International Journal of Environment, Agriculture and Biotechnology

Vol-7, Issue-1; Jan-Feb, 2022

Journal Home Page Available: https://ijeab.com/

Journal DOI: 10.22161/ijeab

Peer Reviewed

Pathophysiologic Mechanism of Erythrocyte

Morphological Alteration in Freshwater Fish Channa
punctatus Under Exposure of Nigrosine Black (Acid Black-
2 a Tanning Industry Dye)
Jyoti Prakash Srivastava

Department of Zoology, DBS College, Kanpur-208016, Uttar Pradesh, India

Email: [email protected]

Received: 10 Dec 2021; Received in revised form: 05 Feb 2022; Accepted: 11 Feb 2022; Available online: 19 Feb 2022
©2022 The Author(s). Published by Infogain Publication. This is an open access article under the CC BY license
(https://creativecommons.org/licenses/by/4.0/).

Abstract— The present study deals with the pathophysiological effects of Nigrosine black on the
morphology of fish's erythrocyte. An exposure of 1/5th LC50 of Nigrosine black (378 mg/liter) was
produced abnormal morphology in fish blood erythrocytes. After 14 days toxicant produced a spherical
shape of erythrocyte with cytoplasmic vacuolation around the periphery of the cytoplasmic membrane of
erythrocytes. After the third-week cytoplasmic vacuolation was appear around the lateral side of the
nucleus. After the 35th day of the experiment, cytoplasmic vacuolation increased around the nucleus, while
in a few erythrocytes, nuclei also showed their acentric condition. Chronic toxicity test (1/20th of LC50 i.e.
94.5 mg/ litter), produced cytoplasmic vacuolation and acentric nucleus condition in fish erythrocyte that
enhanced after two weeks. Degeneration and fragmentation of cytoplasmic membrane of erythrocytes
appeared after the 4th to 6thweek of the experiment. Schistocytosis has appeared after the 60th day of the
experiment along with a few ghost nucleuses. Pathophysiological condition of erythrocytes showed that it
may produce alteration in cytoskeleton protein formation, disturbance in ion transport, gas transport,
immune responses, deficiency of G6PD, increased lipid peroxide formation, altered ion permeability of cell
membrane, and failure of tubulin polymerization in fishes.
Keywords— fish blood erythrocytes, Third-week cytoplasmic vacuolation, acentric nucleus condition,
cytoplasmic vacuolation, altered ion permeability.

I. INTRODUCTION located near the side of river Ganga at Jajmau area. The
Dyes are the main constituent of tannery large amount of tannery effluent containing the Nigrosine
industries effluents (Kavitha and Ganapapthy 2015 and black produced every day in tannery poured into river
Angelika et al.2020) along with other toxic chemicals. Ganga and groundwater of Jajmau area. The water is an
Annually production of dyes is approximately 384 metric important constituent of fish and the presence of such
tonnes in India in 2020. In leather industries, chemicals may cause physiological problems in them.
approximately 10-12 per cent dye is used and 2-5 per cent The blood of fish is a fluid tissue and more
disposed of after the tanning process. important and sensitive tissue for various toxicant
Nigrosine Black (Acid Black-2, C.I. No50420, reflections. Erythrocytes of fish are nucleated hence its
and C22H14N6Na2O9S2) is one of the developed dyes used play an important role in physiology, immune system,
in the tanning industry for the colouring of hides. Kanpur protein signalling and haemostatic condition along with
is an industrial city and about above the 259 tanneries is respiration. Few Authors (Jagruti and Anita,2015, Randhir

