Histopathology of the Foot, Gill and Digestive Gland Tissues of Freshwater Mussel, Lamellidens marginalis Exposed to Oil Effluent

Abstract

Abstract
We investigated the histopathological alterations in the tissues of freshwater
mussel, Lamellidens marginalis in response to oil effluent. Based on the
previous acute toxicity, two sub lethal [1/4th (11.88ppt) and 1/10th (8.55ppt)]
concentrations of oil effluent (hydrocarbon) were prepared and exposed to
mussels. In a first series of experiment, animals were exposed/accumulated
for 30 days [Ist, 8th, 15th, 22nd and 30th days] by two sub lethal concentrations
of oil. In a second series of experiment, oil exposed animals were thereafter
transferred to clean water and kept in it up to 30 days [Ist, 8th, 15th, 22nd and
30th days] to assess the recovery pattern (depuration). At seven-day intervals,
histopathological alterations were analyzed in foot, gill and digestive gland
tissues of mussel. First series of experimental observation showed remarkable
damages in foot (disorganized outer epithelium, necrosis of the cell, the formation
of lumina, disorganized muscle bundle); in gill (disruption of gill filaments,
odema formation, necrosis, dis-aggregated cilia) and in digestive gland (stoma,
detached glandular epithelium, vertical clefts, presence of leucocytes, dense
accumulation of luminal material) and also oil effluent inducement are confirmed
with the aforementioned results. At second series of experiment, it was found
that oil effluent tended to accumulate in tissues in a duration-dose-dependent
manner. Tissue burden by oil effluent of mussels completely were restored at
30th day. The present experimental findings may be of early warning signals of
oil effluent pollution. In conclusion oil effluent are highly toxic to the Lamellidens
marginalis.
Keywords: Oil effluent; Accumulation and depuration; Histopathology of
foot; Gill; Digestive gland; Lamellidens marginalis

Full text

Citation: Balamurugan S and Subramanian P. Histopathology of the Foot, Gill and Digestive Gland Tissues of
Freshwater Mussel, Lamellidens marginalis Exposed to Oil Efuent. Austin J Environ Toxicol. 2021; 7(1): 1033.
Austin J Environ Toxicol - Volume 7 Issue 1 - 2021
ISSN: 2472-372X | www.austinpublishinggroup.com
Balamurugan et al. © All rights are reserved
Austin Journal of Environmental Toxicology
Open Access
Abstract
We investigated the histopathological alterations in the tissues of freshwater
mussel, Lamellidens marginalis in response to oil efuent. Based on the
previous acute toxicity, two sub lethal [1/4th (11.88ppt) and 1/10th (8.55ppt)]
concentrations of oil efuent (hydrocarbon) were prepared and exposed to
mussels. In a rst series of experiment, animals were exposed/accumulated
for 30 days [Ist, 8th, 15th, 22nd and 30th days] by two sub lethal concentrations
of oil. In a second series of experiment, oil exposed animals were thereafter
transferred to clean water and kept in it up to 30 days [Ist, 8th, 15th, 22nd and
30th days] to assess the recovery pattern (depuration). At seven-day intervals,
histopathological alterations were analyzed in foot, gill and digestive gland
tissues of mussel. First series of experimental observation showed remarkable
damages in foot (disorganized outer epithelium, necrosis of the cell, the formation
of lumina, disorganized muscle bundle); in gill (disruption of gill laments,
odema formation, necrosis, dis-aggregated cilia) and in digestive gland (stoma,
detached glandular epithelium, vertical clefts, presence of leucocytes, dense
accumulation of luminal material) and also oil efuent inducement are conrmed
with the aforementioned results. At second series of experiment, it was found
that oil efuent tended to accumulate in tissues in a duration-dose-dependent
manner. Tissue burden by oil efuent of mussels completely were restored at
30th day. The present experimental ndings may be of early warning signals of
oil efuent pollution. In conclusion oil efuent are highly toxic to the Lamellidens
marginalis.
Keywords: Oil efuent; Accumulation and depuration; Histopathology of
foot; Gill; Digestive gland; Lamellidens marginalis
Research Article
Histopathology of the Foot, Gill and Digestive Gland
Tissues of Freshwater Mussel, Lamellidens marginalis
Exposed to Oil Efuent
Balamurugan S1* and Subramanian P2
1Department of Zoology, Arignar Anna Government Arts
College, India
2Department of Animal Science, Bharathidasan
University, India
*Corresponding author: Balamuirugan S, Department
of Zoology, Arignar Anna Government Arts College,
Musiri-621 211, Tiruchirappalli-District, Tamilnadu, India
Received: December 04, 2020; Accepted: December
30, 2020; Published: January 06, 2021
Abbreviations
EP: Epithelium; MU: Muscle Tissue; CI: Cilia; NE: Nucleus; BS:
Blood Sinus; MU: Muscle Tissue; NE: Necrosis of Epithelial Tissue;
GF-Gill Filaments: CR: Chitinus Rod; FL: Frontal Lateral Cilia; FC:
Frontal Cilia; IS: Interlamellar Space; IJ: Interlamellar Junction;
WC: Water Chamber; MU: Muscle Tissue; SC: Supra Brachial
Chamber; GF: Gill Filaments; FL: Frontal Lateral Cilia; DD: Digestive
Diverticula; LU: Lumen; ST: Stomach
Introduction
Researchers with dierent expertise have converged towards
a common interest for understanding and solving the problems
associated with the occurrence of toxic level of contaminants in the
environment, giving raise to the spectacular development of research
in the eld of Environmental Contamination and Toxicology,
which has emerged as a multidisciplinary science resulting from
the integration of classical disciplines such as toxicology, cell and
molecular biology, physiology, ecology, chemistry, etc [1]. Uptake
and accumulation of xenobiotics in the tissues of aquatic organisms
occur from the sediment, contaminated water column and food
chain [2] that cause deleterious eects. Incorporation of even very
low levels of toxicants in the body of aquatic organisms causes various
biochemical, physiological and hematological alterations in vital
tissues [3]. Most common usage of the term biomarker has been for
biochemical, physiological or histological indicators of their exposure
to or the eects of xenobiotic chemicals at the sub-organismal or
organismal level [4]. Most of the monitoring programmes are conned
to the chemical analysis of accumulated substances, but sometimes
include toxic responses, for instance histopathological eects [5,6] or
physiological/biochemical responses [7]. When Polycyclic Aromatic
Hydrocarbon (PAHs) exposed animals, several deleterious eects
such as DNA damage [8]. As an indicator of exposure to contaminant,
histology represents a useful tool to assess the degree of pollution,
particularly for sub lethal and chronic eects [9]. Histological
changes appear as a medium-term response to sub-lethal stressors,
and histology provides a rapid method to detect eects tissues and
organs [10]. Summary of some relevant earlier literature on marine
as well as freshwater mussels histopathological observations with
various toxicant exposure results are compiled (Table 1). In molluscs,
especially in Lamellidens marginalis histological injuries, in response
to the exposure to oil euent, remains unexplored. Gills are the vital
organs, which come into direct contact with water and are indicative
of any environmental stress and also in shes gills are the major vital
respiratory organs [11]. e numerous lamellae along the double
row of lament attached to the gill arch are aected by toxicants

