. Following two immunizations with kehole limpet hemocyanin (KLH), serum IgM titer displayed suppressed values at 0.01 ppm CUS04 and enhanced levels to 1.0 ppm CUS04, for the primary response. For secondary response, fish exposed to low and high concentrations of CUS04 revealed increased titers of IgM.Fish exposed to 0.0001 and 0.01 ppm ML for 96 hoursshowed no change for IgM titers when compared with control.After two immunizations with KLH, IgM titers had increasedlevels for ML treated fish at the primary response. For the . secondary response, fish exposed to 0.01 ppm ML showed increased levels of IgM titers and no change in fish exposed to 0.0001 ppm ML.Fish exposed to 0.0001 and 0.01 ppm PO for 96 hours revealed an increase for IgM titer at both of two concentrations After the fish were immunized twice, the IgM titer showedenhanced levels for treated fish at the primary and secondar. |