International Journal of Oceans and Oceanography
ISSN 0973-2667 Volume 12, Number 1 (2018), pp. 53-65
© Research India Publications
http://www.ripublication.com
Clarias batrachus: Bioindicator to monitor the quality
of water in marine communities of South India
Gnana Veera Subhashini1*, Beesetti Swarna latha1,
Mavuluri Jayadev1 and G. Bharath2
1
Baba Clinical and Genomic Research Centre, Chennai 600113, Tamilnadu, India.
2
Department of Chemical Engineering, Khalifa University of Science and
Technology, P.O. Box 127788, Abu Dhabi, United Arab Emirates.
* Correspondence: Dr. N. Gnana Veera Subhashini
Abstract
Successful fish rearing and production of healthy fish, study of environmental
risk factors and stress factors is highly essential. In the present study, the
significant effects of ozone against the exposure of various environmental
factors on walking catfish, Clarias batrachus was divulge in terms of
haematological and biochemical parameters such as total cholesterol,
triglycerides, High-density lipoprotein (HDL), Low-density lipoprotein (LDL),
Very low-density lipoprotein (VLDL), total cholesterol/HDL, Total RBC’s,
Haemoglobin (Hb), Packed cell volume (PCV) and Blood Glucose. These
parameters were analysed statistically and results showed drastic changes in
lipid and hematological profile of Clarias batrachus. Taken together, our data
suggest that Clarias batrachus could be a bioindicator to monitor the quality of
lake water and also health status of fish in marine communities of South India.
Keywords: lead, ozone, UVA, UVA-ozone, haematological and biochemical
parameters, walking catfish, Clarias batrachus.
INTRODUCTION
Rearing good quality farm fish is a colossal challenge in the present days and depends
on various natural factors and mankind activities. Man-made pollutants like heavy
metals enter the ecosystem, gets distributed and accumulated in the organs and tissues
of organisms (1). They might be essential in trace amounts but are toxic at higher
54 Gnana Veera Subhashini et al
concentration (2). The distribution of heavy metals in the water bodies causes
deleterious effects on the aquatic life like delayed development of larva, alters
metabolism of brain and skeletal muscles and fish death due to their persistent and non-
bio degradable nature of heavy metals (3). Heavy metal intrusion leads to reduction of
ozone level in stratosphere thereby increasing the levels of ultraviolet radiation (UVR)
which ultimately results in hypoxia and hypercapnia in fishes. Environmental
vacillations create hormonal and biochemical alterations that determines the quality and
health status of fish. Fishes accumulate heavy metals in their body at dangerous level.
The food chain in the ecosystem passes the energy as well as toxic metals to the higher
levels. In humans, heavy metal like lead causes learning dysfunction, mental retardation
and loss of coordination (4)
Clarias species (Walking Cat Fish) is a widely distributed fish which constitutes one
of the major fisheries in Asia and Africa. Among which Clarias batrachus is one of the
commonly reared fish in Asian countries and is an edible fish found in ponds, streams
and rivers. Clarias batrachus is popular for its tasty flesh, rapid growth and high market
price. Heavy metals like Lead and environmental aspects like UV and Ozone disturb
the ecosystem. Lead is a potentially toxic chemical that might be directly ingested by
man or indirectly through aquatic animals like fish and shell fish (5). Like other heavy
metals, lead at high concentration act as poison. In fish, exposure to chemical pollutants
can induce either increase or decrease in hematological levels (6). Conversely, Ozone
acts as a good disinfectant of water and is a very powerful bactericide and virucide and
unlike other agents, it leaves no desirable residues but tends to be toxic at ground level
(7).
UV exposure during early stages of development can cause developmental delays or
lethality that will affect recruitment to adult populations. UV stress results in an inedible
replacement species or a significant decline in productivity (8). Blood parameters truly
reflect physical and chemical changes occurring in an organism (9). Walking catfish,
Clarias batrachus are mostly encountered in muddy or swampy water of high turbidity.
They have high tolerance level and can adapt to mixt physiological changes for instance
highly vascularized gills serves as an accessory breathing structures (10). Thus, walking
catfish is chosen for this study by exposing them to different environmental factors.
They tolerate to the adverse conditions by activating adaptive biochemical and
physiological mechanisms that include changes in endocrine or metabolic systems (11,
12). The best way to monitor physiological and pathological changes in fishes is to
study hematological parameters. The blood indices reveal the status of health in fishes.