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Marine Mussels Pollutants Tissues Responses/Effect Year Reference
Mytilus
galloprovincialis Metals Gill, digestive gland brown cells, metal burden in tissues 1997 [42]
Perna indica Heavy metals Gill, digestive galnd Clumping of ciliary, damage of gill laments, dislodged
epithelial cells, disintegration of digestive tubules 2005 [46]
Crenomytilus
grayanus Heavy metals and pesticides Digestive gland Heavy vacuolization of digestive cells, desquamation
of digestive cells of tubules, necrosis, Edemata, lysis of
vesicular cells and of muscle bers
2006 [17]
Perna viridis Heavy metals Gill Loss of cilia, epithelial cell damage, swollen lumen,
elongation of gill laments. 2008 [40]
Mytilus edulis Heavy metal Gill, Digestive gland
and adductor muscle Inammation and necrosis. 2010 [44]
Gafrarium
divaricatum (clams) xylene, benzene and gear oil-WSF Hepatopancreas Cell debris, fusion of Nuclei, interruption of lumen line,
disintegration of epithelial cells, necrosis, iated epithelial
layer, detachment of epithelial cells. 2011 [53]
Perna viridis Heavy metal Gill, Digestive gland
and adductor muscle
digestive epithelium, hemocytic inltration in the gills
and myodegeneration in the muscle tissue, necrosis
and digestive tubule thickness 2012 [45]
Mytilus
galloprovincialis PAH Digestive gland Altered diverticula, damages in digestive tubule 2013 [47]
Ruditapes
decussatus Anthropogenic activities Gill ,Digestive gland intertubular tissue necrosis, lesions such as digestive
tubule (diverticula) 2013 [52]
Mytilus
galloprovincialis Industry Efuent (Iron, paper
Harbour, cement etc.,) Gill, Hepatopancreas dejeneration, cell loss and necrose, lumen enlargement,
Cilia loss and fusion, haemocytic inltration, vacuolar
degeneration 2016 [48]
Mytilus
galloprovincialis Cadmium Digestive gland lumen of digestive tubules, increase of the atrophic
tubule 2016 [51]
Mytilus spp. Mixture of Microplastics Gill, Digestive gland Necrosis, atrophies, lumen enlargement 2019 [39]
Mytilus
galloprovincialis Insecticide Gill, Digestive gland Vacuolation, epithelial alterations, lipofuscin aggregates,
presence of brown cells, digestive tubule alterations,
hypertrophy, hyperplasia. 2020 [36]
Freshwater Mussel
Lamellidens
marginalis Oil efuent (hydrocarbon) Foot, Gill, Digestive
gland
Foot-disorganization ofouter epithelium, necrosis of
the cell, formation of lumina, disorganisation of muscle
bundle, Gill-disruption of the epithelium, oedima
formation and necrosis, cilia appeared disaggregated
Digestive gland- stroma, detached glandular epithelium,
dense accumulation leucocytes, integrity of the
epithelium, vertical clefts.
2020 *Our Results
Lamellidens
marginalis Heavy metals Foot, Hepatopancreas splitting of muscle bundles, loss of connective tissue,
oedema ,atrophy of muscle bundles, Cell necrosis,
damage to the intertubular connective tissue 2008 [26]
Lamellidens
marginalis Insecticide Gill The bulging of primary lament gill tips, curling of
secondary lament, fusion of gill lamellae, hyperplasia,
necrotic and clavate globate lamellae of the gills 2011 [33]
Lamellidens
marginalis Dimethoate Hepatopancreas Distruption in digestive tubules, disrupted epithelial
lining and necrotic tissue in the lumen, Hypertrophic
nucleus, Necrotic tissue 2011 [54]
Lamellidens
marginalis Dimethoate Gill Disruption in epithelium, damage in epithelial lining,
nuclear hypertrophy etc. 2012 [34]
Lamellidens
marginalis Pesticide Gill, Digestive gland
gill exhibiting reduced space between water channel
and interlamellar junction ,intense inltration of hyper
chromatic anaplastic cells , tissue swelling ,irregular
shaped branchial laments, digestive gland exhibiting
hepatic tubules with disintegrated epithelial cells and
inltrated basophilic cells
2012 [35]
Dreissena
polymorpha Fluoride Gill, Digestive gland scattered pyknotic nuclei, condensed nuclei, altered
morphology of the cells 2012 [50]
Lamellidens
marginalis Monocrotophos Foot Hyperplasia of marginal pedal glands, distruption of
nuclei of the epithelial glands. 2015 [27]
Lamellidens
marginalis Mercury choloride Gill Hypoplasia of epithelial cells, gill laments altered,
oedematic, necrotic and vacuolated epithelium 2016 [41]
Anodonta cygnea Heavy metals Gill, digestive gland
and gonads
Gills-lamellar fusion, dilated hemolymphatic sinus,
clumping, and generation of cilia and hemocytic
inltration digestive gland-inammation, hydropic
vacuolation, and lipofuscin pigments, and gonads-
atresia, necrosis, granulocytoma, hemocytic inltration
2018 [49]
Unio Pictorum Pesticide Gill damaged cilia, epithelium rupture, damaged epithelium 2019 [37]
Lamellidens
marginalis Pesticide Gill Damaged ciliated epithelium, Elongated gill lament,
Delaminated ciliated epithelium, gill epithelium ruptured
with damaged ciliary lining 2019 [38]
Table 1: Summary of Relevant Earlier Literatures on Freshwater and Marine Mussels Histolagical Changes with Various Xenobiotic Exposures and Comparison with
Our Results.

