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APCBEE Procedia 10 (2014) 31 – 36

ICESD 2014: February 19-21, Singapore

Integrated Environmental Risk Assessment Using Biomarkers in

Marine Plants of the Gulf of Mannar, India
N. Nagarani and M. Anand
Department of Marine & Coastal studies, Madurai Kamaraj University, Madurai-625021, India.

Abstract

The purpose of this study was to explore a possible relationship between inorganic metals and oxidative stress in marine
plants of south east coast of India. The investigation was carried out during the low tides in Pudumadam, Mandapam,
Pamban and Keelakarai coastal areas. The distribution of elements such as phosphorus, nitrogen, lead (Pb), manganese
(Mn), iron (Fe), copper (Cu), and zinc (Zn) were determined in the marine plants and its surrounding seawater. An atomic
absorption spectrometer, Varian Model SPECTRAA was used to determine the level of heavy metals. The impact of
metals on the metabolism of the plants was determined by the level of antioxidant response. Low levels of Mn, Cu were
observed in Pudumadam species. The thiobarbutric acid reactive substances formation in the marine plants showed a
positive correlation with the trace element selected sites, which indicates the contamination of sewage and industrial
pollution in these waters.

© 2014The
© 2014 Published by Elsevier
Authors. Published B.V. Selection
by Elsevier B.V. This and/or peer
is an open review
access articleunder
under responsibility
the CC BY-NC-NDof Asia-Pacific
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(http://creativecommons.org/licenses/by-nc-nd/3.0/).
Chemical, Biological & Environmental Engineering Society
Selection and peer review under responsibility of Asia-Pacific Chemical, Biological & Environmental Engineering Society
Keywords: Gulf of Mannar; heavy metals; marine plants; oxidative stress

1. Introduction

Marine flora are the extraordinary sustainable resources in the coastal ecosystem which have been used as
a source of food, agriculture, medicine etc. The production or growth of the marine plants was affected by
natural calamities and anthropogenic activities [1]. Heavy metals and pesticides are the major pollutants in the
marine environment due to human activities. The ocean provides a sink for these pollutants. Among which

Corresponding author.
E-mail address: nagaranikannan@yahoo.co.in.

2212-6708 © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/3.0/).
Selection and peer review under responsibility of Asia-Pacific Chemical, Biological & Environmental Engineering Society
doi:10.1016/j.apcbee.2014.10.010
32 N. Nagarani and M. Anand / APCBEE Procedia 10 (2014) 31 – 36

heavy metals are non-biodegradable and are strongly phytotoxic (cadmium, lead, mercury etc.,) because of the
generation of reactive oxygen species (ROS), which react with macromolecules causing lipid peroxidation,
membrane damage, metabolite degradation, inactivation of enzymes, and finally cell death [2]. This study was,
therefore, designed to investigate (i) the extent of heavy metals accumulated in seagrass species and (ii) to
examine changes in anti-oxidant enzyme activities (SOD, CAT, GSH and GPX) and lipid peroxidation in
relation to element accumulation. The ultimate focus of the present study is to examine if aquatic pollutants
present in the selected coastal regions are generating biological responses in the seagrass. To accomplish this
objective, biomarker responses were measured in seagrass species collected along the coasts of gulf of mannar
region.

2. Experimental Method

2.1. Study site

The fresh seagrass of Halodule uninervis (Forsskål) Ascherson (HU), Syringodium isoetifolium (Ascherson)
Dandy (SI) and Cymodocea serrulata (R. Brown) Ascherson (CS) were collected from Kellakarai (KKR),
Pamban (PBN), Mandapam (MDM) and Pudumadam (PDM) coasts of the Gulf of Mannar region, Tamilnadu,
India during April 2013.

2.2. Sample Collection and Preparation

Collected seagrass samples were immediately brought to the University Research laboratory in new plastic
bags containing filtered sea water in ice-cold conditions (4°C). Plants were washed thoroughly with tap water
to remove epiphytes, sediments and shade dried for metal analysis.

