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International Journal of Pharmaceutical
Biological and Chemical Sciences
e-ISSN: 2278-5191
International Journal of Pharmaceutical, Biological and Chemical Sciences (IJPBCS)
e-ISSN: 2278-5191 | OCT-DEC 2013 | VOLUME 2 | ISSUE 4| 26-30 www.ijpbcs.net or www.ijpbcs.com
Research Article
Pag
e26
EFFECT OF CADMIUM ON GROWTH, CHLOROPHYLL A AND BIOMASS
PRODUCTION OF TETRASELMIS Sp
K. Manimaran & V. Ashok Prabu*
CAS in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai-608 502
*Corresponding Author Email: vprabu37@gmail.com
INTRODUCTION The effects of heavy metal toxicity on animals, plants
and humans have been extensively investigated. Microalgae, the primary producers at the base of the
aquatic food chain, are the first target affected by heavy
metal pollution, the trace concentrations of heavy metals
are necessary as co-factor of enzymatic reactions, but
high level of them could be extremely toxic [15].
Cadmium is used in different industrial processes as
production of television tube phosphores, alloy
preparation, metal plating, nuclear reactor shields and
rods, pigments, stabilizers, batteries, etc. A high
concentration of cadmium in mine drainage has also
been reported. The concentration of this element occurs at ultra trace levels in natural environments, but its
increasing utilization in industrial processes has led to
increased levels in aquatic environments [9]. This
observation has created the necessity for the
development of techniques to remove this metal from
the environment [14, 17, 18]
In general the growth inhibition test with phytoplankton
is not much easy because maintenance of culture and
conduct of experiments are very critical. So, there has
not much development in this field in worldwide besides
OECD, USEPA and EPS have been developed a growth inhibition test procedures [10,16]. Moreover the growth
inhibition tests have been conducted with the freshwater
green alga, Selenastrum capricornutum [4] and the
marine diatom, Phaeodactylum tricornutum [16]. The
toxicity studies are restricted only on marine
phytoplankton especially diatoms which are the
dominant among the marine phytoplankton. The cell
density has been taken as a growth parameter for the
toxicological studies by most of the researchers [5]
because cell density only has showed linear response
with different concentrations of toxicant.
The green microalgae from the genus Tetraselmis sp
(Prasinophyceae) used in this study are essential in aquaculture because of their high nutritional value and
ease of culture. They are cultured on a large scale and
contributed to the food chain. Bioaccumulation and
toxicity effect of metal ions on T. chuii have been
studied [2, 19]. The aim of this study was to test the
effect of cadmium on growth rate biomass and
chlorophyll a content of the marine microalga
Tetraselmis sp.
MATERIALS AND METHODS Microalgal Species
The non-axenic culture of marine microalgae
Tetraselmis sp was obtained and the stock culture
maintenance and also the experiments were carried out
in Algal Culture Laboratory, CAS in Marine Biology,
Faculty of Marine Science, Annamalai University. The
filtered seawater from Parangipettai coast, enriched with
f/2 medium nutrients [6] was used for all the
experiments.
EXPERIMENTAL SETUP All the experiments were conducted in 250 ml conical
flasks with 100 ml of 2-3 days aged exponentially
grown algal cultures. The standard growth inhibition test
procedures were followed by OECD [10]. Totally three
sets of experiment were conducted with seven different
concentrations of the heavy metal, cadmium (0.2, 0.4,
ABSTRACT:
Short-term experiments were carried out to examine the toxicity of cadmium chloride (CdCl2) on marine microalgae
Tetraselmis sp. The analyzed growth rate, growth inhibition, biomass and chlorophyll content was analyzed in microalgal
cells. Tetraselmis sp grown at different concentration of cadmium (0.2, 0.4, 0.8, 1.6, 3.2, 6.4 and 12.8 mg L-1) for 96 hours.
