الفهرس 
Material and MethodsOnly 14 pages are availabe for public view
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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.