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[12]. Molluscs are widely used in dierent biomonitoring projects
and their histopathological analysis provides information about the
general health of the animals and contaminant-specic changes in
the tissues. Although laboratory as well as eld studies suggest that
pollutants cause toxic eects to molluscs, the histopathological eects
of chemical contaminants have not generally been measured [13]. Blue
mussels can retain on their gills, including oil particles have observed
[14]. Like numerous bivalves, they concentrate many xenobiotics in
their tissues and have been used extensively for biomonitoring of
pollutants [15] but there is inadequate contribution on freshwater
mussels. Gills [16] are suitable organs for histological examination
in order to determine the eect of pollution. Histological changes
occurs in the bivalves especially in the hepatopancreas (digestive
gland) as they are the metabolically active sites and are responsible
for food collection, absorption, digestion, enzymatic activity as well
as accumulation and biotransformation (detoxication) of various
organic and inorganic toxic substances upon exposure to the organic
and inorganic contaminants in the water. Pathological changes in the
vital tissues of bivalves have been reported aer pollutant exposure
[17,6]. Owing to their poor existence and meagre information about
the histopathology in invertebrates remarkably in freshwater mussels.
is present study attempts to understand the pathological injuries
in mussels. erefore, present investigation were examined during
acumulation (30 days) and depuration (recovery) period (30 days)
in response to sub lethal concentration of 1/4th (11.88ppt) and 1/10th
(8.55ppt) of oil euent in freshwater mussel tissues of foot, gill
and digestive gland. e aims of present study were to observe (1)
histopathological damages in foot, gill and digestive gland tissues of
mussels during accumulation period of both sublethal concentrations
of oil euent in comparison to control (2) whether these
histopathological damages in various tissues of mussels recoverd/
restored in the depuration period in comparison to control mussels
and (3) whether these alteration/damages would serve as a biomarker
to detect the accumulated oil euent (hydrocarbon) in this species.
Materials and Methods
Animals
Almost uniform size fresh water mussel Lamellidens marginalis
(total length 6-7 cm and weight 25-27 g) were collected from the River
Cauvery (Tiruchirappalli, India) and maintained in the laboratory.
Acute toxicity experiment
e aqueous oil euent originated from the coal conversion plant,
turbine section of boiler units in the Boiler plants of Bharat Heavy
Electricals Limited (BHEL) situated 14 km away from Tiruchirappalli,
are collectively released into a drainage canal. It consisted mainly
of hydrocarbons. Initial experiments were conducted to assess the
minimum concentration of oil euent to obtain maximum mortality,
for freshwater mussel, Lamellidens marginalis, over a 96-hr exposure.
Aer conrming the minimum concentration, 10 animals in 5L of
tubs (each) and exposed to various concentrations of oil euent,
ranging from 4ppt to 16ppt for a period of 96-hr to ascertain LC50
concentration. In addition, a control was also maintained. e 96-
hr LC50 values with 95% condence limits were calculated using
National Crop Production Centre Technical Bulletin [18].
Exposure experiment
Based on the 96-hr LC50 value of oil euent, sublethal
concentrations of 1/4th (11.88ppt) and 1/10th (8.55ppt ) of LC50 were
prepared and used for histopathology. In this study, two sets of 10-l
plastic tubs were used. In each tub, mussels were exposed to 11.88ppt
or 8.55ppt of oil euent. A control was also run simultaneously
without the addition of oil euent. At seven days interval, mussels
were sacriced for histological analysis. Aer 30 days, the treated
mussels were released into freshwater 30-day depuration (recovery)
study was conducted. Four mussels were randomly chosen and
removed from each of the two tubs (n=8) for dissection.
Preparations of tissue samples
Control set of animals tissues were sacriced for Ist, 8th, 15th, 22nd
and 30th days of foot, gill and digestive gland tissues of mussels. At
every seven days intervals, accumulation and depuration (recovery)
period of Ist, 8th, 15th, 22nd and 30th days of foot, gill and digestive
gland tissues of mussels from both exposures were sacriced for the
evaluation of histological analysis.
Paran method: For the paran method, the above specied
tissues were xed in Bouin’s uid, and embedded in paran. Serial
sections were obtained at 3-5 µm thickness using a Leica (Germany)
microtome with provision of disposable blade. Serial sections stained
in haematoxylin and eosin [19] and mounted in DPX mountant for
microscopical observations.
Microscopic analysis: For light microscopic observation Carl-
Zeiss (Germany) Axioskop 2-research microscope was used, and the
images were captured in a computer using Carl-Zeiss (Germany)
Axiovision Soware and the images processed using the same
soware. e histopathological changes in the tissues of experimental
as well as control mussels were recorded and compared.
Results
Histology of foot
Foot is the locomotory organ chiey employed for burrowing
and is formed of an outer dense epithelium, which is grown into tall
folds (villous); the epithelium lies top of the cells the musculature,
variety of protractor, retractor muscles. Blood sinuses are found
between the ne muscles. e muscular wall of the foot surrounds
the coelomic phase which itself is lined by coelomic epithelium. In the
outer epithelium, the cells dier in height and length of the nucleus.
In some of the cells the nucleus is extremely elongated. e outer
borders of the epithelium have dense cilia (Figures 1-4).
Histopathology of foot
During the exposure of both sublethal concentrations (1/4th and
1/10th) of oil euent little changes were observed in the foot tissues
of mussel at Ist day. However, from the 8th day onwards the outer
Figure 1: Haematoxylin and eosin stained parafn sections of control mussel
Lamellidens marginalis foot tissue. (X100, 400).

