Abstract
BIOLOGIA (PAKISTAN) PKISSN 0006 – 3096 (Print)
December, 2019, 65 (II), Online ISSN 2313 – 206X (On-Line)
Author’s Contribution: A.S., Did experimental work; D.H., Provided the facility for experimental set up and helped for the determination of
acute and chronic exposure; U.R., Helped in the histological studies of fish; S.M., Supervised research, helped in formulating the work,
experimental design and wrote up of manuscript
Histological responses in Intestine, Kidney and Liver tissues of Labeo rohita during acute and
chronic exposure to Pesticide, Chlorpyrifos
ADEEBA SYED1
, DILAWAR HUSSAIN2
, UZMA RAFI1 & SUMAIRA MAZHAR1*
1Department of Biology, Lahore Garrison University, Phase-VI, DHA, Lahore Pakistan
2Department of Zoology, Government College University, Lahore-54000 Pakistan
ARTICLE INFORMAION ABSTRACT
Received: 27-07-2018
Received in revised form:
26-07-2019
Accepted: 18-09-2019
Aim of the present study was to examine the acute and chronic exposure
of pesticide, chlorpyrifos (CPF) to the fresh water fish Labeo rohita.
During acute exposure, fish were exposed to different concentrations of
CPF ranging from 0, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03,
0.04 and 0.05 mg/L for 96 hrs in glass aquaria. The 96 hrs LC50 value of
CPF for Labeo rohita was found to be 0.01 mg/L. During chronic
exposure fish were subjected to 1/3rd, 1/5th, 1/7th and 1/9th of LC50 for 30
days. At the end of the trials, tissues from various organs like intestine,
liver and kidney were collected and sections were examined under digital
microscope. Pronounced histological changes like necrosis, infiltration,
atrophy, shrinkage and degeneration of intestine was observed for
different CPF concentrations. Kidney sections of Labeo rohita under
different CPF concentrations exhibited nuclear hypertrophy, vacuolar
degeneration of glomeruli, and occlusion of tubular lumen, cloudy swelling
degeneration and hyaline droplets degeneration. In the liver tissue
prominent histological changes observed including hepatic cell
degeneration, nuclear hypertrophy, bile stagnation, irregular shaped cells,
degeneration in the liver parenchymal cells, nuclear and cytoplasmic
degeneration. Therefore, we here conclude that Chlorpyrifos adversely
affects the major organs of Labeo rohita (Rohu).
Keywords: Labeo rohita, Chlorpyrifos, Acute, Chronic, Histology
*Corresponding Author:
Sumaira Mazhar:
smz.mmg@gmail.com
Original Research Article
INTRODUCTION
Chlorpyrifos is extensively used, second largest and
highest selling organophosphate pesticide, used to
control pests, which cause severe damage to crops
for more than ten years (Rao et al., 2003).
Extensive use of CPF boosts the toxicity level in
aquatic life thereby has severe effects on fish.
Previous studies showed slight and chronic effects
of CPF for different species, e.g., Channa
punctatus, Cyprinus carpio, Oreochromis
mossambicus, Cirrhinus mrigala (Ali et al., 2012;
Banaee et al., 2013; Padmanabha et al., 2015;
Anita et al., 2016). Day-by-day uses of pesticides
increase due to the high demand; pesticides are
used widely in agriculture, forestry, and public
health and in veterinary practices. Hence, it is
essential to study the instant and chronic effects of
pesticides on fish, which supply the protein-source,
an essential part of human diet (Ali et al., 2009). It
has been reported that fish are highly sensitive to
aquatic pollution and showed strict physiological
changes when they are exposed to sub lethal
concentrations of toxicants (Ufodike & Omoregie,
1991). CPF is a crystalline organophosphate pesticide. Several billion fishes are died
due to the chlorpyrifos according to a recent report
(AbdelHalim et al., 2006). In less alkaline soil, CPF
has two months and in an average soil, CPF has
half-life of 30 days and indoors, CPF can persist for
weeks and months (Arcury et al., 2007). CPF enters
into water via air drift or surface runoff and then
deposited in different aquatic organisms,
particularly fish (Varo et al., 2002). CPF has lethal
and sub lethal levels of toxicities in aquatic
environment. Lethal levels cause mass mortalities
in fish and sub lethal toxicities induce
morphological, neurobehavioural, oxidative,
biochemical, histopathological, haematological and
developmental alterations (Sunanda et al., 2016).
