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Metallothionein Assay for Bio-Monitoring Trace

Metal Stress in Cichlids (Oreochromis niloticus


and Oreochromis mossambicus) Thriving in
Contaminated Wastewater-Fed Fishponds in East
Calcutta Wetlands, a Ramsar Site.

Utpal Singha Roy


Department of Zoology,
Durgapur Government College,
JN Avenue, Durgapur – 713214,
West Bengal, India
Email: srutpal@gmail.com
East Calcutta Wetland – only Ramsar
site in West Bengal

 Biggest urban wetland ecosystem of around


12500 ha.

 Receives effluents from around 538


tanneries and 5500 other small-scale
industries.

 Inputs: Composite industrial wastewater


and municipal sewage (around 50,000 m3/d)
and solid wastes 2500 MT/d.
Importance of this Ecosystem

Economic
 Fish production from 286
wastewater fed fish ponds
spread over total 30% of
wetland area: producing
nearly 10,915 metric tons
of fish annually.

 Vegetable production (by


using wastewater) around
370,650Kg/ha/Y.

 Employment opportunity-
2 persons/ha [about
50,000 agro-workers].
Importance of this Ecosystem
Ameliorative
 Ability to modify broad
spectrum of
contaminants (acting
as a kidney of
Nature).

 Harbors a rich
diversity

 Water quality
improvement
Selection of study sites - ECW
Uncontaminated fishpond
Three natural-water-
fed fishponds, free
from any
contamination,
located at
Ashoknagar, West
Bengal, India
(about 60 km west
of the
contaminated
sampling sites).
Objectives of the study
 To evaluate trace metal (Cr,
Mn, Fe, Cu, Zn and Pb)
concentrations in
wastewater-fed fishponds.

 To study sequestration
pattern of trace metals in
different fish tissues (liver,
gill, muscle and brain) using
Oreochromis niloticus and
O. mossambicus.

 To evaluate the possible


trace metal stress in
different fish tissues by
means of protein estimation
(Total protein and
Metallothionein).
Trace metal analysis

Detection of Cr, Mn, Fe, Cu,


Zn and Pb was done by
atomic absorption
spectrophotometer
(Perkin-Elmer AAnalyst-
100 with interfacing
AAwinlab Software), using
element-specific hollow
cathode lamps in the
default conditions, in
flame absorption mode
both for water and fish
tissues.
Metallothionein estimation
 Standard ELISA protocol
(Derango and Page, 1996) was
applied to measure the
metallothionein content. Primary
antibody against tilapia
metallothionein (Ab 36882) and
HRP tagged secondary antibody
(Ab 6721) were purchased from
Abcam, UK.

 An automatic micro titer plate


ELISA reader (LabLife-elite 96s
Microplate ELISA reader, Wipro,
India) was used to measure the
absorbance at 650 nm.

 A standard curve was generated


each time the assay was
performed by metallothionein
standard (Sigma M 7641, USA).
Measurement of Total Protein

The protein content of


the various fractions
was calculated after
Lowry et al. (1951)
using bovine serum
albumin as standard
and absorbance was
recorded by using a
UV-Vis
spectrophotometer
(Perkin Elmer Lambda
25).
Elemental profile of fishponds (conc.
Expressed as mg L-1 ± SD )
Elements
Fish
pond
Cr Mn Fe Cu Zn Pb

0.16±0. 0.56±0. 2.26±0. 0.26±0. 2.10±0. 0.12±0.


U
05 16 43 11 23 02

0.37±0. 1.98±0. 4.58±0. 0.35±0. 4.69±0. 0.32±0.


C
13 29 72 16 67 10

U: uncontaminated; C: contaminated
Trace metals (mg Kg-1 dw ± SD) in different
body organs of Oreochromis niloticus
Tissue
Elements
examined
Cr Mn Fe Cu Zn Pb
U 3.08±0.64 10.32±1.8 1423.56±186 83.40±12.47 117.36±24 0.62±0.24
Liver
C 34.68±4.8 23.29±4.0 2245.68±356. 769.41±104 179.18±36 45.92±14.7

