Download synopsis

SYNOPSIS
T
he thesis entitled “Comparative study on chronic toxicity of Metanil Yellow
on a teleostean cat fish, Heteropneustes fossilis (Bloch) and albino rat,
Rattus norvegicus” incorporates of the results derived from the studies carried
out in the research laboratory, Department of Environmental Science, The
University of Burdwan, during the period from February 2009 to October 2012.
Thesis embodies 238 pages including 3 standard curves, 16 figures, 13 tables
and 63 photomicrographic plates. Chronic toxic effects of Metanil Yellow (food
colour as well as additive) for the exposure periods of 30 and 45 days have been
investigated on both the cat fish and albino rat by applying a sublethal
concentration of 2.0 g/l and 3.0 g/kg body weight respectively. Toxic effects are
mainly investigated on the gill, skin, some regions of alimentary canal
(buccopharynx, oesophagus, stomach, intestine and rectum) as well as in liver,
kidney, spleen, brain and testis of H. fossilis. But in case of R. norvegicus toxicity
is mainly observed on the tongue, some regions of alimentary canal
(oesophagus, stomach, intestine, duodenum, ileum, colon and rectum) as well as
in liver, kidney, spleen, brain and testis.
1. Different non-permitted colours have shown different toxic effects on different
organisms. Metanil Yellow is one among them. It has shown its chronic toxic
effects on the teleostean cat fish, H. fossilis and on albino rat, R. norvegicus.
2. The physicochemical parameters of aquarium water have been measured
following APHA, 1998. The parameters which have been chosen for analysis
of water are pH, temperature, dissolved oxygen (DO), conductivity, total
alkalinity, total hardness and chloride. The analysis has been carried out both
for control and treated (30 and 45 days) water. Temperature has been found
to become same in all the aquaria, pH has been changed after treatment. DO
in case of control and treated water havs also been well marked. Changes in
conductivity are well marked experimentally. Variations in total alkalinity are
also noticed during control and after treatment. Significant changes in the
values of total hardness and in chloride have found after treatment.
[1]
SYNOPSIS
3. The weight of the cat fish (H. fossilis) has been found to be reduced after
treatment.
4. Histologically, each gill of teleostean cat fish, H. fossilis consists of a longer
lower limb and shorter outer limb. Each gill also consists of gill arches which
bear number of gill rackers and gill filaments, i.e., primary and secondary gill
lamellae. The pillar cells are found in the gill of H. fossilis. The mucosa of the
buccopharynx of H. fossilis has several layers of stratified epithelial cells, club
cells, mucus-secreting cells and a few taste buds. In H. fossilis the
oesophageal mucosa cosists of stratified epithelial cells and has a large
number of mucus-secreting cells. The gastric mucosa consists of compactly
arranged columnar epithelial cells and the tubular gastric glands are also
present at basal portion of gastric mucosa. Histologically, the mucosal surface
of intestine contains simple columnar epithelium. The mucosal folds consist of
mucus-secreting and columnar absorptive cells. The villi of the intestine is
covered with thin top plate. The mucosa of intestine of H. fossilis is made up
of simple, long columnar epithelial cells with basally or centrally placed
nucleus. The mucosal folds of the rectum are short and broad and have a
large number of mucus-secreting cells. The lamina propria is distinct in case
of rectum. The liver of H. fossilis is composed of a large number of hepatic
cells with centrally placed nucleus and granular cytoplasm. Central vein is
also found in liver. Histologically, the posterior or trunk kidney is made up of a
large number of nephrons, each consists of renal corpuscle. The internal
proximal segment has prominent brush border with nucleus. The distal portion
of the tubules is slightly ciliated with prominent nucleus. The spleen of H.
fossilis consists of white and red pulps. The cerebellum portion of the brain of
H. fossilis consists of numerous brain cells. Each testis of H. fossilis consists
of spermatogonia, primary spermatocytes with darkly stained nucleus.
The toxic effects of Metanil Yellow after an exposure to sublethal
concentrations of 2.0 g/l for 30 and 45 days on the gill, skin, buccopharynx,
oesophagus, stomach, intestine, rectum, liver, kidney, spleen, brain and testis
of H. fossilis have been recorded. The most significant changes have been
[2]
SYNOPSIS
shown in the gill rackers and in gill filaments. The damages have also been
shown in the primary and in secondary lamellae. Mucous cells have been
found to damage after treatment. Degeneration has also been found in
stratified epithelial cells and in the taste buds of buccopharynx. Pathological
lesions are the most significant in the mucosa of buccopharynx and
oesophagus of H. fossilis. Shrinkage of stratified epithelial cells and
deterioration of the basement membrane and submucosa have been
recorded experimentally. The columnar epithelial cells and the gastric glands
of the stomach of H. fossilis have been exhibited pathological lesions.
