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Effects of barley grass powder (Horidium Vulgare) on Behavioral and Histological alterations of Nile Tilapia exposed to Chloropyrifos insecticide

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hloropyrifos is an organophosphate insecticide widely applied in agriculture and aquaculture, It is very highly toxic to freshwater fish and to aquatic invertebrates. Tilapia fish (Oreochromis niloticus) (40±5gm and 12.5±0.5cm) exposed to1/10 LC50 (0.036 mg/l) for one week and 1/20 LC50 (0.018 mg/l) of Pestban for four and eight weeks showed nervous manifestations, loss of equilibrium and abnormal swimming as swim to the water surface and fall vertical to the bottom .Different pathological tissue alterations were shown in some organs after exposure to high and low doses of Pestban .Gills showed hyperplasia, edema and partial sloughing of secondary lamellae. Liver showed congestion of central vein, and vacuolar degeneration. Kidney showed alternation areas of activation, depletion of hemopoietic element and condensed glomerular tuft with edema in Bowaman’s capsule .Brain showed congestion of meningeal blood vessels, cerebral blood vessel with of pronounced malacia in the spinal cord, vacuolar degeneration of optic lobe, focal necrosis in the inner granular layer of cerebellum and the Purkinje cells suffer from pyknosis .The residual analysis revealed increased residues of chloropyrifos in gills that than in the muscle. Addition of barley grass powder 10% and 20% to the fish food revealed mild improvement of the behavioral disorders and pathological tissue alterations compared with Pestban treated group moreover there was decrease of chloropyrifos residues in both gills and muscle. These findings suggest that barley grass powder is positively effective in Pestban toxicity in fish.
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Egypt. J. Aquat. Biol. & Fish., Vol. 15, No. 3: 349- 362(2011) ISSN 1110 – 6131
The 15
th
Conf. of the Egypt. Soc. of Fisheries Development in cooperation with
The 4
th
Global Fisheries & Aquaculture Research Conf.
www.esfhd.eg.net
Effects of barley grass powder (Horidium Vulgare) on Behavioral and
Histological alterations of Nile Tilapia exposed to Chloropyrifos insecticide
Nabela, I. E.
1
, Abou Hadeed, A. H.
1
, Saleh, F. M. Sakr
2
, Farag, M. Malhat
3
and
Samah, A. A.
2
1-Dept. of Forensic Med.Toxicology, Faculty of Veterinary Medicine, Zagazig University
2-Dept. of fish diseases, Central Lab. for Aquaculture Researches (Abbassa) Sharkia
Government.
3-Dept.of pesticide Residues and Environmental Pollution, Central Agricultural Pesticide
Laboratory, Agriculture Research Center, Dokki, Giza, Egypt.
ABSTRACT
hloropyrifos is an organophosphate insecticide widely applied in agriculture and
aquaculture, It is very highly toxic to freshwater fish and to aquatic invertebrates.
Tilapia fish (Oreochromis niloticus) (40±5gm and 12.5±0.5cm) exposed to1/10 LC
50
(0.036
mg/l) for one week and 1/20 LC
50
(0.018 mg/l) of Pestban for four and eight weeks showed
nervous manifestations, loss of equilibrium and abnormal swimming as swim to the water
surface and fall vertical to the bottom .Different pathological tissue alterations were shown in
some organs after exposure to high and low doses of Pestban .Gills showed hyperplasia,
edema and partial sloughing of secondary lamellae. Liver showed congestion of central vein,
and vacuolar degeneration. Kidney showed alternation areas of activation, depletion of
hemopoietic element and condensed glomerular tuft with edema in Bowaman’s capsule
.Brain showed congestion of meningeal blood vessels, cerebral blood vessel with of
pronounced malacia in the spinal cord, vacuolar degeneration of optic lobe, focal necrosis in
the inner granular layer of cerebellum and the Purkinje cells suffer from pyknosis .The
residual analysis revealed increased residues of chloropyrifos in gills that than in the muscle.
Addition of barley grass powder 10% and 20% to the fish food revealed mild improvement
of the behavioral disorders and pathological tissue alterations compared with Pestban treated
group moreover there was decrease of chloropyrifos residues in both gills and muscle.
These findings suggest that barley grass powder is positively effective in Pestban toxicity in
fish.
Keywords: Pestban, Tilapia, behavior, histopathology, residue.