U 1.69±0.71 14.58±1.7 543.21±86.47 17.56±2.12 122.67±24 0.21±0.09


Gill
C 4.54±1.42 27.24±3.8 742.61±38.99 24.01±4.07 87.21±12.5 8.74±1.69

U 0.56±0.29 1.21±0.41 137.04±18.82 6.61±1.06 36.34±12.0 0.18±0.06


Muscle
C 1.82±0.53 5.47±1.39 94.15±21.27 10.39±1.71 42.58±9.37 8.54±0.87

U 0.24±0.11 15.27±2.3 294.59±25.57 4.30±0.63 59.92±10.2 0.12±0.10


Brain
C 0.78±0.29 19.54±3.8 889.34±176.4 7.74±1.28 69.23±12.9 3.11±0.79

U: uncontaminated; C: contaminated
Trace metals (mg Kg-1 dw ± SD) in different
body organs of Oreochromis mossambicus
Tissue
Elements
examined
Cr Mn Fe Cu Zn Pb
U 1.32±0.27 8.53±1.89 1207.67±156.7 88.43±9.24 91.34±29.61 0.41±0.12
Liver
163.76±44.6
C 25.55±4.6 14.65±3.8 1639.47±224.4 415.50±94.68 27.91±8.52
8
117.54±19.3
U 1.98±0.58 18.76±1.9 216.57±41.25 17.37±2.87 0.22±0.13
4
Gill
C 5.03±1.77 23.20±2.3 340.96±67.71 24.01±4.07 93.76±10.72 23.24±3.71

U 0.71±0.39 0.79±0.24 56.49±8.41 2.46±0.88 28.14±8.27 0.17±0.10


Muscle
C 1.04±0.48 1.50±0.32 84.01±14.13 6.61±1.06 49.46±17.12 7.08±1.56

U 0.31±0.12 16.43±3.3 469.63±124.83 1.46±0.67 56.16±9.47 0.11±0.09


Brain
C 0.96±0.37 18.69±4.7 1087.36±205.5 6.72±0.92 74.96±14.59 3.40±1.53

U: uncontaminated; C: contaminated
Metallothionein content (μg g-1 of wet tissue; mean ±
SD) in different body organs of Oreochromis niloticus,
collected from both uncontaminated and contaminated
(wastewater-fed) fishponds.
Metallothionein content (μg g-1 of wet tissue; mean ±
SD) in different body organs of Oreochromis
mossambicus, collected from both uncontaminated and
contaminated (wastewater-fed) fishponds.
Total protein content (mg g-1 of wet tissue; mean ± SD)
in different body organs of Oreochromis niloticus,
collected from both uncontaminated and contaminated
(wastewater-fed) fishponds.
Total protein content (mg g-1 of wet tissue; mean ± SD)
in different body organs of Oreochromis mossambicus,
collected from both uncontaminated and contaminated
(wastewater-fed) fishponds.
Correlation of metallothionein with metals for all the
organs (pooled data) wastewater-fed and freshwater-fed
Oreochromis niloticus and Oreochromis mossambicus.
Marked correlations (*) are significant at p < 0.05.
Elements
Fish species
Cr Mn Fe Cu Zn Pb

Oreochromis U 0.98* -0.09 0.92 0.96* 0.69 0.96*


niloticus

C 1.00* 0.37 0.91* 0.99* 0.96* 1.00*

Oreochromis U 0.73 -0.10 0.64 0.89 0.63 0.96*


mossambicus

C 1.00* 0.07 0.70 0.99* 0.95* 0.82*

U: uncontaminated; C: contaminated
Highlights…………
 Fish tissues collected from contaminated
specimens cultured in East Calcutta Wetlands
contained significantly higher metal concentration
when compared with uncontaminated fish
tissues.

 The potential stress due to exposure to this


adverse ambient condition was reflected in lower
total protein concentration in all the wastewater-
fed fish tissues.

 Despite this stressful ambient environment, fish


yield in wastewater-fed fish ponds in ECW was
2.85 metric ton per ha of water body per annum
Highlights…………

 Fish species thriving at ECWs adopted in a


manner to express higher metallothionein to
arrest excess elements in the system that would
otherwise interfere with the physiological
processes.

 Liver was the site for highest metallothionein


synthesis and thereby arrested highest portions
of elements entered in fish biosystem to maintain
the proper metal homeostasis and lessen the
stress, otherwise intolerable.
 Author thankfully
acknowledges the
infrastructural support
extended by the Director of
Public Instruction and the
Director of Technical Education
Govt. of West Bengal, India.

 Author also expresses their


kindest thank and
acknowledges to CSIR for
providing necessary funds.