CPF also disturbs steroid hormone production and
has harmful effect on reproductive system of fish
(decreased serum estrogen and testosterone
levels), developmental stages and neurobehaviour
2 A. SYED ET AL BIOLOGIA PAKISTAN
(Levin et al., 2004). Several studies have proven
that by inhibiting brain acetylcholinesterase (AChE)
CPF is noxious to living organisms including fish
(Kwong., 2002; Singh & Singh 2008; Xing et al.,
2012; Mishra & Devi 2014). Tissue histology is
extensively used to study the effect of
contamination and toxicity in organisms (Cengiz &
Unlu, 2003). Histopathological changes can also be
used as biomarkers to check the contamination in
fishes both in laboratories and field studies
(Thophon et al., 2003; Schwaiger et al., 1997). The
coverage of sub lethal concentration of CPF led to
the reduction in the level of total protein and
glycogen, concentrations of pesticide also raise the
glucose level which to lethargy (Kadam & Patil,
2013; Majumder & Kaviraj, 2017). CPF also
disturbs chemical composition of fish which leads to
cell damages and is responsible for mortality of fish
(Khan, 2017). Dursban and Lorsban insecticides
are the active form of CPF (Kienle et al., 2009).
Presently, varieties of organophosphate having
chemical, physical and biological properties are
used for agricultural purposes (Kumar, 2012). Due
to direct contact with environment (water) fish gills
are primary site of noxious action of many
waterborne pollutants (Olson, 2002). Acute toxicity
test is the best way to check the toxicity of
organisms and the ecosystem as a whole. These
tests are helpful in creating knowledge about
potential destructive effects of such industrial
discharges to the environment (Adedeji et al., 2008;
Onyedineke et al., 2010). Fish as a source of food
has been documented all over the world (Tacon &
Metian, 2013). Proteins have a key role in human
diet for appropriate growth and other essential
activities. Fish is regarded as an excellent source of
protein for human diet (WHO, 2007). In developing
countries like Pakistan, fish manufacture sector is
very significant not only as a major source of animal
protein to guarantee food security but also to
recover the value of food and raise protein supply in
the food chain (Sheikh & Sheikh 2004; Bacha et al.,
2011). Thus, the present study was designed to
evaluate the effects of CPF under acute and
chronic exposure on the Labeo rohita and its effects
on liver, kidney and intestinal tissues.
MATERIALS AND METHODS
Experimental fish
Healthy fingerlings of Labeo rohita were
purchased from Manawa Fish Hatchery, Lahore
and brought to the fish experiment room, Animal
House in Department of Zoology at Government
College University, Lahore. Prior to the start of the
experiment, fish were acclimatized to laboratory
conditions in round tanks for 15 days. Fish were fed
with commercial pelleted diet at 5% body weight,
twice daily.
Determination of LC50 for Labeo rohita
Labeo rohita were starved for 24hrs before
start of the experiment. Eleven different
concentrations of CPF (0.005, 0.006, 0.007, 0.008,
0.009, 0.01, 0.02, 0.03, 0.04 and 0.05 mg/L) were
prepared in ten equal sized aquaria, in addition to
that one test aquarium kept for the control. Each
aquaria contained 10 L water with 15-fishes/
aquarium. The fishes were exposed to the prepared
test solutions for 96 hrs. Dead fish were watched
and removed per day till the end of the fourth day.
By the end of the fourth day, the mortality
percentage was calculated according to the profit -
analysis method. The experiment was repeated
three times and the average of LC50 value for CPF
was recorded as 0.01mg/L for 96hrs.