INTRODUCTION
The application of various pollutants like pesticides in the aquatic environment is
known to cause several structural and functional changes in the biota. A major part of the
world’s food is being supplied from fish source, so it is essential to secure the health of
fishes (Goldberg and Spooner, 1983). Recent years marked a major shift in human
understanding the role of food products in maintaining their proper health status (Snedecor
and Cochran, 1969). However, it is impossible to completely remove all pesticide residues
from foods. Therefore it is ideal if pesticide residues can be degraded into non-toxic
C
Nabela, I. E. et al.
350
materials in foods (Durham et al., 1999). Chloropyrifos (O, O- diethyl- O -3, 5 -6–trichlor -
2-pyridyl phosphorothioate) (Fig. 1) is a broad-spectrum organophosphate insecticide (OP)
and commercially used for more than a decade to control foliar insects that affect agricultural
crops (Rusyniak and Nanagas, 2004). Chloropyrifos passes via air-drift or surface runoff into
natural waters, where it is accumulated in different organisms living in water, especially fish,
thus making it vulnerable to several discernible effects (Kavitha and Rao, 2008). Barley
Grass provides balanced nutrition including an abundance of minerals (potassium, calcium.
magnesium, iron, copper, phosphorus, manganese and zinc) , 18 amino acids , antioxidant
enzymes, beta-carotene and vitamins B1, B2, B6 and C, , It is also an excellent source of
Chlorophyll which helps to gently detox form the environmental pollutants (Hagiwara etal.,
1979 )
Fig1. Chemical structure of Chlorpyrifos
MATERIAL AND METHODS
Study was conducted on Oreochromis niloticus freshwater fish, (40±5g, and
12.5±0.5cm). Fish were transferred to Central Laboratory for Aquaculture Research (CLAR)
Abbassa, Sharkia Governorate with in plastic bags. The bags were cut open and the fish were
disinfected with 0.1% KMnO4 solution for few seconds and acclimitized under laboratory
conditions for two weeks in 1000 liter water tanks, filled with well aerated tap water and fed
on basal fish diet under laboratory condition PH 7.1-7.5, temperature at 26± 5°C before
transferring them to the experimental. Barley grass green plant with 30cm lenght was
collected from different area of Sharkia Governorate then slowly dried at Muffle Furnace
without delay (within 60 minutes of being cut) at temperature 38
o
C to preserve all nutrients
and to maintain the maximum potency of its vitamins, amino acids, enzymes, chlorophyll
and other ingredients contents. Then grind, using electric blender, resulting in a new
generation micro-fine powder, (David 2006).chemical analysis of barley grass powder was
estimated (AOAC 1990) as shown in Table 1.
Table 1: Chemical analysis of barley grass powder
Protein % Fat % Fiber %
Ash %
30 7 15 16
The basal control diet was formulated (Kim et al., 2003) from practical ingredients
to satisfy all known nutrient requirements of Nile tilapia. Barley grass diet 10% or 20%
barley grass powder experimental diets were formulated to meet the requirement of
experimental fish (NRC, 1993). Both types of the experimental diets (control diet and barley
Effects of barley grass powder (Horidium Vulgare)
351
grass diet) were prepared at the Fish Nutrition Department, Central laboratory for
Aquaculture Research.
Experimental design
Four hundred and fifty fish of O. niloticus were divided into four groups as shown in
Table 2.
Behavioral observation items was done with all fishes through out the experiments
according to (Machado and Fanta2003). At the end of 1
st
week and eight weeks tissue
samples (gills, liver, kidney and brain) for histopathological study preserved in 10% neutral
buffered formalin (Carleton et al 1967). Another sample from (gills and muscle) were taken
at the end of one week, four and eight weeks were preserved at -20
o
C till residual analysis
then extraction and determination by GLC (gas liquid chromatography) (Li et al., 2009).
Statistical analysis was performed using the one way analysis of variance (ANOVA) of SPSS
according to (Snedecor and Cochran 1969).