The growth rate was reduced and doubling time was increased with increased metal concentration. Our data statement that the
maximum cell density 6.93 ± 0.010 × 105 cells mL-1 was reached in 0.2 mg L-1 of cadmium followed by 6.91 ± 0.010 × 105
cells mL-1 at control and minimum cell densities (1.54 ± 0.00 × 105 cells ml-1) were found at 12.8 mg L-1 of Cd. The higher
amount of chlorophyll a and biomass were obtained in 0.2 mg L-1 of cadmium. The 96-h median inhibited concentration (96-h
IC50) of cadmium for Tetraselmis sp was 3.18 mg L-1. The results showed the microalgae can tolerate little high amount of
heavy metal stress in the presence of required cadmium concentration in the environment.
KEYWORDS: Biomass, Cadmium, Chlorophyll a, Microalgae, Tetraselmis sp, Toxicity
K. Manimaran and V. Ashok Prabu*; EFFECT OF CADMIUM ON GROWTH, CHLOROPHYLL A & BIOMASS PRODUCTION …
International Journal of Pharmaceutical, Biological and Chemical Sciences | e-ISSN: 2278-5191 | OCT-DEC 2013 | VOLUME 2 | ISSUE 4 | 26-30| www.ijpbcs.net
Pag
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0.8, 1.6, 3.2, 6.4 and 12.8 mg L-1) for 96 hours. The
experiment and each concentration were maintained in
triplicate. During the experiment, the cell density was
estimated at every 24 hours interval and the physico-
chemical parameters viz. temperature of 26 ± 1º C,
salinity of 30‰ and the light intensity of 5000 ± 450 Lux were maintained. The temperature was measured by
a mercury celsius thermometer, pH by electronic pH
meter (ELICO, Model LI 120), Salinity estimated using
hand refractometer (ERMA, Japan) and the light
intensity with digital Lux meter. The cell density was
estimated by manual counting under light microscope
and expressed as cells ml-1.
Determination of Chlorophyll ‘a’
The 10 ml of cultures were harvested by centrifugation
at 5000 rpm for 5 minutes. Five ml of 90 % acetone was
added to each pellet and kept in refrigerator for 24 hrs
under dark condition. It was again centrifuged at 4000 rpm for 10 minutes. The clear supernatant was made up
to 10 ml using 90 % acetone. The absorbance was
measured in UV-Vis spectrophotometer (Shimadzu,
UV-1800) at three wave lengths of 660, 645 and 630 nm
using 90 % acetone as blank. The concentration of
chlorophyll a was then calculated using the extinction
values as described by [13].
Growth rate
The growth rate was calculated using the following
formula [10],
Where,
N0 – Number of cells in time zero
Nx - Number of cells in time x
t0 – starting time (0)
tx – time X (in days)
Doubling time The doubling time was calculated by the following
formula,
Where,
N0 – Number of cells in time zero Nt – Number of cell in time t
t – Time in hours
The results were presented in doubling time (DT) in
hours.
Percentage of growth inhibition
Percentage of growth inhibition was calculated by the
formula of,
Median inhibitory concentration (IC50)
Growth rate and percentage of growth inhibition were
calculated using cell density and time interval. The
percentage of growth inhibition with respect to control
in each concentration was used to calculate IC50 with the
use of Probit analysis software [10].
Metal Analysis
The dissolved concentrations of initial cadmium was
analyzed by ICP-OES (Inductively Coupled Plasma-
Optical Emission Spectrophotometer). The multi
elemental standard solution IV (Lot No. 90336403), Merck, Mumbai, India was used for standardization of
instrument with the detection levels of 4.6 ppb and 2.7
ppb (diluted samples) for Cd respectively.