It also provides required information on metabolic disorders, deficiencies and chronic
stress. Exogenous factors like stress, management and diseases also contribute
significantly for the change in blood composition. In response to handling and hypoxia,
glucose, cholesterol and cortisol levels were observed to fluctuate (13). Industrial waste
expelled into the nearby waterbodies leads to pollution of aquatic ecosystems. The
Clarias batrachus: Bioindicator to monitor the quality of water in marine…. 55
present study aims to evaluate the effect of pollutants like heavy metal lead and UVA
exposure on various hematological and biochemical indices such as hemoglobin (Hb),
Total Erythrocyte Count (TEC), Mean Corpuscular Hemoglobin (MCH), LDL, HDL,
Triglycerides and Oxygen Carrying Capacity (OCC) in marine communities. Blood cell
responses are indicators of changes in the internal and external environments of
animals.
MATERIALS AND METHODS:
SAMPLE COLLECTION:
15 adult Clarias batrachus were collected from three different lakes such as
Chembarambakkam (Fresh water lake in Tamilnadu), Madhuranthagam (lake in
Tamilnadu) and Pulicat (brackish water lake in Andhra Pradesh) in India, of which 10
fish were collected from each lake and then transported to Baba Clinical and Genomic
Research Centre, Taramani, Tamilnadu. These sources were selected as they are in their
natural habitat. The fish (180-230g each) were kept in well aerated 60 liters tanks, filled
with dechlorinated water and kept on a 12:12 hrs. photo period. After 2 weeks of
acclimatization, fishes were fed with commercial food (Taiyo grow, Chennai). Fishes
were classified into five groups: (a) Control (b) Heavy metal (c) Ozone with Heavy
metal (d) Ultraviolet A with Heavy metal and (e) Combination of Ultraviolet A and
ozone with Heavy metal in which each group has fish collected from different lakes.
The ozone and Ultraviolet A were exposed for one hour per day for the respective
group. The water used was characterized by pH of 7.6, temperature of 24 ± 1.2°C
and 6.3 ± 0.2 mg/l of oxygen under identical laboratory conditions.
STOCK PREPARATION:
Lead nitrate was commercially purchased and used as source of Heavy metal. The
concentration of lead beyond 5.6mg/l is lethal. Hence, we used lead at concentration far
below the lethal dose 1mg/l
OZONE TREATMENT:
Kent ozone generator was selected as ozone source (by corona discharge method).
Ozone in the range of 200mg/hr. was supplied to the acclimated fish per day. After the
onset of experiment ozone exposure was continued for 4 days.
56 Gnana Veera Subhashini et al
UV A SOURCE:
UV A lamps were used as a UV A source (SANKYO DENKI (F8T5BLB)) with a range
of 352nm.
HEMATOLOGICAL AND BIOCHEMICAL ANALYSIS:
The blood was drawn from the caudal vein of fish into heparinized tube after being
anesthetized with 2-phenoxy-ethanol (Sigma-Aldrich, MO, USA) at a concentration of
250 mg/L. The concentration of the hemoglobin was analyzed immediately. The blood
sample for serum analysis were centrifuged at 4000 rpm for 10 minutes and stored at
-20ºC until analysis. The hematological and biochemical analysis such as total
cholesterol, triglycerides, High-density lipoprotein (HDL), Low-density lipoprotein
(LDL), Very low-density lipoprotein (VLDL), total cholesterol/HDL, Total RBC’s,
Packed cell volume (PCV) and Blood Glucose were analyzed in an auto biochemistry
analyzer (Fuji Dri-Chem 3500i, Tokyo, Japan). Mean corpuscular hemoglobin (MCH),
Mean corpuscular hemoglobin concentration (MCHC) Mean corpuscular volume
(MCV) and Oxygen carrying capacity were calculated using the formulae described
earlier (14).
STATISTICAL ANALYSIS:
The hematological profiles were analyzed statistically by calculating the Mean±
Standard error. Graph Pad PRISM software and one-way ANOVA was used for
statistical analysis of data. Statistical significance was evaluated by calculating P-
values. Differences where P<0.05 were considered statistically significant. (*P<0.05,
**P<0.01, ***P<0.001).
RESULTS AND DISCUSSION:
HEMATOLOGICAL AND BIOCHEMICAL ANALYSIS:
The mean values of hematological and biochemical parameters of Clarias batrachus
when exposed to different conditions (a) Control (b) Heavy metal (c) Ozone with Heavy
metal (d) Ultraviolet A with Heavy metal and (e) Combination of ultraviolet A and
ozone with Heavy metal were calculated and analyzed. The fish exposed to the
environmental factors had significant difference (<0.05) with the control ones. No
mortality was observed.