2.3. Determination of Metal concentrations of native seagrass species

Perkin Elmer atomic absorption spectrometer model was used to analyze the concentration (mg/Kg) of
heavy metals (Mn, Pb, Zn,Fe, and Cu) in the different seagrass samples. The concentrations of Cu, Zn, Pb,
Mn and Fe were determined according to the standard double acid digestion methods analyzed using an
atomic absorption spectrometer. Standards were made using certified solutions (Merck, UK) acidified with
HNO3 to the same pH as the samples. Results are expressed as the means ± S.E. of three replicate samples.

2.4. Biochemical analysis

Fresh tissue was homogenized in ice-cold 0.1 M Phosphate buffer at pH 7.8 containing 1 mM EDTA, 1
mM β-mercaptoethanol and 5 ml of 4% polyvinylpyrrolidone/g FW. The homogenate was filtered through a
nylon mesh and centrifuged at 20,000 g at 4°C. The supernatant was used for measuring enzyme activity.
MDA activity was determined to indicate the level of lipid peroxidation of seagrass species as described by
Buege and Aust [3]. The antioxidant enzyme response was measured using Double beam UV spectrometer
(Model 2201; Systronics) following the methods viz., Catalase [4]; Superoxide dismutase [5]; Gluthathione
[6]; Glutathione peroxidase [7] and Ascorbate peroxidase [8]. All Results were expressed as mean ± standard
error. Data were analyzed through ANOVA with 95% significance level.

3. Results and Discussion

 N. Nagarani and M. Anand / APCBEE Procedia 10 (2014) 31 – 36 33

The physico-chemical characterisation of water at various study sites of Gulf of Mannar is depicted in
Table 1. The surface water temperature varied from 30 to 32oC during the collection of seagrass samples. All
the study sites showed similar trends with similar parameter changes. Generally, surface water temperature is
influenced by the intensity of solar radiation, monsoon, evaporation, organic matter, water inflow etc. [9].
With a higher concentration of phosphate and nitrite in Keelakarai and Pamban coast compared to the other
two sites. The mean DO for polluted waters in gulf of mannar was previously reported as 3.79 ml/l [9].
Phosphate and nitrate concentrations were less and nitrite was higher when compared to the previous report
[10]. Nitrite is reactive and nitrates are non-reactive forms of nitrogen. Nitrite when enters the biological
systems it can oxidize the antioxidants. Which are considerably more toxic to aquatic life than nitrate [11].
Table 1. Physico-chemical parameters

Study Temp Nitrite Turbidity Nitrate Inorganic PO4

Sites (°C) Salinity (ppt) pH DO (mg/L) (μM) (μM) (μM)
KKR 31 34 7.9 3.97 4 5 3.99 5
PDM 32 32.9 7.85 3.93 3.9 6 3.93 4
MDM 32 33.2 7.8 3.99 3.88 7 3.94 4.22
PBN 30 34 7.98 2.9 4.32 8 3.91 5.2

