INTRODUCTION
Water is one of the most important and abundant compound of the
ecosystem and is essential for existence of life. "Water is the best of all
things", said the eminent Greek philosopher Pindar. Though uttered two
and a half millennium ago, the importance of this statement is evident
even now as we stepped into 21st century. The availability of adequate
water in terms of both quantity and quality is essential for human
existence. Earlier the importance of water was given only from the point
of view of quantity. Historically civilizations developed around water
bodies that were utilized to support agriculture and transportation besides
for human consumption. Recognition to the importance of water quality
came more slowly.
The natural water bodies, both lotic and lentic are most important
sources of water to sustain the life. The whole human kind needs water
for sustaining life; the provision of a safe drinking water supply is a high
priority issue for safeguarding the health and well-being of humans (Van
Leeuwen, 2000; WHO, 2011) and is an important development issue at
national, regional and local level (WHO, 2011). The supply of quality
water remains a major challenge for humanity in the twenty-first century
(Schwarzenbach et al., 2010). Water quality is important for drinking,
agriculture, aquaculture and industrial use. The aquatic resources of the
country are its national wealth. Water resources need special interest for
conservation, development and management for best and sustainable
utilization.
Aquatic ecosystems are progressively coming under permanent
pressure of anthropogenic pollutants. Water constitutes the “trouble spot”
of all ecosystems (Brandys et al., 1999). The heavy metal contamination
of aquatic ecosystems is a worldwide problem (Jayakumar et al., 2008).
The natural aquatic systems are extensively contaminated with heavy
1
metals released from anthropogenic activities (Dirilgen, 2001; Vutukuru,
2005). Municipal wastes, industrial effluents, herbicides, pesticides and
chemical fertilizers have entered the water bodies and degraded the water
quality. The water pollutants contain various organic, inorganic,
degradable and non-degradable matter, heavy metals, chlorinated
hydrocarbons etc. Among the various kinds of pollutant, heavy metals are
considered to be serious contaminants of aquatic system due to their
“conservative nature”, they are either not broken down at all, or they are
broken down over long period of time that they essentially become
permanent additions to the aquatic environment (Sarabject and Dinesh,
2007; Gad and Saad, 2008; Jadhav et al., 2010). Heavy metal
contamination is also described as a ticking environmental bomb
(Bohlmark, 2003). Research has shown that metals have the ability to
bioconcentrate in organisms directly from the water, and bioaccumulated
and biomagnified heavy metals within food chains, cause higher trophic
organisms to become contaminated with higher concentrations of
chemical contaminants than their prey (Hargrave et al., 2000 and Lee et
al., 2000; Boran and Altinok, 2010; Shariati et al., 2011).
The term “heavy metals” refers to any metallic element that has a
relatively high density and is toxic at low concentration (Lenntech, 2004).
In cooperation with the U.S. Environmental Protection Agency, the
Agency for Toxic Substances and Disease Registry (ATSDR) has
compiled a priority list in 2001 called the “Top 20 Hazardous
Substances”. The heavy metals arsenic, lead, mercury and cadmium
ranked 1st, 2nd, 3rd and 4th in the list respectively.
The most important metals from the point of view of water
pollution are As, Zn, Cu, Pb, Cd, Hg, Ni and Cr (Li et al., 2002). Some of
these metals (e.g. Cu, Ni, Cr and Zn) are essential trace metals to living
organisms, but become toxic at higher concentrations (Albergoni and
2
Piccinni, 1983). Essential heavy metals are commonly considered to be
less toxic than non-essential metals (Batley, 1983); they are toxic when
present in high concentrations in the environment (Preez and Vuren,
1994; Sanders, 1997). Metals such as lead, cadmium, arsenic, mercury,
copper, zinc, nickel, chromium etc do not degrade in general
(Mackeviciene et al., 2002; Golovanova, 2008) therefore, they
accumulate through food chain and are toxic to living organisms.
Consumption of aquatic food highly contaminated with heavy metals may
form a significant pathway to metal contamination in the human being
and creating public health problems wherever man is involved in the food
chain (Otitoloju and Don-Pedro, 2004; Lodhi et al., 2006; Yigit and
Altindag, 2006; Sarabject and Dinesh, 2007; Medeiros et al., 2012).The
toxicant bioaccumulation became a topic of public and scientific concern
early in the 1950s (Barron, 2003). Therefore, heavy metal pollution
poses a great potential threat to the environment and human health.