Chronic exposure of Chlorpyrifos for Labeo
rohita
To determine the chronic toxicity fishes
were collected and randomly divided into five
groups. The first group represented the control and
other four groups were experimental labeled as T1
(control group), T2, T3, T4 and T5 (experimental
groups). LC50 value was recorded as 0.01mg/L.
Each aquarium contained 40 L water and 20 fish
individual were transferred. 1/3rd
, 1/5th, 1/7th and
1/9th of LC50 were considered for chronic study. Fish
were fed with commercial food at least once a day
during the study period (30 days) and fecal matter
was removed daily from aquaria. After 30 days
samples were collected for the histological studies
of liver, kidney and intestine.
Histology of liver, kidney and intestine
For the study of histopathological affects,
tissue specimens from three fish per treatment were
removed by dissecting and preserved the tissues of
kidney, liver and intestine in formalin solution (10%
distilled water, 5% ethanol and 10% formaldehyde).
For histology slide preparation fixed the tissues in
10% buffered formaline for 24hrs and ratio of
formalin and tissue were 10:1 (10ml of formaline
per 1cm3
tissue). After fixation tissues, specimens
were trimmed by using a scalpel to enable them to
fit into an appropriate labeled tissue cassette. The
tissue cassettes were stored in formalin until
processing begins. Tissue processing began under
three different steps, dehydration, clearing and
VOL. 65 (II) HISTOLOGICAL RESPONSES OF LABEO ROHITA AGAINST PESTICIDE, CHLORPYRIFOS 3
embedding of specimens. After tissue processing
tissue specimens were cut into sections and placed
on the glass slides. Most cells were transparent.
Histochemical stains (Haematoxylin and Eosin)
were used to stain the tissue. A cover slip was
mounted over the tissue specimen on the slide by
using optical grade glues to protect the specimen.
RESULTS AND DISCUSSION
Histological changes in intestine
Intestinal sections of fish in the T1 (control
group) exhibited normal structure of intestine tissue
with long and tapering villi with tightly packed sub
mucosal tissues and epithelium, serous membrane,
muscularis layers, stratum compactum, and lamina
propria (Fig. 1a). In contrast, slight changes in villi,
sub-mucosal tissue, necrosis of epithelial cells,
infiltration of lymphocytes into the lamina propria of
fish exposed to 1.1µl CPF were observed (Fig. 1b).
Intestine of fish exposed to 1.4µl of CPF showed
shrinkage of sub-mucosal tissue and the villi
enlarged towards the tip, atrophy of epithelial cells
and shrinkage of sub-mucosal tissues (Fig. 1c).
Ruthless mucosal secretion has occurred due to
suffering which enable the fish to deal with
ecological stress (Samanta et al., 2016). When
exposed to 2.0µl of CPF, there was broadening and
flattening of the villi towards their tips observed,
some sign of deterioration were also visible and
mucosal epithelium collapsed too (Fig. 1d). In the
last group T5 exposed to the 3.3µl of CPF, the
intestine showed severe structural damage and
intestine completely degenerated (Fig. 1e). Intestine
is one of the most important part of a fish digestive
system, performing main role in digestion and
absorption of food materials. It is extremely
sensitive to any toxic material and can be used as a
significant biomarker organ for measurement of
ecotoxicology (Kroon et al., 2017). In this study,
changes in intestinal tissues of L. rohita were
primarily necrosis, hemorrhages, over production of
goblet cells in villi, fusion, detachment and
shortening of villi. When treated with deltamethrin,
leukocytes infiltration, necrosis in gut tissues of
Mosquito fish, Gambusia affinis has been reported
by Cengiz & Unlu, 2006; Exposed to lambdacyhalothrin for 60 days, Cirrhinus mrigala showed
intestinal lesions, eosinophils invasion into the
lamina properia and epithelial cells atrophy were
observed which showed reduction of villi with
inflammation, rupture of cells, disintegration
changes in tips of villi, curved villi, hemorrhage,
necrosis, numerous vacuoles, dilation in the blood
vessels, completely damaged villi and loss of
architecture in a number of fish species (Cengiz
and Unlu, 2006; Velmurugan et al., 2007; Vidhya
and Nair, 2016).