The distribution of metal contents in seagrass species is shown in Table 2. It seems that the tendency of
seagrass to bioconcentrate the metals was exactly the same order in all the selected species, Fe > Zn > Cu >
Mn > Pb. The region wise distribution of metals in the study sites follow as for, Mn and Pb (KKR >
PBN >MDM > PDM); Cu and Zn higher in the order of KKR > MDM >PBN > PDM and Fe were higher in
PBN compared to other study sites (Table 2). Cu and Zn accumulation in the seagrass are less than maximum
permissible limits prescribed for seafood for human consumption (10 mg/kg, 50 mg/kg respectively) in India
[12]. Similar to the studies of Ragupathi Raja Kannan et al., [13] the levels of essential elements like Fe, Mn,
Cu and Zn were higher than the level of non-essential elements like Pb Since, unlike other types of
contaminants heavy metals cannot be eliminated as they occur naturally all over the world. Most of the metals
play a vital role in the biological metabolism such as Fe (hemoglobin, SOD, CAT), Cu (respiratory pigments),
Zn (SOD), Co (Vitamin B12), Mo and Mn (enzyme cofactor) [14]. Compared to the present study higher
levels of Cu and lower level of Fe was reported in marine sponge Haliclona tenuiramosa; seaweed
Cladophora glomerata [15] and Amphiroa fragilissima seaweed reported high Cu and Zn concentrations
[16]. Karthick and his co-workers [17] have reported higher concentration of manganese than P, Zn and Cu in
the seaweeds. The high Fe levels in all the species and sites studied reveals the role of iron for normal growth
of seagrass and biomagnifications or accumulation of iron from the surrounding environment and
contamination from industrial and other operations. Metals such as Pb, Hg, Ni, Cr, As etc., are generally not
required for metabolic activity and produce reactive oxygen species which in turn are toxic to living
organisms damaging lipid, DNA, protein at very low concentrations [2].
Usually plants encounter the oxidative stress upon exposure to pollutant or heavy metals. Which disturbs
their cellular ionic homeostasis through their oxidative defense mechanisms such as enzymes, chelation etc.
[14]. The metabolic activity of seagrass in their native sites is given in table 3. The increase in the reduced
glutathione level in the present study may be due to the synthesis of metal chelator- phytochelations [18]. As
shown in table 3 elevation in the levels of MDA reveals the indication of lipid damage among the species
collected from KKR, PBN and MDM sites when compared to the PDM site (between sites F [1.063, 2.127] =
16.94; P = 0.0489) and between species F (2, 6) = 27.43; P= 0.0010. SOD increase in the seagrass was noted
as 378, 233 and 532% (KKR, MDM, PBN respectively) compared to PDM species. The occurrences of
biochemical’s changes in the seagrass species follow the order of LPO and CAT (SI > HU > CS); GSH and
34 N. Nagarani and M. Anand / APCBEE Procedia 10 (2014) 31 – 36

SOD (HU > SI > CS); APX (CS > SI > HU) and GPX (CS > HU > SI) respectively. The data shows that S.
isoetifolium and H. uninervis was moderate in responding to the environmental changes. The date of enzyme
analysis in species at various sites indicate the impact of the environment in the regulation of metabolisms in
seagrass species. SOD, APX and GPx synthesis were higher in PBN > KKR native species compared to the
other two sites, where Fe and Mn were higher. Mn and Fe are the cofactor for superoxide dismutase. Catalase
activity was higher in MDM and PBN native seagrass species. There are no significant changes in the levels
of GPx and APX in Mandapam native species to that of Pudumadam species.
Table 2. Distribution of metals in segrass (mg/Kg)

Site Sample Mn Cu Zn Pb Fe
S.isoetifolium 2.78 ± 0.613 6.7 ± 0.055 7.3 ± 0.93 1.92 ± 0.001 63.5±0.012
PDM H. uninervis 7.72 ± 0.057 7.01 ± 0.017 11.23 ± 0.037 0.62 ± 0.013 61.23 ± 0.04
C. serrulata 6.16 ± 0.032 8.23 ± 0.09 15.3 ± 0.09 0.61 ± 0.09 60.03 ± 0.38
S.isoetifolium 19.3 ± 0.035 13.23 ± 0.003 16.3 ± 0.03 5.6 ± 0.6 74.23 ± 0.03
KKR H. uninervis 13.23 ± 0.722 15.23 ± 0.005 21.3 ± 0.034 4.5 ± 0.311 88.23 ± 0.034
C. serrulata 17.23 ± 0.567 17.23 ± 0.034 20.78 ± 0.06 3.9 ± 0.3 72.47 ± 0.002
S.isoetifolium 11.23 ± 0.873 10.475 ± 0.045 13.23 ± 0.01 1.15 ± 0.01 73.4 ± 0.01
MDM H. uninervis 10.23 ± 0.049 5.502 ± 0.031 26.17 ± 0.02 0.98 ± 0.002 81.3 ± 0.2
C. serrulata 12.87 ± 0.44 7.183 ± 0.005 28.98 ± 0.12 0.83 ± 0.001 69.34 ± 0.034
S.isoetifolium 13.2 ± 0.45 8.23 ± 0.001 8.21 ± 0.45 2.22 ± 0.04 80.23 ± 0.64
PBN H. uninervis 15.6 ± 0.07 7.8 ± 0.02 19.38 ± 0.05 1.67 ± 0.64 90.23 ± 0.016
C. serrulata 15.9 ± 0.036 8.42 ± 0.08 17.59 ± 0.9 0.89 ± 0.01 83.56 ± 0.05