Stomach shows degeneration resulting into vacuolation in the tunica propria
and submucosa. The columnar epithelial cells of intestine and rectum have
also been undergone degeneration and the necrosis of thin top plate has also
been well marked. Brush borders of the intestine have shown damages.
Necrosed lamina propria of the intestine and rectum of H. fossilis are also
evident due to Metanil Yellow toxicity. Liver cord disarray, degeneration of
hepatocytes, pycnotic nuclei have been noticed in H. fossilis. Most
pronounced effects of Metanil Yellow have been found in kidney. Hypertrophy
of Bowman’s capsule along with glomerular shrinkage and vacuolation have
been found. Necrosis of haematopoietic tissues have also been found after
treatment.
Disorganization
of
the
splenic
cords
have
been
found
experimentally but marked depletions of the white pulp and red pulps have
been found after 45 days treatment. Brain cells have been found to damage
after treatment. The sublethal concentration of Metanil Yellow has been found
to cause degeneration in the seminiferous tubules as well as spermatogonia.
Vacuolation is found in the sertoli cells after 45 days treatment.
5. In case of H. fossilis SEM study has shown that each gill arch bears two rows
of gill filaments – the primary gill lamellae which contains alternatively
arranged secondary lamellae on its both the sides. The mucous cells are
found in the interlamellar epithelium of H. fossilis. Stratified epithelial cells
have been found in gill tissue of H. fossilis. SEM study has been revealed the
presence of mucous glands on the skin which secrete the slippery mucus. In
[3]
SYNOPSIS
H. fossilis, the buccopharyngeal epithelium has been appeared as oval and/or
pentagonal in shape and the microridges on the apical surface of the
epithelial cells have been arranged in a concentric whorl. The mucosal
surface of oesophagus contains elongated pentagonal and/or hexagonal
stratified epithelial cells surfaced with linearly arranged microridges. The inner
surface of stomach of H. fossilis is lined with columnar epithelial cells which
are densely packed with stubby microvilli. Gastric pits have been encircled by
the epithelial cells which are well marked in this region. Irregular and zig-zag
type of infoldings have been found in the intestine of H. fossilis. The columnar
epithelial cells of intestine are covered apically with microvilli. The rectal folds
have been anastomosed with each other to form the shallow irregular
depressions. The luminal surface of the rectum has round or oval columnar
epithelial cells along with short microvilli.
Microanatomical changes are observed in the gill, skin and in the various
regions of the alimentary canal of H. fossilis after applying a sublethal dose of
Metanil Yellow for the periods of exposure of 30 and 45 days. Clubbing ends
of the lamellae and fusion of adjacent lamellae of gill of H. fossilis have been
observed after treatment. Severe loss of microridges and disruption in the
stratified epithelial cells has also been well marked. Disruption and dilation of
mucous cells of skin of H. fossilis and excessive secretion of mucus have
been found after treatment. In buccopharynx and oesophagus shrinkage of
oval or pentagonal stratified epithelial cells and disruption as well as necrosis
in the regular arrangement of the microridges has been found. In the stomach
of H. fossilis, the disarrangement of mucosal folds, destruction of epithelial
cells, fragmentation of microridges and also excessive secretion of mucus are
well marked after treatment. The mucosal folds of the intestine have been
disrupted. The columnar epithelial cells along with densely packed microvilli
have been damaged. Disarrangement of the microridges of the epithelial cells
and excessive secretion of mucus have been observed after treatmernt.
6. The localization and the chemical nature of acid and neutral mucin in the
intestine and rectum of H. fossilis have been investigated by employing PAS[4]
SYNOPSIS
AB tests. AF-AB test has also been applied in the intestine of H. fossilis for
detecting the acid and neutral mucins. The present investigation has shown
that the connective tissues of lamina propria and submucosa are PAS
positive while the muscularis layers have shown negative reaction. In case of
rectum of H. fossilis, mucous cells have shown the positive reaction to PASAB while the brush borders of the rectal villi have shown the moderate
reaction. In case of intestine, the mucous cells of the intestine of H. fossilis
are well stained with AF-AB reaction. The submucosal connective tissues and
columnar epithelial cells of H. fossilis are positive to AF-AB test while brush
border and luminal mucin are showing moderate reaction to this test.