RESULTS AND DISCUSSION As The effect of cadmium on growth rate, biomass and
chlorophyll a content of marine microalgae (Tetraselmis
sp.) was observed. The Maximum cell density of 6.93 ±
0.010 × 105 cells mL-1 was observed in 0.2 mg L-1 of cadmium followed by 6.91 ± 0.010 × 105 cells mL-1 at
control and minimum of 1.54 ± 0.00 × 105 cells ml-1 at
12.8 mg L-1 of cadmium. The cell density was gradually
decreased with increasing except 0.2 mg L-1
concentrations of Cadmium after 96 hours (Table 1).
The growth rates of Tetraselmis sp were observed as
1.35 μ day -1 in 0.2 mg L-1 of cadmium followed by 1.34
and 0.01 μ day -1 at control and 12.8 mg L-1 of cadmium
concentrations respectively. The growth rates were also
decreased as cadmium concentration in the medium
increased. The maximum growth rates were obtained in 0.2 mg L-1 of cadmium and control cultures (Fig 1). The
IC50 value calculated for these microalgae after 4 days of
exposure to cadmium was 3.18 mg L-1. (Fig 2). The
doubling time was increased with increasing metal
concentration for 96 hours. However, the effective
concentration value (EC50) for Tetraselmis suecica was
7.9 mg Cd/l after six days of metal exposure under these
culture conditions. Cadmium uptake by the microalgae
T. suecica followed an initial rapid phase of uptaking
during the first days of culture, reached a maximum and
thereafter, there was a decrease or stabilization of the
amount of total removed cadmium [11] The biomass of Tetraselmis sp in seven different
concentration of cadmium was presented in (Table 1)
the species were shown different growth characteristics
among the treatments. The higher biomass of
Tetraselmis sp (3.041 ± 0.028 and 2.913 ± 0.232 mg
Wet wt. /ml of culture) were grown in 0.2 mg L-1 of
cadmium and control respectively. Whereas, the lower
biomass of Tetraselmis sp (0.013 ± 0.001 mg Wet wt.
/ml of culture) was observed in 12.8 mg L-1 of cadmium.
The chlorophyll a content was decreased with increasing
concentration of cadmium except 0.2 mg L-1 concentrations of Cd after four days (Fig 3). The higher
amount of chlorophyll a (0.856 ± 0.028 and 0.825 ±
K. Manimaran and V. Ashok Prabu*; EFFECT OF CADMIUM ON GROWTH, CHLOROPHYLL A & BIOMASS PRODUCTION …
International Journal of Pharmaceutical, Biological and Chemical Sciences | e-ISSN: 2278-5191 | OCT-DEC 2013 | VOLUME 2 | ISSUE 4 | 26-30| www.ijpbcs.net
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0.029 pg. cell-1) was observed in 0.2 mg L-1 of cadmium
and control respectively. The low amount of chlorophyll
a (0.059 ± 0.014 pg cell-1) was observed in highest
concentration of cadmium (12.8 mg L-1). [3] Statement
was reduction of chlorophyll a contents is a common
symptom of heavy metals toxicity. This may be attributing to inhibition of reduction steps in the
biosynthetic pathways of the pigment. The decreases of
Chl a and total Chl indicated a decline in the antenna
size of the photosynthetic reaction center complexes.
[12] showed that in Chlorococcum sp. AZHB the
decrease of Chl a correlated to the increasing
concentrations of Cu or Cd treatment. The decrease of
chlorophyll, accompanied by the degradation of the
chloroplast structure, in Chlorella sorokiniana heavy
metal polluted indicates that the photosynthetic
apparatus in these cells could be disrupted. Therefore,
the decrease of growth occurring in the algae Cd or Pb
treated could be ascribed to the reduction of the
photosynthetic activity [1]. [7] have reported IC50 values
for Cd(II) (0.05-7.5 mg l-1) for the marine microalgal
species, C. calcitrans, I. galbana, T. tetrathele and Tetraselmis sp., after 96 h based on O.D. measurements
and cell counting, either 24-well plates or 250-ml
Erlenmeyer flasks being used. This all statement was
carried out for safe limits value of Cadmium, had given
to Tetraselmis sp through the calculated medium
inhibiting concentration based on the growth rate with
help of probit analysis software and analyzed dissolved
concentration of Cadmium in medium with help of
Inductively Coupled Plasma-Optical Emission
Spectrophotometer.