Histological changes in kidney
Renal sections of fish were exposed to T1
(control group) exhibits normal architecture of renal
tubules and showing Renal Corpuscles (showing
glomerulus & Bowmen’s space) Proximal tubule
and distal tubule (Fig. 2a). Cloudy swelling of
epithelial cells of renal tubules, renal tubules with
dilated lumens and occlusion lumens, fragmentation
of glomeruli and renal corpuscles (showing
glomerular expansion & absence of Bowman’s
space) and nuclear hypertrophy were observed, T2
exposed to 1.1µl of CPF (Fig. 2b). Decreases in the
tubular lumen may be due to the cloudy swelling of
the epithelial cells of the renal tubules, which could
be a reversible change. Also, the dilation in the
tubules lumen may be due to the marked decrease
in the length of the epithelial cells as a result of
epithelial tubules degeneration (Issa et al., 2011)
whereas in the present study, the recognized
homogenous eosinophilic deposits within the
tubular lumens could be attributed to the protein
leakage into the filtrate due to the glomerular
disease as described by Roberts (2001). T3 treated
with 1.4µl of CPF and showed shrinkage and
vacuolar degeneration of glumeruli and Vacuole
formation (Fig. 2C). T4 exposed to 2.0µl of CPF and
showed cloudy swelling of epithelial cell of renal of
renal tubules with narrowing lumens, renal tubules
with degenerated epithelia and occlusion lumens
(Fig. 2D). T5 subjected to exposure of 3.3µl of CPF
and showed renal tubules with degenerated
epithelial cells and dilated lumens, complete
destruction of tubule architectures, hyaline droplets
degeneration and vacuole formation (Fig. 2e).
These results partially agrees with (Hossain et al.,
2002), where in more pathologies were found in B.
gonionotus. This may be due to the use of
pesticides at sub-lethal concentrations, compared
to the doses used in the present study. Oropesa et
4 A. SYED ET AL BIOLOGIA PAKISTAN
al. (2009) documented more or less similar findings
for Cyprinus carpio. Although kidneys do not
possess high levels of xenobiotic metabolizing
enzymes as does the liver, many of the enzymatic
reactions occurring in the liver have been shown to
occur in the kidney (Mohssen, 2001). And kidney
receives the bulk of the post branchial blood flow
therefore its tissue play an important function in the
detoxification and elimination of aquatic
contaminants in fish (Durmaz et al., 2006).
Histological changes in liver
The liver tissues (Hepatic cell and Granular
cytoplasm) of fish in T1 (control group) showed
normal morphology because they were not exposed
to any CPF intoxication (Fig. 3a). The fish exposed
to 1.1µl of CPF, showed almost normal pattern in
liver cells with very slight degenerative changes in
cell arrangements and nuclear hypertrophy, bile
stagnation and vacuole formation (Fig. 3b). This
group T3 represents 1.4 µl exposure of CPF. The
liver tissue showed initial stage of cirrhosis and
vacuolization of cytoplasm and eosinophilic
granules and irregular shaped cells (Fig. 3c). The
fish exposed to 2.0µl of CPF showed degeneration
in the liver parenchymal cells, which was
pronounced with severe damage and drastic
karyolysis and necrosis were observed in some
regions (Fig. 3d). Fish exposed to 3.3µl CPF has
showed pronounced degeneration of liver tissues,
vacuolization was severe, cirrhosis was remarkable,
necrosis and karyolysis were highly pronounced
and nuclear degeneration, cytoplasmic
degeneration and melanomacrophases aggregate
(Fig. 3e). In the present study common liver
abnormalities were observed: loss of parenchymal
architecture, fatty degeneration, vacuolar
degeneration, atrophy and necrosis of hepatic and
pancreatic cells with leucocytic infiltration. These
results are in harmony with the previous studies of
Tilak et al., (2005) and Kunjamma et al., (2008).