Table 2: Experimental Design
Groups Sub groups No.of fish Treatment Duration
Group I 90 Control (-ve) basal diet 1 , 4 and 8 weeks
Group II 90 Control (+ve) barley grass powder
diet (10%, 20%) 1 , 4 and 8 weeks
a 30 1/10 of 96-hrs LC
50
of pestban
According to (Abo Hadeed et al.,
2011)
1 week Group III
b 60 1/20 of 96-hrs LC
50
of pestban 4 and 8 weeks
a 1 30 1/10 of 96-hrs LC
50
of pestban +10%
barley grass powder diet
a 2 30 1/10 of 96-hrs LC
50
of pestban +20%
barley grass powder diet
1 week
b1 60 1/20 of 96-hrs LC
50
of pestban +10%
barley grass powder diet
Group IV
b2 60 1/20 of 96-hrs LC
50
of pestban +20%
barley grass powder diet
4 and 8 weeks
RESULTS AND DISCUSSION
Behavioral changes observed on O. niloticus during exposure to pestban 1/10 LC
50
(0.036 mg/l) showed tremors, loss of escape reflex and loss of equilibrium during exposure
to pestban 1/20 LC
50
(0.018mg/l) for four and eight weeks all mentioned above responses
were observed, beside swimming of the fish to the water surface and then falling vertically to
the aquarium bottom, loss of feeding and showed respiratory manifestation characterized by
fish raised to the surface of the water and gasping ,improvement of behavior disorder in
barley grass powder treated groups. These signs are closed to those reported by (Gehad
2010) in O. niloticus exposed to CPF. This could be attributed to the inhibition AChE
activity (de Aguiar et al., 2004) or due to stress induced by toxicant and accumulation of
acetylcholine (ACh) which explained interrupted coordination between the nerves and
muscular junctions (Kavitha and Rao 2008). While improvement in barley grass powder
treated groups may be returned to its chlorophyll which helps in the detoxification process by
chelating and adsorbing toxins from the blood (Terry 2010).
Gills of O. niloticus during exposure 1/10 LC
50
of pestban showed mild hyperplasia in
the secondary lamellae, edema (Figs. 3&4) and telangectasis (Fig. 5)and during of exposure
1/20LC
50
of Pestban showed hyperplasia especially at the tip of lamellae, congestion of
branchial blood vessele and partial sloughing of the secondary lamellae (Fig. 6). Addition
Nabela, I. E. et al.
352
barley barley grass powder 10%, 20%for both treated groups slight hyperplasia& edema in
the secondary lamellae were noticed (Fig. 7) comparing with control (Fig. 2).Similar finding
is supported by (Rao et al., 2003a) mentioned that Oreochromis mossambicus exposed to 26
µg/litre Chloropyrifos noticed necrosis and abnormalities in the gill lamellae. The epithelial
hyperplasia was known as a protective and defense mechanism of fish gills against harmful
pollutants while sloughing of some secondary lamellae may be due to the simple response to
cellular necrosis. Congestion, edema and hemorrhage in gill may be attributed to increase the
permeability of blood vessel, due to the destruction of cement substance connecting the
endothelial cells leading to escape of protein and consequently decrease the colloidal osmotic
pressure at extravasations fluids of RBCs (hemorrhage, by dipodies) (Roberts, 1978).
The common liver pathological changes observed in the present study were
congestion of the hepatoportal vien (Fig. 9), hepatic sinusoids (Fig. 10) during of exposure
1/10 LC
50
of pestban and vacuolar degeneration (Fig. 11), dissociation of pancreatic acini
were noticed during exposure 1/20 LC
50
of pestban. Addition barley grass powder 10%,20%
for both treated groups showing congestion of hepatoportal veins &activation of pancreatic
acini (Figs. 12,13) comparing with control groups showing normal exostructure of hepatic
cell, the connective tissue of liver expressed normal and central vein free from red blood
cells (Fig. 8).The degenerative changes of the hepatocytes were probably induced by the non
lethal damage to cells by the free radicals failure sodium-potassium pump leading to entrance
of sodium into the cell followed by water leading to cellular swelling, cytoplasmic
vacuolation and rupture (Roberts, 1989).
kidney of O. niloticus during exposure 1/10 LC
50
Pestban showed hypercelluarity in
glomeruli (Fig. 15), and during exposure 1/20 LC
50
Pestban showed edema in Bowman
,
s
capsules, condensation of glomerular tuft (Fig. 16) and degenerative changes in renal
tubules(Fig. 17), Addition barley grass powder 10%, 20% for both treated groups normal
renal tubule with focal activation of hemopioptic element (Figs 18and19) comparing with
control groups showed intact renal tubules (Fig. 14),with hypercelluarity of glomeroli. Focal
areas of activation of hemopoietic elements were encounted.