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epithelium was thoroughly disorganized resulted in necrosis of the
cell and formation of lumina both on top of the musculature, with
exposure for longer durations the muscle bundle themselves were
disorganized in both sublethal concentration of oil euent exposure
(Figures 5-11). During the depuration (recovery) process, the fresh
water mussel brought about almost complete restorations of the
histo-architecture of the foot tissues (Figures 12-21).
Histology of gill
e gill of the mussel is formed of an outer and inner lamellae
called ctenidium, each folded to form the outer and inner lamellae.
e lamellae are connected by inter-lamellar junctions, which contain
blood vessels. e gill lament constitutes a longitudinal array and
Figure 2: Haematoxylin and eosin stained parafn sections of control mussel
Lamellidens marginalis foot tissue. (X100, 400).
Figure 3: Haematoxylin and eosin stained parafn sections of treated mussel
(1/4th accumulation-Ist, 8th and 15th days) foot tissue. (X400).
Figure 4: Haematoxylin and eosin stained parafn sections of treated mussel
(1/4th accumulation-Ist, 8th and 15th days) foot tissue. (X400).
Figure 5: Haematoxylin and eosin stained parafn sections of treated mussel
(1/4th accumulation-Ist, 8th and 15th days) foot tissue. (X400).
Figure 6: Haematoxylin and eosin stained parafn sections of treated mussel
(1/4th accumulation-Ist, 8th and 15th days) foot tissue. (X400).
Figure 7: Haematoxylin and eosin stained parafn sections of treated mussel
(1/4th accumulation-22nd and 30th days) foot tissue. (X400).
Figure 8: Haematoxylin and eosin stained parafn sections of treated mussel
(1/4th accumulation-22nd and 30th days) foot tissue. (X400).
Figure 9: Haematoxylin and eosin stained parafn sections of treated mussel
(1/10th accumulation- 8th, 15 th and 22nd days) foot tissue. (X400, X400, X100).
Figure 10: Haematoxylin and eosin stained parafn sections of treated
mussel (1/10th accumulation- 8th, 15th and 22nd days) foot tissue. (X400, X400,
X100).
Figure 11: Haematoxylin and eosin stained parafn sections of treated
mussel (1/10th accumulation-8th, 15 and 22nd days) foot tissue. (X400, X400,
X100).