Histological changes in the liver could be attributed
to the fact that, the liver is the major site of
detoxification (Nagai et al., 2002), it is expected that
the toxicant insecticide would reach there in
abundance for detoxification and disposal
(Mushigeri & David, 2005). Appearance of lipidosis
and hepatocyte hypertrophy in zebrafish was
reported by Zodrow et al. (2004). Oropesa et al.,
(2009) found necrotic foci and lipid droplets in liver
of Cyprinus carpio, whereas histological analysis of
Silver catfish (Rhamdia quelen) has showed
vacuolation in the liver after exposure to the
herbicide, clomazone (Crestani et al., 2007). Tissue
histology is a helpful means to identify the level of
pollution and it is considered as an indicator of
exposure to pollutants for sublethal and chronic
effects of fish (Cengiz and Unlu, 2003).
Histopathological changes found in the present
study accorded with the previous studies for liver,
kidney and intestine of fish (Labeo rohita) treated
with CPF. Moreover, present results demonstrated
that as the concentration of CPF increased more
distribution were observed in fish organs.
CONCLUSION
We conclude that CPF is highly toxic to
Labeo rohita and its chronic exposure resulted in
significant alterations in histology, which can
ultimately affect the nutritional quality of L. rohita.
After chronic study of the present study shows that,
CPF adversely affects the intestine of fish. Higher
value of chronic study, i.e., 3.3µl has the
degenerated effects on intestine of fish.
Fig. 1a: Micrograph showing Intestinal villi of Labeo rohita
fed control diet (T1). Cross section of intestine without any
exposure to CPF showing normal structure of Intestine
E: epithelium; LP: lamina propria; SC: stratum
compactum; ML: muscularis layers; SM: serous
membrane
E
LP
pp
SC
SM
ML
VOL. 65 (II) HISTOLOGICAL RESPONSES OF LABEO ROHITA AGAINST PESTICIDE, CHLORPYRIFOS 5
Fig. 1b: Micrograph showing intestinal villi of Labeo rohita in
experimental group T2. Slight changes occur in Intestinal villi
NEC: necrosis of epithelial cells; ILLP: infiltration of lymphocytes
into the lamina propria
Fig. 1c: Micrograph showing intestinal villi of Labeo rohita in
experimental group T3. Shrinkage of sub-mucosal tissues is quite
visible
AEC: atrophy of epithelial cells; SST: Shrinkage of sub-mucosal
tissues
Fig 1d: Micrograph showing intestinal villi of Labeo rohita in
experimental group T4. Mucosal epithelium is collapsed.
MEC: Mucosal epithelium collapsed.
Fig. 1e: Micrograph showing intestinal villi of Labeo rohita in
experimental group T5. Intestine complete degeneration
ICD: Intestine complete degeneration.
Fig. 1f: Micrograph showing internal structure of Kidney
of Labeo rohita in experimental group T5.Renal tubule
with degenerated epithelial cells and dilated lumen,
complete destruction of tubule architecture.
HDD: Hyaline Droplets Degeneration, VF: Vacuole
Formation
Fig. 2a: Micrograph showing internal structure of Kidney of
Labeo rohita fed control diet (T1) showing normal architecture,
renal tubule.
RC: Renal Corpuscle (showing glomerulus & bowmen’s space),
PT: Proximal Tubule; DT; Distal Tubule
NEC
ILLP
AEC
SST
MEC
ICD
RC
PT
DT
VF
HDD
6 A. SYED ET AL BIOLOGIA PAKISTAN
Fig 2b: Micrograph showing internal structure of kidney of Labeo
rohita in experimental group T2. Cloudy swelling of epithelial cells
of renal tubule, renal tubule with dilated lumen and occlusion
lumen and fragmentation of glomeruli.