Clarias batrachus: Bioindicator to monitor the quality of water in marine…. 57
HEMATOLOGICAL PARAMETERS
The variations of the mean values for the hematological parameters after exposure are
given in Table 1.
Table 1: Hematological parameters of Clarias batrachus with different treatments.
(b) Heavy (c) Ozone+ (d) UV A + (e) UV A + Ozone
Parameters (a) Control
metal Heavy metal Heavy metal + Heavy metal
Haemoglobin
7.8 ±0.124 8.7 ±0.234 6.83 ±0.221 9 ±0.323 9.3 ±0.331
(gms/dl)
Total RBC
2.6 ±0.126 3.43 ±0.321 2.2 ±0.332 3.43 ±0.234 1.36 ±0.176
(millions/cum)
PCV (%) 23.66 ±0.198 23.57 ±0.099 25.56 ±0.228 27.5 ±0.564 26.46 ±0.336
MCV(μm3) 91 ±0.168 68.7 ±0.289 116.18 ±0.189 80.17 ±0.254 194.55 ±0.321
MCH (pg) 30 ±0.321 25.36 ±0.203 31.045 ±0.126 26.23 ±0.211 68.38 ±0.321
MCHC (g
32.96 ±0.387 36.91 ±0.121 26.72 ±0.254 32.72 ±0.478 35.147 ±0.178
dL−1)
OCC (Vol%) 9.75 10.875 8.5375 11.25 11.625
Altered hematocrit, red blood cell count and hemoglobin levels were reported in a fish
when subjected to stress (15, 16). The Clarias batrachus showed significant increase
in haemoglobin levels when exposed to (b) Heavy metal (lead) (d) Ultraviolet A with
Heavy metal and (e) Combination of ultraviolet A and ozone with Heavy metal for 96
hours. Clarias exposed to (c) Ozone with Heavy metal showed decrease in hemoglobin.
The Heavy metals and UVA deplete the dissolved oxygen in water bodies and thus this
increased hemoglobin might be essential for uptake of adequate oxygen. The UVA
induces photochemical reaction and thus bleaches the available oxygen in water source
(Fig 1A). Total RBC count was increased in (b) Heavy metal and (d) Ultraviolet A with
Heavy metal treated fish but there is a drastic fall in RBC count in (e) Combination of
Ultraviolet A and Ozone with Heavy metal treated fish. The low oxygen tension in the
water treated with (b) Heavy metal and (d) Ultraviolet A with heavy metal necessitated
the increase in circulating RBC number. But the fish treated with (c) Ozone with Heavy
metal showed no considerable change in RBC count. The drastic fall of RBC count in
the case of (e) Combination of ultraviolet A and ozone with heavy metal is due to
58 Gnana Veera Subhashini et al
hemolysis induced by the combined toxic effect of Heavy metal and UVA (Fig 1B).
These conditions lead to anemic condition in fish by decreasing the total RBC count.
We observed no significant difference in packed cell volume on (b) Heavy metal
treatment. But on exposure to (c) Ozone with Heavy metal, (d) Ultraviolet A with
Heavy metal and (e) Combination of ultraviolet A and ozone with heavy metal there is
an increase in hematocrit (Fig 1C).
MCV, MCH and MCHC refer to calculated secondary red cell indices. They indicate
the size and content of hemoglobin in erythrocytes. High MCV signifies macrocytic
condition and low as microcytic condition (17). In the present study exposure to (b)
Heavy metal and (d) Ultraviolet A with Heavy metal showed microcytic condition
whereas exposure to (c) Ozone with Heavy metal and (e) Combination of ultraviolet A
and ozone with heavy metal showed macrocytic condition (Fig1D). The average
content of hemoglobin in erythrocytes is expressed as MCH and MCHC. Only the fish
exposed to combination of ultraviolet A and ozone with Heavy metal showed increase
in MCH (Fig 1E). Fish exposed to (b) Heavy metal showed increase in MCHC value
and those exposed to (c) ozone with Heavy metal showed reduced MCHC value (Fig
1F).