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adjacent laments are connected by inter-lamentor junctions. e
gill lament is lined by an epithelium formed of a single row cells,
short cuboidal adiphase and tall columnar towards the tip. ree
types of cilia are associated with gill lament, they are, tall lateral-cilia,
tall latero-frontal cilia and short frontal -cilia at the tip. e stroma
underlying the epithelium bridges of connective tissue containing
Figure 12: Haematoxylin and eosin stained parafn sections during
depuration period of mussel (1/4th depuration-Ist day) foot tissue. (X400).
Figure 13: Haematoxylin and eosin stained parafn sections during
depuration period of mussel (1/4th depuration-8th, 15th, 22nd and 30th days) foot
tissues. (X400, X1000).
Figure 14: Haematoxylin and eosin stained parafn sections during
depuration period of mussel (1/4th depuration-8th, 15th, 22nd and 30th days) foot
tissues. (X400, X1000).
Figure 15: Haematoxylin and eosin stained parafn sections during
depuration period of mussel (1/4th depuration-8th, 15th, 22nd and 30th days) foot
tissues. (X400, X1000).
Figure 16: Haematoxylin and eosin stained parafn sections during
depuration period of mussel (1/4th depuration-8th, 15th, 22nd and 30th days) foot
tissues. (X400, X1000).
Figure 17: Haematoxylin and eosin stained parafn sections during
depuration period of mussel (1/10th depuration-Ist and 8th days) foot tissues.
(X400).
Figure 18: Haematoxylin and eosin stained parafn sections during
depuration period of mussel (1/10th depuration-Ist and 8th days) foot tissues.
(X400).
Figure 19: Haematoxylin and eosin stained parafn sections during
depuration period of mussel (1/10th depuration-Ist and 8th days) foot tissues.
(X100, X400).
Figure 20: Haematoxylin and eosin stained parafn sections during
depuration period of mussel (1/10th depuration-15th, 22nd and 30th days) foot
tissues. (X100, X400).
Figure 21: Haematoxylin and eosin stained parafn sections during
depuration period of mussel (1/10th depuration-15th, 22nd and 30th days) foot
tissues. (X100, X400).

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connecting rods for support and also blood vessels (Figures 22,23).
Histopathology of gill
When exposed to both sublethal concentrations of oil euent
caused disruption of the epithelium of the gill laments and all the
versions of the cilia. e epithelium indicated severe pathological
changes of the oedima formation and necrosis. Disaggregated cilia
Figure 22: Haematoxylin and eosin stained parafn sections of control
mussel gill tissues (X400, X1000).
Figure 23: Haematoxylin and eosin stained parafn sections of control
mussel gill tissues (X400, X1000).
Figure 24: Haematoxylin and eosin stained parafn sections of treated
mussel gill tissues (1/4th accumulation-1st day) (X400).
Figure 25: Haematoxylin and eosin stained parafn sections of treated
mussel gill tissues (1/4th accumulation-8th, 15th, 22nd and 30th days and 1/10th
accumulation-Ist day and 8th day). (X400).
Figure 26: Haematoxylin and eosin stained parafn sections of treated
mussel gill tissues (1/4th accumulation-8th, 15th, 22nd and 30th days and 1/10th
accumulation-Ist day and 8th day). (X400).
were appeared (Figures 24-32). During the recovery period, brought
about partial to almost complete restoration of the histo-architecture
of the gill laments. e cilia appeared normal. e epithelium shows
almost free from oedima formation and necrosis (Figures 33-42).
Figure 27: Haematoxylin and eosin stained parafn sections of treated
mussel gill tissues (1/4th accumulation-8th, 15th, 22nd and 30th days and 1/10th
accumulation-Ist day and 8th day). (X400).
Figure 28: Haematoxylin and eosin stained parafn sections of treated
mussel gill tissues (1/4th accumulation-8th, 15th, 22nd and 30th days and 1/10th
accumulation-Ist day and 8th day). (X400).
Figure 29: Haematoxylin and eosin stained parafn sections of treated
mussel gill tissues (1/4th accumulation-8th, 15th, 22nd and 30th days and 1/10th
accumulation-Ist day and 8th day). (X400).
Figure 30: Haematoxylin and eosin stained parafn sections of treated
mussel gill tissues (1/4th accumulation-8th, 15th, 22nd and 30th days and 1/10th
accumulation-Ist day and 8th day). (X400).
Figure 31: Haematoxylin and eosin stained parafn sections of treated
mussel gill tissues (1/10th accumulation-22nd and 30th days). (X400).

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Histology of digestive gland
e digestive gland consists of diverticula and their ducts,
which connected to be, and inter diverticula tissue (Figure 43). e
Figure 32: Haematoxylin and eosin stained parafn sections of treated
mussel gill tissues (1/10th accumulation-22nd and 30th days). (X400).
Figure 33: Haematoxylin and eosin stained parafn sections during
depuration period of mussel gill tissues (1/4th depuration-Ist, 8th, 15th, 22nd
days). (X400).
Figure 34: Haematoxylin and eosin stained parafn sections during
depuration period of mussel gill tissues (1/4th depuration-Ist, 8th, 15th, 22nd
days). (X400).
Figure 35: Haematoxylin and eosin stained parafn sections during
depuration period of mussel gill tissues (1/4th depuration-Ist, 8th, 15th, 22nd
days). (X400).
Figure 36: Haematoxylin and eosin stained parafn sections during
depuration period of mussel gill tissues (1/4th depuration-Ist, 8th, 15th, 22nd
days). (X400). glandular epithelium is formed of dierent types, most of which are
tall columnar. e dierent heights of the cell, render the epithelium
appears live villous folds, lamellae propitious underlies the epithelium.
e lumen of diverticulate contains a few materials, which are likely
the ingested food (Figure 44). e nucleus of epithelium is elongated,
Figure 37: Haematoxylin and eosin stained parafn sections during
depuration period of mussel gill tissues (1/4th depuration-30th days). (X400).
Figure 38: Haematoxylin and eosin stained parafn sections during
depuration period of mussel gill tissues (1/10th depuration-Ist, 8th, 15th, 22nd
and 30th days). (X400).
Figure 39: Haematoxylin and eosin stained parafn sections during
depuration period of mussel gill tissues (1/10th depuration-Ist, 8th, 15th, 22nd
and 30th days). (X400).
Figure 40: Haematoxylin and eosin stained parafn sections during
depuration period of mussel gill tissues (1/10th depuration-Ist, 8th, 15th, 22nd
and 30th days). (X400).
Figure 41: Haematoxylin and eosin stained parafn sections during
depuration period of mussel gill tissues (1/10th depuration-Ist, 8th, 15th, 22nd
and 30th days). (X400).