Sources of Heavy Metal Pollution of Surface Water
Heavy metals are widely distributed in nature; they are persistent
contaminants that enter the environment by natural and anthropogenic
sources.
a) Natural sources of heavy metals
It is generally resumed that the principal natural sources of heavy
metals include chemical and physical weathering of igneous and
metamorphic rocks and soil, wind-borne soil particles, sea salt spray,
volcanoes, and wild forest fires, decomposition of plant and animal
detritus precipitation, bacterial activity and atmospheric fallout (Nriagu,
1989; Kennish, 1992; Florea and Busselberg, 2006).
A global assessment of natural sources of atmospheric heavy
metals has been made by Nriagu (1989). He stated that the biogenic
sources can account, on average, for over 50% of the Se, Hg, and Mo,
3
and from 30 to 50% of the As, Cd, Cu, Mn, Pb, and Zn, released annually
to the atmosphere from natural sources. Volcanic emissions can account
for 40-50% of the Cd and Hg and 20-40% of the As, Cr, Cu, I, Pb, and Sb
released annually from natural sources. Sea salt aerosols seem to account
for <10% of atmospheric heavy metals from natural sources. Finally, soilderived dusts can account for over 50% of the total Cr, Mn, and V
emissions, as well as for 20-30% of the Cu, Mo, Ni, Pb, Sb and Zn
released annually to the atmosphere. In general, soils and sediments tend
to reflect the composition of their parent material. Soils and sediments in
mineralized areas, therefore, usually have the highest concentrations of
the corresponding metals. For example, rocks with high Hg content
usually occur in areas of crustal instability where volcanic and
geothermal activity is high.
b) Anthropogenic sources of heavy metals
i) Agricultural sources of heavy metals
The inorganic and organic fertilizers are the most significant source
of heavy metals to agricultural soil. The increase in heavy metals
pollution of agriculture soil depend on the rate of application of the
supplier with its elemental concentration, and soil characteristics to which
it was applied. Several pesticides with heavy metal are used to control the
disease of grain and fruit crops and vegetables, are sources of heavy
metals pollution to the agricultural soil (Verkleji, 1993; Ross, 1994). The
orchards where these compounds have been used commonly resulted into
pollution of orchard soil with high level of heavy metals such as zinc,
copper, lead, arsenic, iron, and mercury (Ross, 1994). Inorganic fertilizers
mainly phosphate fertilizer has variable level of lead, cadmium, nickel,
chromium, and zinc depending on their sources. Super phosphate is an
important fertilizer used at the time of plantation of paddy seedlings. It is
estimated that is contain 3mg Cd/kg super phosphate (Pillai, 1985). Soil
4
contamination by heavy metals may also be from irrigation water sources
such as deep wells, river, lakes, and irrigation canals and also due to
application of soil amendments such as compost refusing and nitrate
fertilizer (Ross, 1994). Cd enrichment also occurs due to manure and
limes (Nirugu and Pacnya, 1988). The pesticides and fertilizers applied in
restricted area contaminate the soil and may carry away by rains and
floods to larger water bodies.
ii) Industrial sources of heavy metals
Heavy metal pollution of the biosphere has accelerated
dramatically since the beginning of the industrial revolution (Sayyed and
Sayadi, 2011). `Heavy metals are normal constituents of freshwater
environment that occur as a result of pollution principally due to the
discharge of untreated wastes into rivers by many industries (Fakayode
and Onionwa, 2002; Fakayode, 2005). Industrial sources of metals are the
mining, spoil heaps and tailing, transport of ores, smelting and metal
finishing and recycling of metals. High temperature processing of metals
like smelting and casting, releases metal in particulate and vapors form in
the environment. Vapors form of heavy metal like cadmium, arsenic,
copper, lead and tin combines with water in the atmosphere to form
aerosols. These may be dispersed by wind and precipitate in rainfall
causing contamination of soil or water bodies. Water pollution of heavy
metals occurs due to different type of processing in refineries. Energy
providing power station such as petroleum combustion, coal burning
power plants, nuclear power stations and high tension lines add many
heavy metals such as Se, B, Cd, Cu, Zn and Cs to the environment
(Verkleji, 1993). Processing of plastics textile, microelectronics, wood
preservation and paper processing these are other industrial sources
(Cetesb, 1992). Ultimate disposal of treated and untreated waste effluents
contains toxic metals as well as metal chelates (Santos et al., 2005) from
5
different industries and chemical plant. Cadmium is used in pesticides,
batteries, rubber processing and production of pigments (Kidambi et al.,
2003). Cd levels in the environment vary widely and are depending on
the presence of industrial sites, extensive agricultural activities or dump
sites (Tsukahara et al., 2003).