Brain of O. niloticus during exposure 1/10 LC
50
Pestban revealed congestion of
meningeal blood vessels (Fig. 21) and cerebral blood vessel (Fig. 22) in 1/20 LC
50
pestban
showing the spinal cord showing pronounced malacia (Fig. 23) vacuolar degenration of optic
lobe ( Fig. 24) and focal necrosis in the inner granular layer of cerebellum (Fig. 25), The
Purkinje cells suffer from pyknosis (Fig. 26). The treated group with barely 10% showed
meninges free from red blood cells (Fig. 27) while moderate congestion of cerebral blood
vessels was encountered (Fig. 28) in group treated with 20% barely comparing with control
normal brain was noticed (Fig. 20).
The recorded pathological alteration in the present study may be belonged to the
generation of reactive oxygen species (ROS) as O
-
,H
2
0
2
, and OH
.
due to Chloropyrifos
applications (Gultekin et al., 2000, Altuntas and Delibas, 2002) formation of the toxic
compounds malonaldehyde which resulting of exposed fish species to Chloropyrifos
(Kavitha and Rao, 2008and Kavitha and Rao, 2009) lead to damage tissue.
Addition of barley grass powder treated groups revealed an improvement
histopathological lesions in gills, Liver, kidney and brain which may be returned to 2”-O-
Effects of barley grass powder (Horidium Vulgare)
353
GIV glycosylisovitexin Flavonoids, present only in young barley grass, is an extremely
effective as antioxidant and preventing free-radical oxidation of lipids found in the skin and
blood, enhances the antioxidant actions of vitamin C and also helps prevent the formation of
the toxic compounds malonaldehyde and acetaldehyde, which are known to denature
proteins and DNA (Osawa et al., 1992). Beside its anti-oxidative, anti-inflammatory, anti-
allergic, anti-carcinogenic and anti-ulcer properties (Kubota et al., 1983).
The present results study revealed that gill tissues showed higher CPF
bioconcentration than muscle tissues. This result on the harmony with (Rao et al., 2003b and
Rao et al., 2005) who recorded accumulation CPF in viscera>head>body in Oreochromis
mossambicus, mosquito fish, (Gambusia affinis) to CPF respectively.CPF bioconcentration
in gills and muscle in the order of accumulation as fish exposed to 1/10 LC
50
of pestban for
one week >1 /20 LC
50
of pestban for eight weeks > Fish exposed to 1 /20 LC
50
of pestban for
four weeks this may be attributed to difference in dose and period of exposure . Addition
barley grass powder 10% and20% to fish food resulted in decrease residues of CPF in gills
and muscle which was effective in case of 20% this agree with (Durham et al., 1999) found
that young barley grass extracts induced complete degradation CPF after incubation of the
individual pesticides with a 3% solution of barley grass juice. There are several mechanisms
for barley to chelate or neutralize the pesticides or their toxic effects due to the presence
flavonid,chlorophyll, beta carotine (Terry 2010 and Hagiwara et al., 1979).
CONCLUSION
Ten pecentage and 20% barley grass powder slight improvement alteration
behavior, pathology while 20% was effectively decrease residual accumulation of CPF in
gills and muscle counteract its pathological alterations.
Table 3: Bioconcentration of CPF (µg/g) in gills and muscle of the Nile tilapia (O. niloticus) after
exposure 1/10 LC
50
(0.036mg/l) Pestban, concomitant with 10, 20 % barley for one week and
after exposure 1/20 LC
50
(0.018mg/l) Pestban, concomitant with 10, 20 % barley for four,
eight weeks.
Means in the same column carrying different superscript are significant at P<0.05. a : increase, b,c: decrease
Organs Group DOSE Period of exposure gills(µg/g tissue) Muscle (µg/g tissue)
III( a) 1/10LC
50
of pestban 3.841±0.040a 0.6152±0.05a
IV(a1) 1/10LC
50
of pestban
+10%barley 3.706±0.016b 0.554±0.002b
IV( a2) 1/10LC
50
of pestban
+20%barley
1 week
3.565±0.024c 0.26 ±0.016c
III(b) 1/20 LC
50
of pestban 0.299±0.034a 0.0715 ±0.045a
IV(b1) 1/20LC
50
of pestban
+10%barley 0.232±0.027b 0.0598±0.0034b
IV( b2) 1/20LC
50
of pestban
+20%barley
4 week
0.1839±0.01c 0.0451±0.003c
III(b) 1/20 LC
50
of pestban 0.522 ±0.021a 1.317±0.034a
IV( b1) 1/20LC
50
of pestban
+10%barley 0.421±0.023b 0.033±0.002b
IV( b2) 1/20LC
50
of pestban
+20%barley
8 week
0.221±0.015c 0.0281±0.003c
Nabela, I. E. et al.