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and Banerjee 2016, Avni and Alkesh 2021) were observe II. MATERIAL AND METHODS
numerical and morphological anomalies in fish 2.1 Test Animal
erythrocytes under different chemical exposure. According
Fish Channa punctatus of both sexes with varying
to Vosyliene (1996), the basic quantitative red blood
weight were collected from local fish farmers from Kanpur
parameter in fish tends to remain stable due to
city and disinfected by dipping them in 0.01% KMNO4
considerable compensatory potential but the morphological
solution. After collection, the fish were maintained in
alteration in erythrocytes is a biomarker of environmental
laboratory aquaria for about 10 days for acclimatization
impact in fish. When these chemicals alter the morphology
following the method of Dehadrai (1971).
of erythrocytes, they affect the fish’s entire physiology,
which is bad for their population. Fish were kept in a large size aquaria (2.5`x1`x1`)
contain100 liter of water in each. Commercial fish food
There is copious literature on the effect of
was supplied daily with water was 1/10th of their body
chemicals on fish erythrocytes but no case reported on the
weight. The water was changed daily with aeration.
effect of Nigrosine on fish erythrocytes morphology.
Keeping this point of view in mind we decided to observe 2.2 Biochemical parameters of the water
the effect of Nigrosine black dye on the erythrocyte samples used in experiment and their methods of
morphology of freshwater fish Channa punctatus. analysis.

PARAMETER METHOD OF ANALYSIS VALUE REFERENCES

pH Digital pH meter 7.36 ± 1.97
Total hardness EDTA titrimetric method 283.22 ± 4.57 mg/l APHA (2005)
Dissolved oxygen Wrinkle method 8.2 ± 0.44 mg/l APHA(2005)
Turbidity Nephlometric 281 ± 3.85 mg/l APHA (2005)
O
Temperature Digital thermometer 28.87 ± 1.47 C APHA (2005)

2.3 Test chemical used in present study. 2.5 Blood Sampling and the Making of Blood
The test chemical is used in the present studies is Smear
Nigrosine Black (Acid Black-2, C.I. No50420, As per the above-fixed schedule, 6 fishes
C22H14N6Na2O9S2) obtained from a local vendor. of each group were anesthetized and sacrificed. Blood of
2.4 Experiment design and schedule of fishes was collected from the caudal peduncle in a
treatment. heparinized vial for haematological analysis. Blood smears
of fish blood were prepared by taking a drop of blood on a
Ludmila (1996) was reported LC50 (96 hrs.) of
glass slide and smeared smoothly and air-dried. Now
Nigrosine black for freshwater fish Poeciliareticulata is
smear blood slide was fixed with methanol after that blood
1890 mg/l. On the basis of this information, the following
smear was stained by May Grünwald stain for 5 minutes
exposure duration and concentrations were used with the
after that stained slide was washed with 6.8 Ph buffer
control group.
solutions for 1 minute. After wash with buffer solution
1. 1/5th concentration of LC50 i.e. 378 mg/liter blood smear was re-stain with 5% Giemsa stain for 10
for 35 days exposure (sub-acute). minutes, followed by a 10-second wash with Ph. neutral
2. 1/20th concentration of LC50 i.e. 94.5 mg/ water. After washing the slide, let it air dry, then use one
litre for 56 day exposure (chronic). drop of mounting medium on the slide and place a
coverslip on it, and then it was photo grouped.
3. Exposed fish in normal water used as
controlled with the whole duration of the 2.6 Analysis of nuclear and cellular
treated experiment. morphology of erythrocytes

All parameters for study in fish will measure at 7, Morphological analysis of fish erythrocytes were
21, and 35 days for sub-acute while 15, 35, 45, and 56 done according to method of Jahanet al.,(2019).
days for chronic toxicity test along with control, each
group have 10 fishes.