iii) Other sources of heavy metals
Other sources of heavy metal contain refuse burning, landfills and
transportation (Automobiles, diesel-powered vehicles and aircraft). Two
main anthropogenic sources that pollute the water and soil are fly ash
produce due to coal burning and the deterioration of commercial waste
product, which add Cr, Cu, Pb and galvanized metals (Al-Hiyaly et al.,
1988). Coal burning contributes heavy metals such Cd, Hg, Mn, Ni, etc,
oil burning adds V, Fe, Pb and Ni to the environment (Verkleji, 1993).
Metal release during the transportation vehicles include Ni, and Zn from
tires, Cd and Cu primarily from diesel engines, Zn from aerosol
emissions, lubricants and Al from catalyst, which are antiwar protestants
for vehicles emits Cd, Cr, Hg, Ni, Pb, and Zn particularly in case of
leaded gasoline has been main source of Pb in the environment. Addition
of municipal wastes to water bodies generates significant concentration of
Zn, Pb, Al, Sn, Fe and Cu. Human activities, such as industrial and traffic
emissions and different land-use practices may increase heavy metal into
aquatic ecosystems (Nriagu and Pacyna, 1988; Mukherjee, 1989). Heavy
metals are carried to the water bodies through atmospheric discharge.
Addition of municipal wastes generates significant concentration of Zn,
Pb, Al, Sn, Fe and Cu. Domestic sewage is also reported to carry heavy
metals (Shrivasatava et al., 2003). Mining and the related operations are
the most important anthropogenic sources of heavy metals (Vanderlinden
et al., 2006; Conesa et al., 2007).
6
Fossil fuel combustion and cement production contribute to
significant mobilization of metals. Electroplating processes and thermal
power station also generate large volumes of liquid waste containing
metals. Domestic sewage is yet another source in the urban regions of
riverine system. The use of metal containing pesticides in agriculture and
leaded petrol in motor vehicles results in atmospheric pollution by
mercury and lead respectively. Oil and coal particularly from some
localities contain significant quantities of heavy metal. Oil is rich in
vanadium, nickel, molybdenum and mercury (Nair, 1984). Contamination
of water bodies and soil can also take place through runoff from mine
waste, corrosion of metals, dusts produce during the transport of crude
ores and leaching of heavy meals to soil and ground water.
Heavy Metals in Soil Sediments
It has been recognized that aquatic sediments absorb constant and
toxic chemicals to levels several times higher than the water column
concentration (Linnik and Zubenko, 2000; Casper et al., 2004). Halcrow
et al., (1973) has reported that heavy metal concentrations in soil
sediments increase with the decrease of the particle size and increase of
organic matter content. Metals which accumulate in the soil sediments
may turn into factors hostile the ecosystem well-being and may constitute
a danger and risk factor for the ecosystem (Shrivastava et al., 2003; Wildi
et al., 2004). Soil sediments comprises the most important sink of metals
and other contaminants; it can act as a non-point source and has the
potential to release the sediment-bound metals and other pollutants to
overlying waters, and in turn unfavorably affects aquatic organisms
(Wang et al., 2004). Heavy metals accumulate particularly in the soil
sediments they cannot be biologically degraded like organic pollutants.