Histochemical localization of glycogen has been shown in the stomach and
liver of H. fossilis by Best’s Carmine. Moderate amount of glycogen is well
marked in the columnar epithelial cells of the stomach of H. fossilis. Most
glycogen has been noticed in the cytoplasm of the Best’s Carmine test but
ground connective have shown weak content of glycogen.
Sublethal concentration of Metanil Yellow for 30 and 45 days has shown the
variations of acid and neutral mucopolysaccharide contents in the intestine
and rectum of H. fossilis. Significant reduction in the neutral and acid mucin
have not been detected in the secretory and non-secretory mucous cells,
brush borders as well as lamina propria of the intestine of H. fossilis by
following PAS-AB method after treatment. The apical brush border and the
basal region of the intestinal villi have shown intense reaction of PAS-AB test
while the secreted luminal mucin has shown AB positive reaction after
treatment with Metanil Yellow. An exposure of Metanil Yellow causes the
reduction of AF-AB reaction in the mucous cells of intestine of H. fossilis. The
columnar epithelial cells and disrupted lamina propria are well marked but
showing weak reaction to this test. The absence of glycogen in the gastric
glands of stomach has also been well marked after treatment. The amount of
glycogen has been found to be decreased in the hepatic cells of H. fossilis
owing to Metanil Yellow toxicity.
[5]
SYNOPSIS
7. Maximum protein content is well marked in the stomach, liver and muscle of
H. fossilis. Moderate amount have been found in the anterior and middle
intestine while lowest amount has been found in the posterior intestine. The
maximum reduction in protein content has been noticed in the stomach and
liver of H. fossilis.
8. The pH in stomach of H. fossilis shows acidic in nature, near about 4.7, while
in intestine and rectum it is near about 8.2 and in liver and pancreas it varies
from 7.4 to 8.3. The maximum amylase activity is shown in the middle
intestine and pancreas of H. fossilis while moderate quantity of amylase is
found in the stomach and liver of the same. Lowest activity is well marked in
the oesophagus and muscle of H. fossilis. The highest protease activity is
detected in the stomach and pancreas while moderate activity in the anterior
and middle intestine and in liver. Lowest activity is detected in the muscle of
H. fossilis. The lipase activity shows maximum in the stomach and pancreas
of H. fossilis, and moderate in the intestinal parts while oesophagus and
muscle of H. fossilis exhibit low lipase activity.
Maximum reduction in amylase activity is noticed in middle intestine and
pancreas of H. fossilis after treatment with Metanil Yellow. But there is no
marked reduction found in oesophagus and stomach. Due to Metanil Yellow
toxicity protease activity has been found to reduce mainly in the stomach and
pancreas of H. fossilis. But less reduction occurs in the liver of H. fossilis.
Protease activity has also been reduced in anterior, middle and posterior
intestine. Lipase activity has become reduced maximally in the stomach and
pancreas. Decrease in the lipase activity has also been well marked in the
anterior, middle and posterior intestine. There is no significant alteration in the
lipase activity as found in oesophagus and liver of H. fossilis.
9. The weight of albino rat has been reduced after treatment.
10. Histologically, the tongue of albino rat (R. norvegicus) consists of mucous
membrane. The dorsal surface of tongue is irregular with a number of
papillae. Conical and elongated filiform papillae are present in the tongue.