Table 3. Biochemical response of seagrass in their native sites

GSH SOD CAT GPx APX

Site Species LPO (μmole/Cm) (μM) (U/mL) (U/mL) (U/mL) (mM/cm)
SI 23.96 ± 0.1 10.55 ± 0.001 1.17 ± 0.001 2.51 ± 0.01 0.047 ± .011 0.030 ± 0.05
I HU 35.91 ± 0.02 97.98 ± 0.03 1.52 ± 0.01 2.15 ± 0.02 0.070 ± 0.031 0.0037 ± 0.01
CS 76.47 ± 0 .01 124.98 ± 0.001 0.56 ± 0.01 1.42 ± 0.03 0.176 ± 0.01 0.079 ± 0.001
SI 89.26 ± 0.871 23.34 ± 0.01 5.6 ± 0.01 2.73 ± 0.01 0.36 ± 0.01 0.171 ± 0.01
II HU 79.19 ± 0.03 140.1 ± 0.9 6.7 ± 0.001 2.14 ± 0.63 1.03 ± 0.11 0.41 ± 0.02
CS 176.23 ± 0.11 167.2 ± 0.89 8.1 ± 0.011 1.901 ± 0.03 3.61 ± 0.2 1.3 ± 0.05
SI 67.23 ± 0.04 9.23 ± 0.01 3.9 ± 0.01 3.4 ± 0.01 0.06 ± 0.01 0.045 ± 0.01
III HU 69.68 ± 0.5 83.23 ± 0.9 2.56 ± 0.02 2.9 ± 0.05 0.09 ± 0.05 0.05 ± 0.01
CS 111.20 ± 0.87 101.98 ± 0.01 1.91 ± 0.5 0.9 ± 0.001 0.44 ± 0.2 0.39 ± 0.002
SI 88.5 ± 0.76 19.89 ± 0.45 7.3 ± 0.06 4.23 ± 0.07 0.9 ± 0.01 2.3 ±0.1
IV HU 91.3 ± 1.2 91.87 ± 0.91 9.08 ± 0.3 2.9 ± 0.011 1.72 ± 0.08 0.82 ± 0.003
CS 156.96 ± 0.95 123.8 ± 0.99 9.2 ± 0.07 1.9 ± 0.02 2.4 ± 0.11 2.07 0.11
 N. Nagarani and M. Anand / APCBEE Procedia 10 (2014) 31 – 36 35

4. Conclusion

There is a growing concern that the natural cycling process of metals are being disturbed by anthropogenic
activities, especially the growth of industrial, domestic and urban and discharge of its effluents. At the same
time due to over exploitation of resources, the marine biodiversity of the Gulf of Mannar is facing the danger
of shrinking. There are number of fisheries industries located at Mandapam, Ramnad which discharge the
wastes into the sea [19]. Ultimately, this study focus on measuring the level of elements that might induce
irrevocable ecological changes to aquatic ecosystems. Until now the levels of toxicity were moderate and it
was progressing towards the danger. Efforts can be made to control the activities that release pollutants
unnaturally into the environment from both public and government.

Acknowledgment

The authors would like to thank the Council of Scientific and Industrial Research, India for financial
support to complete the work.

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