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spindle shaped and darkly staining, it is located at dierent heights of
the cells. e folds of epithelium produce pockets in the prole of the
lumen (Figure 45).
Histopathology of digestive gland
When exposed to both subletahal concentrations of oil euent,
produced gross changes in the epithelium of the glands as well as
Figure 42: Haematoxylin and eosin stained parafn sections during
depuration period of mussel gill tissues (1/10th depuration-Ist, 8th, 15th, 22nd
and 30th days). (X400).
Figure 43: Haematoxylin and eosin stained parafn sections of control
mussel Lamellidens marginalis digestive gland tissue. (X100, X400, X100).
Figure 44: Haematoxylin and eosin stained parafn sections of control
mussel Lamellidens marginalis digestive gland tissue. (X100, X400, X100).
Figure 45: Haematoxylin and eosin stained parafn sections of control
mussel Lamellidens marginalis digestive gland tissue. (X100, X400, X100).
Figure 46: Haematoxylin and eosin stained parafn sections of treated
mussel digestive gland tissue. (1/4th accumulation-Ist, 8th and 15th days)
(X400).
the stroma. e glandular epithelium was invariably detached from
the stroma (Figure 46-65). is stroma adds dense accumulation
leucocytes from Ist day of both sublethal concentrations. e integrity
of the epithelium was thoroughly disrupted and a major change was
consisted of vertical cles. Another important feature was noticed
towards the dense accumulation of the luminal material, which also
Figure 47: Haematoxylin and eosin stained parafn sections of treated
mussel digestive gland tissue (1/4th accumulation-Ist, 8th and 15th days) (X400).
Figure 48: Haematoxylin and eosin stained parafn sections of treated
mussel digestive gland tissue. (1/4th accumulation-Ist, 8th and 15th days)
(X400).
Figure 49: Haematoxylin and eosin stained parafn sections of treated
mussel digestive gland tissue. (1/4th accumulation-22nd and 30th days) (X400).
Figure 50: Haematoxylin and eosin stained parafn sections of treated
mussel digestive gland tissue. (1/4th accumulation-22nd and 30th days) (X400).
Figure 51: Haematoxylin and eosin stained parafn sections of treated
mussel digestive gland tissue. (1/10th accumulation-Ist, 8th, 15th and 22nd days)
(X400).

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contain leucocytes, which were otherwise conned to stroma. During
the recovery (depuration) period, the fresh water mussel brought
Figure 52: Haematoxylin and eosin stained parafn sections of treated
mussel digestive gland tissue. (1/10th accumulation-Ist, 8th, 15th and 22nd days)
(X400).
Figure 53: Haematoxylin and eosin stained parafn sections of treated
mussel digestive gland tissue. (1/10th accumulation-Ist, 8th, 15th and 22nd days)
(X400).
Figure 54: Haematoxylin and eosin stained parafn sections of treated
mussel digestive gland tissue. (1/10th accumulation-Ist, 8th, 15th and 22nd days)
(X400).
Figure 55: Haematoxylin and eosin stained parafn sections of treated
mussel digestive gland tissues (1/10th accumulation-30th days). (X400).
Figure 56: Haematoxylin and eosin stained parafn sections of treated
mussel digestive gland tissue. (1/4th accumulation-Ist, 8th, 15th, 22nd and 30th
days) (X1000).
about a gradual restoration of the organization of the digestive gland
and nature of the epithelium. e day 15th onwards the glandular
Figure 57: Haematoxylin and eosin stained parafn sections of treated
mussel digestive gland tissue. (1/4th accumulation-Ist, 8th, 15th, 22nd and 30th
days) (X1000).
Figure 58: Haematoxylin and eosin stained parafn sections of treated
mussel digestive gland tissue. (1/4th accumulation-Ist, 8th, 15th, 22nd and 30th
days) (X1000).
Figure 59: Haematoxylin and eosin stained parafn sections of treated
mussel digestive gland tissue. (1/4th accumulation-Ist, 8th, 15th, 22nd and 30th
days) (X1000).
Figure 60: Haematoxylin and eosin stained parafn sections of treated
mussel digestive gland tissue. (1/4th accumulation-Ist, 8th, 15th, 22nd and 30th
days) (X1000).
Figure 61: Haematoxylin and eosin stained parafn sections of treated
mussel digestive gland tissue. (1/10th accumulation-Ist, 8th, 15th, 22nd and 30th
days). (X1000).