UV exposure is known to increase the oxygen carrying capacity whereas ozone reduced
oxygen carrying capacity in fish. In our studies, we observed a strong correlation
between hemoglobin levels and oxygen carrying capacity. In (b) Heavy metal exposed
fish, OCC is increased and we observed significant reduction in the fish exposed to (c)
Ozone with Heavy metal (Fig 1G).
Clarias batrachus: Bioindicator to monitor the quality of water in marine…. 59
Figure 1: Hematological parameters of Clarias batrachus with different treatments.
60 Gnana Veera Subhashini et al
BIOCHEMICAL PARAMETERS:
The variations of the mean values for the biochemical parameters after exposure are
given in Table 2.
Table 2: Biochemical parameters of Clarias batrachus with different treatments
Parameters (a)Control (b)Heavy metal (c)Ozone+ Heavy (d)UV A + Heavy (e)UV A + Ozone
metal metal + Heavy metal
Blood glucose 174.79±0.264 150.9±0.31898 165.4±0.52098 181.74±0.261 139.9±0.1117
(mgs/dl)
cholesterol 170.06±0.479 135.54±0.98 330.34±0.953 203.21±0.967 213.08±0.755
(mgs/dl)
Triglycerides 202.69±0.812 150.21±0.924 100.44±0.346 30.31±0.5776 102.04±0.637
(mgs/dl)
VLDL (mgs/dl) 40.43±0.366 30.89±1.1061 22.05±0.969 6.3±1.271 20.39±1.164
LDL (mgs/dl) 85.5±0.841 63.61±0.695 257.25±0.531 146.18±0.545 146.9±0.665
HDL (mgs/dl) 45.34±0.504 42.21±0.239 53.28±0.9070 51.08±1.135 47.88±0.742
Total cholesterol/ 3.74±0.053 3.15±0.022 6.203±0.088 3.98±0.109 4.45±0.0555
HDL
HDL/LDL 0.53±0.264 0.663±0.354 0.2±0.144 0.34±0.454 0.32±0.434
LDL/HDL 1.88±0.121 1.506±0.253 4.82±0.369 2.86±0.423 3.06±0.354
Triglycerides/HDL 4.47±0.134 3.5±0.365 1.8±0.249 0.59±0.289 2.13±0.356
Increase in plasma glucose level is an indicator of sympathetic activation during stress
(18). Increased glycogenolysis or a decreased clearance of glucose from the blood was
reported to be the source for increase in plasma glucose concentrations in confined
tilapia (19). The catfish showed significant increase in glucose concentration of blood
exposed to (d) Ultraviolet A with Heavy metal whereas a significant reduction is
observed when exposed to (b) Heavy metal, (c) ozone with Heavy metal and (e)
Combination of ultraviolet A and ozone with Heavy metal compared to control group
(Fig 2A). The increased levels of glucose in blood might be due to the stress hormones
that promote glucose production through glycogenesis and glycogen lysis pathways.
The increased glucose concentration is essential to meet the energy demand during
Clarias batrachus: Bioindicator to monitor the quality of water in marine…. 61
stress. The reduction in glucose level might be due to the depletion of reserved energy.
Under stress condition, glucose is rapidly consumed for the effective functioning of
nervous system (CNS) and also to maintain homeostasis.
Lipoproteins in the form of very low-density lipoprotein (VLDL), low density
lipoprotein (LDL) and high-density lipoprotein (HDL) mediate the transport of lipids
and other lipid soluble components from intestine to peripheral tissues (20).
Variation in the lipid profile leads to dreadful effects in the body. We observed
significant increase in the cholesterol levels when Clarias batrachus fish exposed to (c)
Ozone with Heavy metal (d) Ultraviolet A with Heavy metal and (e) Combination of
Ultraviolet A and ozone with Heavy metal. This increase might be due to mobilization
of lipid either through oxidation or by instauration of lipid molecules. But there is
significant reduction of cholesterol levels in (b) Heavy metal (Lead) exposed fish (Fig
2B).
Cholesterol, phospholipids and triglycerides constitute total lipids. Hence the variations
in these components affect the total lipid levels. We observed a steep decline in the
triglyceride levels on exposure to (b) Heavy metal (c) Ozone with Heavy metal and (d)
Ultraviolet A with Heavy metal. Even the Clarias batrachus exposed to (e) combination
of ultraviolet A and ozone with Heavy metal also showed decrease in triglyceride level
(Fig 2C).