RC: Renal corpuscle (showing glomerular expansion & absence
of bowmans space) , NH : Nuclear Hypertrophy
Fig. 2c: Micrograph showing internal structure of Kidney of
Labeo rohita in experimental group T3.Showing shrinkage and
vacuolar degeneration of glomeruli.
VF : Vacuole Formation; VDG: Vacuolar degeneration of
glomeruli
Fig. 2d: Micrograph showing internal structure of Kidney of
Labeo rohita in experimental group T4.Cloudyswelling of
epithelial cell of renal tubule with narrowing lumen, renal tubule
with degenerated epithelia and occlusion lumen.
OT : Occlusion of Tubular lumen , CSD :Cloudy Swelling
Degeneration
Fig. 3a: Micrograph showing Liver of Labeo rohita fed
control diet (T1) showed normal morphology of liver cells.
HC: Hepatic cell, GC: Granular cytoplasm
Fig 3b: Micrograph showing internal structure of Liver of
Labeo rohita in experimental group T2
. Showed normal
pattern in liver with very slight degenerative changes in
cell arrangements
NH : Nuclear Hypertrophy , BS : Bile Stagnation, VF :
Vacuole Formation
Fig. 3c: Micrograph showing internal structure of Liver of
Labeo rohita in experimental group T3. The liver tissue
showed initial stage of cirrhosis and vacuolization of
cytoplasm.
EG: Eosinophilic Granules, ISC: Irregular Shaped cells
RC
NH
VF
VDG
CSD OT
GC
HC
NH
BS
VF
EG ISC
VOL. 65 (II) HISTOLOGICAL RESPONSES OF LABEO ROHITA AGAINST PESTICIDE, CHLORPYRIFOS 7
Fig. 3d: Micrograph showing internal structure of Liver of
Labeo rohita in experimental group T4. Degeneration in
the liver parenchymal cells is pronounced with severe
damage and drastic karyolysis and necrosis is observed
in some regions.
VF : Vacuole formation, DLPC: Degeneration in the
liver parenchymal cells
Fig. 3e: Micrograph showing internal structure of liver of
Labeo rohita in experimental group T5. Showed
pronounced degeneration of liver tissues, vacuolization is
severe, cirrhosis is remarkable, necrosis and karyolysis
are highly pronounced.
ND: Nuclear Degeneration, CD: Cytoplasmic
degeneration , MA : Melanomacrophases Aggregate
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10 A. SYED ET AL BIOLOGIA PAKISTAN
Table 1: Effects of different concentrations of CPF on intestine, kidney and liver of Labeo rohita
Stages
T1 T2 T 3 T4 T5
Intestine
Epithelium
Lamina Propria
Stratum Compactum
Muscularis Layer
Serous Membrane
Necrosis of epithelial cells
Infiltration of lymphocytes
into lamina propria
Atrophy of epithelial cells
Shrinkage of sub-mucosal
tissues
Mucosal epithelium
collapsed Intestine complete
degeneration
Kidney
Renal corpuscle
(Showing glomerulus
& bowmen, s Space)
Proximal tubule
Distal tubule
Renal corpuscle (showing
glomerular expansion &
absence of bowmn, s
space)
Nuclear Hypertrophy
Vacuole formation
Vacuolar degeneration of
glomeruli
Occlusion of tubular
lumen
Cloudy swelling
degeneration
Hyaline droplets
degeneration
Vacuole formation
Liver
Hepatic cell
degeneration
Granular cytoplasm
Nuclear hypertrophy
Bile stragnation
Vacuole formation
Eosinophilic granules
Irregular shaped cells
Vacuole formation
Degeneration in the
liver parenchymal cells
Nuclear degeneration
Cytoplasmic degeneration
Malenomacrophages
aggregate
ADEEBA SYED, DILAWAR HUSSAIN, UZMA RAFI , SUMAIRA MAZHAR. (2019) Histological responses in Intestine, Kidney and Liver tissues of Labeo rohita during acute and chronic exposure to Pesticide, Chlorpyrifos, Biologia – Journal of Biological Society of Pakistan, Volume 65 (II), Issue 2.
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