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architecture was comparable to that of the control mussels, though
the dense accumulation of leucocytes between the stroma and
Figure 62: Haematoxylin and eosin stained parafn sections of treated
mussel digestive gland tissue. (1/10th accumulation-Ist, 8th, 15th, 22nd and 30th
days). (X1000).
Figure 63: Haematoxylin and eosin stained parafn sections of treated
mussel digestive gland tissue. (1/10th accumulation-Ist, 8th, 15th, 22nd and 30th
days). (X1000).
Figure 64: Haematoxylin and eosin stained parafn sections of treated
mussel digestive gland tissue. (1/10th accumulation-Ist, 8th, 15th, 22nd and 30th
days). (X1000).
Figure 65: Haematoxylin and eosin stained parafn sections of treated
mussel digestive gland tissue. (1/10th accumulation-Ist, 8th, 15th, 22nd and 30th
days). (X1000).
Figure 66: Haematoxylin and eosin stained parafn sections during
depuration period of mussel digestive gland tissue. (1/4th depuration-Ist day)
(X1000).
epithelium continued till the 30th day, but the stroma was absent i.e.
completely restored (Figure 66-75).
Figure 67: Haematoxylin and eosin stained parafn sections during
depuration period of mussel digestive gland tissue. (1/4th depuration-8th, 15th,
22nd and 30th days; 1/10th depuration-1st and 8th days). (X1000).
Figure 68: Haematoxylin and eosin stained parafn sections during
depuration period of mussel digestive gland tissue. (1/4th depuration-8th, 15th,
22nd and 30th days; 1/10th depuration-1st and 8th days). (X1000).
Figure 69: Haematoxylin and eosin stained parafn sections during
depuration period of mussel digestive gland tissue. (1/4th depuration-8th, 15th,
22nd and 30th days; 1/10th depuration-1st and 8th days). (X1000).
Figure 70: Haematoxylin and eosin stained parafn sections during
depuration period of mussel digestive gland tissue. (1/4th depuration-8th, 15th,
22nd and 30th days; 1/10th depuration-1st and 8th days). (X1000).
Figure 71: Haematoxylin and eosin stained parafn sections during
depuration period of mussel digestive gland tissue. (1/4th depuration-8th, 15th,
22nd and 30th days; 1/10th depuration-1st and 8th days). (X1000).

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Discussion
Biological, physiology and morphological structure of molluscan
systems were described [20-22]. e normal structure of mussels gill,
foot, digestive gland has been well-described [23,24]. In some
publications, lesions have only been described morphologically [25].
Summary of some relevant earlier literature on marine as well as
freshwater mussels histological observations with various toxicants
and comparison with our present results are compiled (Table 1). In
the present study, when exposed to both sublethal concentrations of
oil euent little changes were observed in mussels foot tissue on Ist
day. From 8th day onwards, outer epithelium of the mussels’ foot
tissue were disorgnaised and resulted in necrosis of the cell. When
Figure 72: Haematoxylin and eosin stained parafn sections during
depuration period of mussel digestive gland tissue. (1/4th depuration-8th, 15th,
22nd and 30th days; 1/10th depuration-1st and 8th days). (X1000).
Figure 73: Haematoxylin and eosin stained parafn sections during
depuration period of mussel digestive gland tissue. (1/10th depuration-15th
22nd and 30th days) (X1000).
Figure 74: Haematoxylin and eosin stained parafn sections during
depuration period of mussel digestive gland tissue. (1/10th depuration-15th
22nd and 30th days) (X1000).
Figure 75: Haematoxylin and eosin stained parafn sections during
depuration period of mussel digestive gland tissue. (1/10th depuration-15th
22nd and 30th days) (X1000).
exposed to the sublethal concentrations of oil euent, for longer
duration’s muscle bundle themselves were also disorgnaised in
mussel foot tissue. is could lead to the failure of a number of
biochemical activities as well as osmo and iono-regulatory functions
of the foot. Present histopathological ndings may explain a defensive
reaction from the mussels under investigation as similar results were
observed by molluscan researchers. us, the present results are in
agreement with [26] who exposed to heavy metals splitting of muscle
bundles in foot tissue were observed in the fresh water mussel,
Lamellidens marginali. Similar ndings [27] were observed disruption
of nuclei of the epithelial glands in foot tissue when exposed to
pesticide. e histopathology of foot indicated that concentration
and duration of exposure period resulted in massive destruction in
normal architecture of foot tissue of mussel. Similarly, the disruption
of the epithelium of the gill laments and all the versions of the cilia
were observed when exposed of both sublethal concentrations of oil
euent. e epithelium indicated severe pathological changes of the
oedima formation and necrosis. e cilia appeared disaggregated
during accumulation of both sublethal concentrations of oil euent.
e gills (ctenidia) of lamellibranch bivalves play a dominant role in
controlling the interaction between the individual and its
environment. A great deal of literature is available on the mechanisms
of food particle retention, as well as on the nature and activities of the
cilary systems of such organs [28]. Histological alterations and
biochemical changes of gill tissues produced by the chemical stress
causes disturbed metabolism, enzyme inhibition, retardation of
growth, fecundity reduction and longevity of the organism, which
will aect balance in the ecosystem [29]. An exhaustive review of
toxicant/ irritant induced changes in the gill, stated that the
inammatory changes tend to be largely non-specic, and seen to
reect physiological adaptation to stresses were made [16]. Tributyltin
(TBT) treated mussel Mytilus galloprovincialis the structure of gill
was destroyed, interlament junctions and cilia disappeared and
lateral and endothelial cells were changed [30]. A wide variety of
histopathological and physiological responses to naphthalene
exposure were described in the mummichog Fundulus heteroclitus
[31]. Structural changes and proliferate lesions of gills in Salmo trutta,
Oncorhychus mykiss, was observed [32] when exposed to sewage plant
euents. When exposed to pesticides, fusion of gill lamellae in
Lamellidens marginalis [33], gill epithelium lining and disruption in
Lamellidens marginalis [34], gill exibits reducing space between
channels, tissue swelling in Lamellidens marginalis [35], vacuolation,
epithelial alterations in Mytilus galloprovincialis [36], damaged
epithelium, cilia, epithelium ruptured in gill of Unio pictorum [37],
elongated gill lament, gill epithelium ruptured with damaged ciliary
lining in Lamellidens marginalis [38] were observed and when
exposed to microplastics, epithelial alterations, necrosis were
observed in Mytilus spp. [39]. When exposed to heavy metals loss of
cilia, epithelial damage, swollen lumen, elongation of gill laments in
Perna viridis [40]; gill laments altered, oedematic, necrotic and
vacuolated epithelium in Lamellidens marginalis [41] were observed.
Hence, the changes in the histopathological structure of the gill can be
use as biomarkers of exposure in the aquatic environment and the
freshwater bivalve Lamellidens marginalis can be considered as a
bioindicator organism to assess the water quality. Similarly, the
digestive gland produced gross changes in the epithelium of the
glands as well as the stroma. e glandular epithelium invariably