[6]
SYNOPSIS
Taste buds are present in the stratified squamous epithelium of these
papillae. Oesophagus consists of thick keratinized stratified squamous
epithelium, the lamina propria and muscularis layer. A few mucous glands are
found in the lamina propria of the oesophagus. The fore-stomach of rat
consists of stratified squamous epithelium, lamina propria, muscularis and the
submucosa. The columnar cells contain microvilli and thin tips containing
mucigen. Gastric glands are found in the stomach of R. norvegicus. The
intestine of albino rat is made up of simple, long columnar epithelial cells each
with basally or centrally placed nucleus. Brush borders and lamina propria are
also present in this region. Finger like intestinal villi are found as an outgrowth
of the mucosa. Duodenum of R. norvegicus has columnar epithelial cells
embraced with microvilli. Absorptive and goblet cells are present in this
region. Ileum of the rat contains same coats and glands as those of the
duodenum. Crypts of Lieberkuhn have been found in both the duodenum and
ileum of rat tissues. Colon contains absorptive cells which have well projected
microvilli. Colon also has Crypts of Lieberkuhn within numerable mucous
cells. Paneth cells are found to be rare in this tissue. The surface epithelium
of rectum contains more goblet cells. Histologically, the liver of R. norvegicus
contains numerous polyhedral hepatic lobules each of them contains central
vein. Liver cells or hepatocytes are polyhedral in shape with one or
occasionally two spherical nuclei with prominent nucleoli. Kidney of rat
contains a cup-shaped hollow structure called Bowman’s capsule whose
concavity is occupied by a globular tuft of glomerulus. Bowman’s capsule is
continuous with cuboidal proximal convoluted tubule. In kidney of R.
norvegicus the distal convoluted tubule contains spherical nuclei. The spleen
contains white pulp and red pulps. Brain of R. norvegicus has Purkinje layer
and granular layer. Each testis of rat contains sertoli cells and spermatogonia.
Each testicular lobule is surrounded to form seminiferous tubules.
After the exposure periods of 30 and 45 days in Metanil Yellow following a
sublethal dose of 3.0 g/kg body weight several toxic effects have been found
in the tongue, oesophagus, stomach, intestine, duodenum, ileum, colon,
[7]
SYNOPSIS
rectum, liver, kidney, spleen, brain and testis of albino rat (R. norvegicus).
The tongue papillae become damage after treatment. Distortion in the taste
buds has also been recorded due to Metanil Yellow toxicity. Changes in the
stratified epithelial cells have been found after 30 days treatment. But
maximum changes are observed after 45 days treatment. Lamina propria and
the mucous glands have been found to become necrosed after treatment.
The mucosal folds of the stomach have been disrupted. Degeneration in
gastric epithelium and gastric glands and excessive secretion of mucus has
been found after treatment. The mucosal epithelium and the connective tissue
of lamina propria of intestine have been found to be distorted due to the effect
of Metanil Yellow. Necrosis in the brush borders and disruption in the
Paneth’s cells has also been well marked. The Crypts of Liberkuhn in case of
duodenum and ileum undergo necrosis due to the toxicity of Metanil Yellow.
Distortion in the microvilli and in the lamina propria of colon and rectum has
also been noticed in R. norvegicus. Damages are well marked in the mucous
cells of the rectum. Hepatocytes and the central vein of liver have been
damaged after treatment. Pycnotic nuclei have also been observed after
treatment. Cystic cells have also been found after 45 days treatment.
Damage in the proximal and distal convoluted tubules is also well marked.
The Bowman’s capsule and glomerulus has been affected due to toxicity of
Metanil Yellow. Significant changes have been noticed in the white and red
pulp regions of spleen of R. norvegicus. Purkinje layer and granular layers of
the brain of R. norvegicus have been damaged due to toxicity. Degeneration
in seminiferous tubules and spermatogonia, and vacuolation in the sertoli
cells has been noticed after treatment. Necrosis in the seminiferous tubules
and pycnosis in the spermatocytes and vacuolation in the sertoli cells have
also been well marked after 45 days treatment.
11. Microanatomical study on R. norvegicus reveals that the muscular tongue
contains papillae which are the elevations of the oral epithelium and lamina
propria. The stratified epithelial cells and several taste buds have also been
well marked in oesophagus. Oval and rounded columnar epithelial cells are
[8]
SYNOPSIS
packed with microvilli and numbers of gastric pits are found in the mucosal
surface of stomach of R. norvegicus. Intestinal villi contain absorptive and
mucous secreting goblet cells while intestinal mucosa is packed with round
columnar epithelial cells. Duodenum of R. norvegicus contains columnar
epithelial cells along with microvilli. Compactly arranged epithelial cells and
mucosal folds are well developed in ileum of R. norvegicus. Plenty of mucus
secreting cells, columnar epithelial cells and mucosal folds have been
detected in the colon. Mucous membrane of rectum has a number of
longitudinal folds. Rectum of R. norvegicus also contains round and oval
columnar epithelial cells.