A similar pattern of reduction was observed in VLDL concentration with treatment.
This is due to the fact that triglyceride fraction determines the VLDL concentration (Fig
2D).
LDL-cholesterol leads to plaque formation when accumulated in the arteries.
Significant elevation in the LDL-cholesterol is observed when treated with (c) Ozone
with Heavy metal (d) Ultraviolet A with Heavy metal and (e) Combination of ultraviolet
A and ozone with Heavy metal. The LDL profile pattern is identical to the cholesterol
levels in blood (Fig 2E)
Elevated HDL levels are noticed in the Clarias batrachus treated with (c) Ozone with
Heavy metal (d) Ultraviolet A with Heavy metal and (e) Combination of Ultraviolet A
and ozone with Heavy metal compared to control (Fig 2F). Heavy metal (lead) induced
decline in HDL levels, increasing the risk of metabolic disorders. Total
cholesterol/HDL ratio determines cardiovascular risk. We observed significant increase
in total cholesterol/HDL ratio when Clarias batrachus exposed to (c) Ozone with
Heavy metal and (e) Combination of Ultraviolet A and ozone with Heavy metal but
showed no remarkable change when exposed to (b) Heavy metal and (d) Ultraviolet A
with Heavy metal (Fig 2G).
We analyzed other atherogenic lipid marker indices like HDL/LDL, LDL/HDL and
Triglycerides/HDL on treatment and noticed significant variation with each condition
(Table 2). All these hematological and biochemical changes reflect the adverse
conditions and prepares the fish to fight and survive unpropitious circumstances.
The current study provides an idea to assess health status of fish and the serious
ecological consequences of pollutants in marine communities. This study provides
62 Gnana Veera Subhashini et al
baseline for grouper culture management in aquaculture. An elevated level of LDL-
cholesterol level is associated with atherosclerosis and peripheral vascular disease.
Commotion in the metabolism of lipids/glucose or blight clearance from plasma causes
liver dysfunction. Hence these pollutants like ozone, Heavy metal (lead) and UVA
exposure affects not only the quality of water but also is hazardous to the major diet in
the form of fish. Under external stress these Heavy metals accumulate in the fish which
further alters the metabolic pathways. As we observed drastic changes in lipid and
hematological profile of Clarias batrachus, it can be used as bioindicator to monitor
the quality of water and also health status of fish in aquatic bodies.
Figure 2: Biochemical parameters of Clarias batrachus with different treatments.
Clarias batrachus: Bioindicator to monitor the quality of water in marine…. 63
In the modern society life couldn’t sustain without the use of lead and its products but
is life threatening and toxic to aquatic and terrestrial life. In spite of ozone and UVA
being used as good disinfectant to water bodies is very harmful when combined with
other pollutants like Heavy metals. On a long term, they may affect the entire life of
fauna and flora of earth including humans if the release of these pollutants is not
controlled.
CONCLUSION:
We studied the hematological and biochemical profiling of fish Clarias batrachus in
response to natural and manmade pollutants. Heavy metal (Lead) treated fish had a
massive impact shown by reducing the levels of total cholesterol, triglycerides, HDL,
LDL, VLDL and Blood Glucose but contradict to it, the levels of hemoglobin and total
RBC were elevated with the control ones. The combination of ozone with Heavy metal
treatment inflated the levels of total cholesterol, HDL, LDL, total cholesterol /HDL and
PCV whereas, the levels of triglycerides, VLDL, hemoglobin, total RBC and blood
glucose were decreased. The effect of UV-A with the Heavy metal had increased the
levels of total cholesterol, HDL, LDL, total cholesterol/HDL, hemoglobin, total RBC,
PCV and blood glucose with the drastic downfall in the triglycerides and VLDL levels.
The tri-combination of UV-A, ozone and Heavy metal increased the levels of total
cholesterol, HDL, LDL, total cholesterol/ HDL, hemoglobin and PCV but decreased
levels of triglycerides, VLDL, total RBC and blood glucose. The significant menacing
effect of environmental risk factors on health profile of fish Clarias batrachus, reveals
the combined toxic effect of heavy metal, ozone and UVA and is likely to be used as
bio indicator to monitor the quality of water in marine communities of South India. In
spite of ozone and UVA being used as good disinfectant to water bodies is very harmful
when combined with other pollutants like Heavy metals. Our studies reveal better
understanding of choice of disinfectant to use in marine communities.
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