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detached from the stroma were observed in mussel, during
accumulation period of oil euent. is stroma adds dense
accumulation of leukocytes from Ist day exposure of both
concentrations. oroughly disrupted integrity of the epithelium was
observed during the accumulation of both sublethal concentrations
of oil euent and a major change was consisted of vertical cles.
Another important feature were observed towards the dense
accumulation of the luminal material, which also contain leukocytes,
which were otherwise conned to stroma. e responses of digestive
gland of mussels to various pollutants exposure of previous results
and our present study are in agreement with the following ndings of
works. Disruption in the epithelial of glands, tissue burden, digestive
tube thickness, lumen enlargement, necrotic tissues by heavy metals
in Mytilus galloprovincialis [42,43], in mytilus edulis [44], in Perna
viridis, Perna indica [45,46], in Crenomytilus grayanus [17]; PAH
induced altered diverticula, damages in digestive tubules in Mytilus
galloprovincialis [47]; industrial euents alters vacuolar degeneration
in Mytilus galloprovincialis [48]; mixture of micro-plastics altered
atrophies, lumen enlargement in Mytilus spp [39]; insecticide
damages digestive tubule alterations, hypertrophy, hyperplasia in
Mytilus galloprovincialis [36]; heavy metals damages hemocytic
inltration digestive gland, inammation in Anodonta cygnea [49],
condensed nuclei, altered morphology of cell in Dreissena polymorpha
[50], increase of atrophic tubule, damages lumen of digestive tubule
in Mytilus galloprovincialis [51]; anthropogenic contaminant induces
diverticula, inter-tubular tissue necrosis in Ruditapes decussatus [52];
xylene, benzene and gear oil-WSF damages cell debris, fusion of
nuclei, disintegration of epithelial cells necrosis in Gafrarium
divaricatum (calm) [53]; pesticide alters disruption in digestive
tubules, epithelial lining, necrotic tissue in the lumen in Lamellidens
marginalis [54] were observed. Disruption of normal lysosomal
function is indicated by the increased fragility of lysosomes in
digestive cells throughout the experiment and this is reected in a
reduced physiological scope for growth [55]. Formation of neoplasams
in the digestive gland in Unio pictorum by subjecting 200-400 ppm
diethyl- and dimethylnitrosamines were observed [56]. Number of
gill mucous cells and the heights of digestive diverticula tubule cells
were dierent, compared with the control, at 2 or 4 weeks, when
exposed to cadmium were observed [57]. During depuration
(recovery) period, the mussel brought about almost complete
restorations of the histo-architecture of the foot tissue. Similarly,
brought about partial to almost complete restoration of the histo-
architecture of the gill laments. e cilia appeared normal. e
epithelium is almost free from oedima formation and necrosis.
Likewise, the mussel brought about a gradual restoration of the
organization of the digestive gland and nature of the epithelium. e
day 15th onwards the granular architecture was comparable to that of
the control mussels, though the dense accumulation of leukocytes
between the stroma and epithelium continued till the 30th day, but the
stroma was absent, completely restored. Such a high level of oil
euent toxicity (concentration and duration) in digestive gland
might be responsible of histological alterations. e digestive gland is
also helpful for metabolism of xenobiotics. e histological damage
to digestive gland tissue of Lamellidens marginalis due to exposure of
oil euent, denitely disturbs its normal functions like secretion,
absorption and storage of nutrient materials and this gland is also
helpful for metabolism of oil euent. Present histopathology
investigation on the freshwater mussel is sparse, to ll up the gap, a
preliminary study has been carried out on the mussel Lamellidens
marginalis.
Conclusion
e results of this study are well under the aims and objective of
the study. However, results of our research enabled us to understand
that histopathological changes in the foot, gill and digestive gland
tissues of freshwater mussel, Lamellidens marginalis and is noted
accumulator organism of oil pollution. e observed damage to foot,
gill and digestive gland tissues due to oil euent and denitely disturbs
its normal functions and storage of nutrient materials. Particularly,
the digestive gland is also helpful for metabolism of xenobiotics in
freshwater mussel Lamellidens marginalis. ese histopathology
might be due to the possible utilization for metabolic purpose and
may be of early warning signals of environmental pollution.
Acknowledgement
Authors wish to thank, Department of Animal Science,
Bharathidasan University, Tiruchirappalli, Tamilnadu, India, for
providing necessary facilities for completion of research work.
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