354
Fig 3: photomicrograph of gills of tilapia
fish exposed to 1/10 LC
50
(0.036
mg/l) pestban for one week
showing mild hyperplasia in the
secondary lamella (H&E., X150)
(arrow).
Fig 2: photomicrograph of gills of control
tilapia fish showing normal for one
week &8 weeks (H&E., X300)
(arrow).
Fig 5: photomicrograph of gills of tilapia
fish exposed to 1/10 LC50 (0.036
mg/l) pestban for one week showing
telangectasis in the secondary
lamellae, (H&E., X600) (arrow).
Fig. 4: photomicrograph of gills of tilapia fish
exposed to 1/10 LC50 (0.036 mg/l)
pestban for one week showing edema
(arrow) and mild hyperplasia in the
secondary lamella (star) (H&E., X300)
.
Effects of barley grass powder (Horidium Vulgare)
355
Fig. 6: photomicrograph of gills of tilapia fish
exposed to 1/20 LC
50
(0.018mg/l)
pestban for 8 weeks showing
congestion of branchial blood vessele
(arrow) and partial sloughing in the
secondary lamellae (star). (H&E.,
X
600
).
Fig. 7: photomicrograph of gills of tilapia fish
treated with 10%,20% exposed to
pestban 1/20LC
50
(0.018mg/l) for one
week, 8 weeks showing slight
hyperplasia in the secondary
lamella(star) & edema (H&E., X300)
(arrow).
Fig. 8: photomicrograph of liver of
control tilapia fish showing
intact liver for one week & 8
weeks (H&E., X600)
Fig. 9: photomicrograph of liver of tilapia
fish exposed to 1/10LC
50
(0.036mg/l)
pestban for one week showing
congestion of hepatoportal vien
(
H
&
E
.
,
X
600
) (
arrow
).
Nabela, I. E. et al.
356
Fig. 10: photomicrograph of liver of tilapia fish
exposed to 1/10 LC
50
(0.036) pestban
for one week showing congestion of
hepatic sinasoid. (H&E., X300) (arrow).
Fig. 11: photomicrograph of liver of tilapia fish
exposed to 1/20 LC
50
(0.018) pestban
for 8 weeks showing vacular
degeneration. (H&E., X300) (arrow).
Fig. 12: photomicrograph of liver of tilapia fish
treated with 10% barley exposed to
pestban 1/10LC
50
(0.036) for one
week, 8 weeks showing congestion of
hepatoportal vien (arrow) &activation
pancritic acini
(
star
) (
H
&
E
.
,
X
600
)
Fig. 13: photomicrograph of liver of tilapia fish
treated with 20% barley exposed to
pestban 1/20LC
50
(0.018) for one
week, 8 weeks showing slight
congestion of hepatoportal vien (arrow)
& activation pancritic acini (star) (H&E.,
X600)
Effects of barley grass powder (Horidium Vulgare)
357
Fig. 14: photomicrograph of kidney of control
tilapia fish showing intact renal tubule
for one week & 8 weeks (H&E.,
X600) (arrow).
Fig. 15: photomicrograph of kidney of tilapia
fish exposed to 1/10LC
50
(0.036 mg/l)
pestban for one week showing
hypercellarity of glomeruli. (H&E.,
X600) (arrow).
Fig. 16: photomicrograph of kidney of tilapia
fish exposed to 1/20 LC
50
(0.018 mg/l)
pestban for 8 weeks showing
degenerrative changes. Of renal tubule
(H&E., X150) (arrow).
Fig. 17: photomicrograph of kidney of tilapia
fish exposed to 1/20 LC
50
(0.018 mg/l)
pestban for 8 weeks showing
condansation of glomerular tuft (star) and
edema in Bowman
,
s capsule. (H&E.,
X600) (arrow).
Nabela, I. E. et al.
358
Fig. 22: photomicrograph of brain of
tilapia fish exposed to 1/10LC
50
(0.036 mg/l) pestban for one week
showing congestion of cerebral blood
vessels
(H&E., X600) (arrow).
Fig. 23: photomicrograph of spinal cord of
tilapia fish exposed to pestban1/20LC
5
(0.018 mg/l) for 8 weeks showing
pronounced malacia (H&E., X600)
(arrow).
