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

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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

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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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0

20

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Control 0.5 1.0 2.0 4.0 8.0 16.0 32.0

% o

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K. Manimaran and V. Ashok Prabu*; EFFECT OF CADMIUM ON GROWTH, CHLOROPHYLL A & BIOMASS PRODUCTION …

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*Corresponding author address:

V. Ashok Prabu* CAS in Marine Biology,

Faculty of Marine Sciences,

Annamalai University, Parangipettai-608 502