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III. RESULT IV. DISCUSSION

The Control group of fishes was showing normal According to Christopher (2004), evolution and
erythrocytes of Channa punctatus, which is oval in shape interpretation of erythrocyte morphology is an important
with a clear cytoplasmic membrane. Pink color cytoplasm factor for a complete blood count. Erythrocyte morphology
filled inside the erythrocytes and oval to round shape may provide important diagnostic information. In the
nuclei were present at the center of erythrocytes. present study, it is tried to obtain sub-acute and chronic
Cytoplasmic membrane structure and the shape of toxicity effect of Nigrosine black dye on erythrocytes
erythrocytes were also normal in the control group. Few morphology of fish Channa punctatus.
elongated shape erythrocytes were appearing to represent Results of chronic and sub-acute toxicity of
more mature cells. Few leucocytes i.e. heterophils were Nigrosine black for fish bring out changes into erythrocyte
also seen in blood smear. (Photo-1) cytoplasmic membrane. It was reported by Christopher
A sub-acute toxicity test (1/5th of LC50 of (2004) that erythrocyte's shape is maintained by protein
Nigrosine black) was produced an abnormal shape of Actin, Spectin, and Ankyrin , which is present in
erythrocytes in fish after the 14th day of the experiment. erythrocytes in a hexagonal lattice web. In the present
Toxicant Nigrocine black has altered the shape of oval study, the changes in the fish erythrocyte plasma
erythrocytes into spherical shape erythrocytes and a few membrane may change the management of above mention
cytoplasmic vacuolation were also seen. Cytoplasmic protein due to exposure to Nigrosine black dye.
vacuolation appeared around the periphery of the It was also observed in the present study that the
cytoplasmic membrane. Some immature erythrocytes were shape of fish erythrocytes also change after exposure to
devoid of nuclei. (Photo -2) Nigrosine black which indicates some alterations in ion
After the 21stday of the experiment, the transporters.Thomas (1998) reported that in fish red blood
cytoplasmic vacuolation were appearing around the cells, several ion transporters play a major role in cell
nucleus of erythrocytes in experimental fish. Few volume regulation and intracellular acid-base regulation.
erythrocytes showed more cytoplasmic vacuolation at both Changes in shape and volume of fish erythrocyte in
terminal ends of the nucleus. Later on, these cytoplasmic experimental fish indicate that toxicant Nigrosine black
vacuoles increased in their size and after fusing with each changes the function of ion transporter in fish.
other, surround the nucleus of the erythrocyte. (Photo.-3) Sanjibet al., (2005) reported that absorption of
After the 35thday, Nigrosine black toxicity toxic substance in fishes change fish erythrocytes plasma
produced progressive cytoplasmic vacuolation in the membrane’s ion permeability and damage the erythrocyte
cytoplasm and around the nucleus. Few erythrocytes were plasma membrane. In the present study, the same finding
showing the eccentric conditions of nuclei i.e. cytoplasm was observed which supports our conclusion.
and nucleus were reach to the opposite pole of Ford (2013) observe in a study that oxidative
erythrocytes. (Photo.-4) hemolysis produced irregular constricted erythrocytes and
The chronic toxicity of Nigrosine black (1/20th of these cells are known as bite and blister cells and this
LC50) produced cytoplasmic vacuolation along with little oxidative hemolysis may also lead to schistocytosis and
degeneration in erythrocytes on the 15th day of the spherocytosis because of deficiency of G6PD. In the
experiment. (Photo.-5) present investigation, Nigrocine black dye also produces
On the 30th day of chronic exposure produced oxidative hemolysis in the blood of experimental fish
degeneration of cytoplasmic membrane which changes the which later on leads to spherocytosis and schistocytosis
shape of erythrocytes. Some hyaline circles also appeared condition of erythrocytes, hence above findings of Ford
around the nucleus (Photo.-6).More cytoplasmic (2013) support our findings.
vacuolation and degeneration of the nucleus appeared on The present study showed that Nigrosine black
the 45thday of the experiment. (Photo.-7) dye brings out abnormalities in the erythrocyte's nucleus of
Schistocytosis conditions of the erythrocytes of fish. Few research workers mention in their study that the
fish were appearing on the 60th day of the chronic nucleus of fish erythrocyte plays a vital role in all
experiment. Degeneration of erythrocytes nucleus was metabolic and genetic activity (Shahjahan et al., 2020)
clearly seen along with ghost nucleus. (Photo.-8) hence alteration in nucleic material indicate that Nigrosine
black toxicant, malfunction the nucleus of erythrocytes
3
also and altered their function.

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Sarderet al., (2002) reported that all nucleated histocompatibility complex (MHC) molecules and
cells are capable of presenting an antigen, through major nucleated RBC can express MHC and these molecules

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