Fig. 24: photomicrograph of brain brain of tilapia
fish exposed to1/20LC
50
(0.018 mg/l)
pestban for 8 weeks showing vacuolar
degeneration of optic lobe (H&E., X480)
(arrow).
Fig. 25: photomicrograph of brain of tilapia
fish exposed to 1/20LC
50
(0.018 mg/l)
pestban for 8 weeks showing focal
necrosis in the inner granular layer of
cerebellum
(H&E., X600) (arrow).
Effects of barley grass powder (Horidium Vulgare)
359
Fig. 26: photomicrograph of brain of tilapia
fish exposed to 1/20LC
50
(0.018 mg/l)
pestban for 8 weeks showing pyknosis
in Purkinje cells (H&E., X480) (arrow).
Fig. 27: photomicrograph of brain of tilapia
fish treated with 10% barley exposed to
pestban 1/20LC
50
for 8 weeks showing
meninges free from red blood cell
(
H
&
E
.
,
X
600
) (
arrow
).
Fig. 28: photomicrograph of brain of tilapia
fish treated with 20%barley exposed to
pestban 1/20LC
50
for 8 weeks showing
moderate congestion of cerebral blood
vessels (H&E., X600) (arrow).
Nabela, I. E. et al.
360
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تاا   ا ا ةرد اك  ا ا ا
نا ىا
ىوا ما 
1
، أ ر 
1
   ،
2
 ج ، د
3
 حو
2
1 - ا ا 
زا  ،ىا اآ ،ماو
2 - ا  ،كا ضا كا ث ىآا ا ، ،زا ا.
3 - ا ىآا ا، ا ث و تاا ت ث  ثا آ ،ت
ا ،ارا
نا )سوورآ ( ا عاراو ارا  ما ىا تا  ه
ا تاو كا   آا تاا  سوورآ و. ةرد رود ارد 
ا ا كا  نا  ما ا    ا ا)45 ±5 و 12.5 ±
0.5( ا1/و 10 عا ة  ا ا  ه0.036 ما/ وا 1/20 
ة  ا ا8,4 ا ه0.018ما /  ا وا 10 %وا20 % ا ةرد
  ا ةرا آ نا    ت ا كا و ا
أ آ ما  لاو ر   ضاأ ةر ك ا صا ت
او او  تا دو    تاآ ا او نا   وا
 ا قاروا طو ا ر ا ا  دو ا  دوأ حراو
ا و ما    تا ن  ا  ىآا را و ا وا   ن
ا  دو راا تاا  و ا  اد و آو نا  أ آ
ا آ ةدز نا   تاآ ا تاو ا   ا  
تا . ا ةراو كا ك    ا كا  تا 
او ا  آ  ا آ    ا ك و ا 20 % 
ا كا  .  ا ةرد     ا  ارود    ا
ا.
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Article
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Sub-lethal studies of chlorpyrifos, O,O-diethyl-O-(3,5,6-trichloro-2-pyridyl) phosphorothioate on mosquito fish, Gambusia affinis were carried out in vivo, for 20 days to assess the locomotor behavior in relation to bioaccumulation and interaction with a targeted enzyme, acetylcholinesterase (AChE, EC: 3.1.1.7). Fish exposed to sub-lethal concentration of 60 Ag/L (1/5 of LC 50) were under stress, and reduced their locomotor behavior like distance travelled per unit time (m/min) and swimming speed (cm/sec) with respect to the length of exposure. The alteration in locomotor behavior of fish may be due to an accumulation of acetylcholine (ACh), a neurotransmitter at synaptic junctions, due to the inhibition of AChE enzyme activity (40 to 55%) in brain and also bioaccumulation of the toxicant in different parts of fish. The bioaccumulation values indicated that the accumulation of chlorpyrifos was maximum in viscera followed by head and body. The average bio-concentration values are 0.109, 0.009 and 0.004 Ag/g for viscera, head and body with depuration rates of 2.24, 1.69 and 0.39 ng/h respectively. It is evident from the results that the sub-lethal concentration [1/5 of LC 50 ; equivalent to Lowest Observed Effect Concentration (LOEC)] of chlorpyrifos can able to alter the locomotor behavior of G. affinis in relation to the length of exposure. The findings revealed that the locomotor activity of test organism could be considered as a suitable marker to evaluate the affect of toxicant even at LOEC levels.
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