Soil sediments acts as indicators of the burden of heavy metals in
an aquatic ecosystem, as they are the principal reservoir for heavy metals
7
(Fitchko and Hutchinson, 1975; Caccia et al., 2003). A quantitative
analysis of soil sediments shows the current features of an aquatic
ecosystem. The capacity of sediment to accumulate pollutants makes
them one of the most significant tools to assess environmental impact on
aquatic ecosystems (Silva and Rezende, 2002). In fact, lake sediments
can serve as an information record of environmental changes through
time (Haworth and Lund, 1984). Monitoring the concentration of heavy
metals in the soil sediments is important since knowledge of the heavy
metal levels in sediments gives essential information about their sources,
degree and distribution of pollution. This is for the fact that sedimentation
has been regarded as one of the major important fluxes in aquatic
ecosystems (Asaolu et al., 1997).
Heavy metals accumulating in the soil sediments can affect
concentrations of heavy metals in the aquatic organisms that inhabit in
these sediments (Pourang, 1996; Yap et al., 2002; Kim and Kim, 2006).
The occurrence of metals in sediments can lead to greater environmental
problem when the polluted sediments are uptake by filter feeder animals.
Hence, eating of such kind of animals may form a significant pathway to
metals pollution and eventually poses greater health risk to human being.
The accumulation of metals in soil sediments has significant
environmental suggestions for local communities, as well as for lake
quality (Demirak et al., 2006).
Heavy Metal Toxicity
a) Effects of heavy metals on environment
The aquatic environment is more susceptible to the harmful effects
of heavy metal pollution because aquatic organisms are in close and
prolonged contact with the soluble metals (Leatherland, 1998). Heavy
metals when enter the aquatic bodies devastating effects on the ecological
balance of the recipient environment and a diversity of aquatic organisms
8
(Sharma and Agarwal, 2005; Vosyliene and Jankaite, 2006; Farombi et
al., 2007). The heavy metal in wetlands can causes serious changes in the
functioning of natural ecosystem, such as changes in the food chain
structure, affecting organ function, reproductive status, population size,
physiological and behavioral changes in aquatic organisms as well as
decrease of biodiversity and extinction of sensitive taxa (Bogatov and
Bogatova, 2009; Bonanno and Lo Guidice, 2010). Even minute amounts
of metals can cause subtle or persistent biological effects that may result
in irreversible long-term changes in organisms. The toxic metals killing
the benthic organisms reduce the food availability for larger animals.
Elevated metal concentrations in aquatic environment may have a direct
toxic effect on macro-invertebrates and their predators or have an indirect
effect on natural community structure, by reducing prey
diversity
(negative effect) or reducing competition within a species (positive
effect), resulting in a trophic cascade (Fleeger et al., 2003; Chapman,
2004). Elevated environmental metal concentrations have also been
linked to altered chemical composition and decreased concentrations of
protein and lipids in benthic macro-invertebrates (Panfoli et al., 2000;
Hamer et al., 2004), thus potentially affecting their nutritional value.
b) Effects of heavy metals on human
Occurrence of toxic metals in lakes, ponds, dams, ditches and river
water affect the lives of local people that depend upon these water
sources for their daily requirements (Rai et al., 2002; Beckett et al.,
2007). Many aquatic organisms have the ability to accumulate and
biomagnifies metals in food chain (Goodwin et al., 2003; Davies et al.,
2006; Casas et al., 2008). Consumption of such aquatic food stuff highly
contaminated with toxic metals may cause serious health hazards through
food-chain magnification (Khan et al., 2000). Metals are nonbiodegradable and are considered as major environmental pollutants
9
causing cytotoxic, mutagenic and carcinogenic effects in animals (More
et al., 2003, Lewis et al., 2004). Heavy metal toxicity is considered to
induce the production of reactive oxygen species and may result in
significant damage to cellular constituents (Pruski and Dixon, 2003). In
the last decade it has been proved that metal carcinogenicity is mediated
by the generation of reactive free radicals (especially hydroxyl radicals,
HO•) and ROS (Kasprzak, 1995). The toxicity due to metal ions is owing
to their ability to bind with protein molecules (Kar et al., 1992) and
prevent DNA and subsequent cell division which potentially causes DNA
damage and carcinogenic effects caused by their mutagenic ability
(Knasmuller et al., 1998).
1. Cadmium toxicity:
According to the Department of Water Affairs and Forestry
(DWAF) (1996), cadmium is a highly toxic metal element to aquatic life.