After treatment the tongue papillae along with taste buds have been
damaged. Necrosis has been shown in the stratified epithelial cells of
oesophagus of R. norvegicus. Disarrangement of mucosal folds and distortion
of microridges of oesophagus and stomach have also been well marked in R.
norvegicus. Arrangement of columnar epithelial cells and excessive secretion
of mucus have been well noticed in stomach. Intoxication of Metanil Yellow
has shown to create lossened structural configuration of columnar epithelial
cells along with microvilli of intestine and duodenum of R. norvegicus.
Mucosal folds have also been found to be damaged after treatment.
Damaged columnar epithelial cells and excessive secretion of mucus have
been observed in ileum and colon of R. norvegicus. The columnar epithelial
cells and mucosal folds of rectum of R. norvegicus have been ruptured due to
toxicity of Metanil Yellow.
12. In case of R. norvegicus maximum PAS-AB reaction has been detected in the
mucous cells of intestine while the columnar epithelial cells are found to
become negative to this test. Brush borders and luminal mucin of the intestine
of R. norvegicus depict the intense PAS-AB reaction. The mucous cells in the
rectum have shown the positive reaction to PAS-AB in R. norvegicus. More
acid mucin containing mucous cells have been found in the rectal region of
albino rat. The brush borders of villi, the columnar epithelial cells and lamina
propria are showing the positive PAS-AB test. The mucous cells of intestine
[9]
SYNOPSIS
of R. norvegicus have exhibited intense AF-AB reaction while the submucosal
connective tissue and columnar epithelial cells have shown weak reaction.
The brush borders and luminal mucin are showing moderate reaction to this
test. The moderate amount of glycogen has been shown in the columnar
epithelial cells, mucosal border and in the gastric glands of R. norvegicus. But
in case of liver of R. norvegicus glycogen content have been focused in the
cytoplasm of the hepatic cells while the ground connective tissue have been
found to become weak to Best’s carmine test.
After treatment with Metanill Yellow profuse quantity of mixture of both acid
and neutral mucin have been detected in the Paneth’s cells and also in the
lamina propria of the intestine of R. norvegicus. During the chronic toxic
effects of Metanil Yellow the intensity of PAS-AB reaction have been found to
be more pronounced in the mucous cells of the mucosa layer of the rectum of
R. norvegicus. Maximum reduction in the intensity of AF-AB test are well
marked in the mucous cells of the intestine of R. norvegicus. The luminal
mucin of the intestine has shown profuse quantity of mucopolysaccharides
while the columnar epithelial cells and disrupted lamina propria has been
positive to this test. Absence of glycogen content has been found in the
columnar epithelial cells of gastric epithelium of R. norvegicus. Amount of
glycogen content have been decreased in the hepatic cells of R. norvegicus
due to the toxicosis of Metanil Yellow.
13. In case of R. norvegicus highest protein content has been found in stomach
and liver. Moderate amount has been found in both anterior and middle
intestine. But due to toxicosis of Metanil Yellow protein content have been
significantly reduced in stomach and liver of R. norvegicus.
14. In case of albino rat (Rattus norvegicus) the intestine and rectum have
alkaline pH (near about 8.3) while liver and pancreas has alkaline pH varies
from 7.3 to 8.5) but stomach is found to be acidic (pH near about 4.9).
Maximum amylase activity has been noticed in pancreas, liver and
oesophagus. Moderate activity has been well marked in anterior and middle
[10]
SYNOPSIS
intestine. But comparatively low amylase activity is recorded experimentally in
the stomach, posterior intestine and muscle. Highest protease activity has
been found in liver and pancreas of R. norvegicus while anterior and middle
intestine and in muscle have shown moderate activity of the aforesaid
enzyme. Maximum lipase activity is observed in the liver and pancreas of R.
norvegicus, but moderate in the anterior and middle intestine.
Owing to the toxicity of Metanil Yellow the amylase activity has been found to
be reduced in oesophagus, liver and pancreas of R. norvegicus. Maximum
reduction in the protease activity has been found in liver and pancreas of R.
norvegicus, but changes are not been well marked in stomach and posterior
intestine. In R. norvegicus lipase activity has been found to be reduced in liver
and pancreas after chronic exposure of Metanil Yellow. But in oesophagus
and muscle the activity of the aforesaid enzyme has not been marked.
Signature of Research Scholar
[RITUPARNA SARKAR]
Signature of Supervisor
[APURBA RATAN GHOSH]
[11]