Cd (mg L-1
) 0 hrs (× 105 cells m L
-1) 96 hrs (× 10
5 cells m L
-1) Biomass (mg Wet wt. / m L
-1)
Control 1.52 ± 0.006 6.91 ± 0.010 2.913 ± 0.232
0.2 1.52 ± 0.006 6.93 ± 0.010 3.041 ± 0.210
0.4 1.52 ± 0.006 6.84 ± 0.025 2.606 ± 0.174
0.8 1.52 ± 0.000 6.59 ± 0.053 1.934 ± 0.086
1.6 1.52 ± 0.012 5.97 ± 0.072 1.395 ± 0.125
3.2 1.52 ± 0.006 4.47 ± 0.123 0.796 ± 0.132
6.4 1.52 ± 0.006 2.63 ± 0.208 0.024 ± 0.010
12.8 1.52 ± 0.006 1.54 ± 0.000 0.013 ± 0.001
Table a: Cell densities and biomass of Tetraselmis sp, exposure to cadmium after 96 hrs.
Fig 1: Dose-Response (growth rate) curve for the effect of cadmium On Tetraselmis sp for 96 hrs
exposure.
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Control 0.20 0.40 0.80 1.60 3.20 6.40 12.80
Gro
wth
rate
(d
ay-1
)
Cadmium (mg L-1)
K. Manimaran and V. Ashok Prabu*; EFFECT OF CADMIUM ON GROWTH, CHLOROPHYLL A & BIOMASS PRODUCTION …
International Journal of Pharmaceutical, Biological and Chemical Sciences | e-ISSN: 2278-5191 | OCT-DEC 2013 | VOLUME 2 | ISSUE 4 | 26-30| www.ijpbcs.net
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Fig 2: Dose-Response (% of growth inhibition) curve for the effect of cadmium On Tetraselmis sp for
96 hrs exposure.
Fig 3: Effect of cadmium on chlorophyll a after 96 hrs
CONCLUSIONS In the present study have been contributed
understanding of the effect of cadmium on population
growth, chlorophyll a content and biomass of the marine
microalgae Tetraselmis sp. According to the results the
cell density was increased in minimum concentration of
cadmium. The 50 % growth inhibition was observed in
3.18 mg L-1 of cadmium after 96 hrs exposure of
Tetraselmis sp. whereas the metal concentration was
increased, the growth inhibition and doubling time were
increased. The Chlorophyll ‘a’ and growth rate of
Tetraselmis sp was regularly affected (excepting control
and 0.20 mg L-1 of cadmium culture) after 96 hrs exposure to cadmium concentration. It suggested that
the toxicity tests conducted with the concentrations of
metal in environment could be more reliable for the
application on environment, development of water
quality criteria and assessment of pollutants.
ACKNOWLEDGEMENTS
We are grateful to Dr. K. Kathiresan, Director, Faculty
of Marine Science, Annamalai University, Parangipettai
and ICMAM-PD, MoES, Govt. of India for providing
opportunity and Facilities to carry out the work.
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K. Manimaran and V. Ashok Prabu*; EFFECT OF CADMIUM ON GROWTH, CHLOROPHYLL A & BIOMASS PRODUCTION …
International Journal of Pharmaceutical, Biological and Chemical Sciences | e-ISSN: 2278-5191 | OCT-DEC 2013 | VOLUME 2 | ISSUE 4 | 26-30| www.ijpbcs.net
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*Corresponding author address:
V. Ashok Prabu* CAS in Marine Biology,
Faculty of Marine Sciences,
Annamalai University, Parangipettai-608 502
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