Cadmium does not break down in the environment, but can change form,
it remain in the body for long periods of time and can bioaccumulate for
several years after exposure to low levels of this metal (Groundwork,
2002).Toxicological responses of Cd exposure contain respiratory
diseases, kidney damage, neurological disorders and several kinds of
cancer (Waalkes et al., 1992). Japanese who consumed Cd polluted rice
and river water over a period of 30 years were found to have accumulated
in their bodies a large amount of Cd that lead to a serious osteoporosislike bone disease known to the Japanese as “itai–itai byo” or “ouch–ouch
disease” (Pan et al., 2010). Over a long period of intake, cadmium may
accumulate in the kidney and liver and, because of its long biological half
life, may lead to kidney damage (Maduabuchi et al., 2006). A recent
report in post menopausal women also indicates bone damage due to Cd
exposure (Watanabe et al., 2004). Industrial cadmium exposure is
extremely relevant, affecting more than 1,500,000 workers a year in the
10
United States alone (Ragan and Mast, 1990). In occupationally and
environmentally exposed population, cadmium is nephrotoxic, including
tubular and glomerular dysfunction (Akesson et al., 2006 and Bernard,
2004), hepatotoxicity (Gubrelay et al., 2004) and skeletal damage
(Akesson et al., 2006) and several types of cancer in organs such as
testes, lung and prostate (Sahmoun et al., 2005) it is also immunotoxic to
the human immune system (Shenker et al., 1998).
2. Zinc toxicity:
Zinc (Zn) is an essential heavy metal (micronutrient) for all
organisms as it forms the active site in various metalloenzymes. The high
concentrations of this metal may exceed this requirement and the
detoxification mechanisms of the animal may be insufficient to manage
with the influx. The zinc will then apply a direct toxic action (Lloyd,
1992).
Excessive intake of zinc may lead to electrolyte imbalance,
nausea, anemia, and lethargy (Onionwa et al., 2001). Excessive
absorption of zinc suppresses copper and iron absorption (Fosmire,
1990).
3. Copper toxicity:
According to EPA (2006), the symptoms of high copper exposure
cause headache, nausea, hypoglycemia, increased heart rate, and anemia.
Excessive copper in children is related with hyperactive behavior,
learning disorders such as dyslexia and infections. The acute exposure to
copper containing dust is manifested by metal fume fever (Křížek et al.,
1997). Acute exposure to copper can cause vomiting, nausea, stomach
cramps and diarrhea. Abdominal pain, cramps, nausea, diarrhea, and
vomiting have been caused by the consumption of beverages containing
high levels of Cu, and liver damage has been seen in individuals with
diseases of Cu metabolism. High doses of copper can cause anemia, liver
and kidney damage, and stomach and intestinal irritation. People with
11
Wilson‟s disease are at greater risk for health effects from over exposure
to copper in human beings.
4. Lead toxicity:
Lead is a highly toxic substance, exposure to which can produce a
wide range of undesirable health effects. Young children below the age of
six are especially susceptible to lead's harmful health effects, because
their brains and central nervous system are still being formed. At high
levels of exposure to a child, it may become mentally retarded, fall into a
coma, and even die from lead poisoning. In adults, lead can increase
blood pressure and cause nerve disorders, fertility problems, muscle and
joint pain, irritability, and memory problems. Lead is known to induce
intellectual performance in children and increased blood pressure and
cardiovascular disease in adult (Commission of the European
Communities, 2001). When a pregnant woman have an elevated lead
level in blood, that lead can easily be passes to the fetus, as lead crosses
the placenta. Lead has been reported in inhibiting heme synthesis, in
decreasing red cell survival, in carcinogenicity and nucleic acid
destabilization (Pyatt et al., 2005). The main characteristics of chronic
lead toxicity are sterility both in males and females, and abnormal fetal
development (Johnson, 1998) it is also immunotoxic to the human
immune system (Shenker et al., 1998). The excessive accumulation of Pb
in human system produces nephritis, a stage of severe contamination of
kidney.
People on globe are under tremendous threat due to undesired
changes in the physical, chemical and biological characteristics of water
(Misra and Dinesh, 1991). Therefore, monitoring of these metals is
important for safety assessment of the aquatic ecosystem and to avoid
human health risk in particular. Monitoring and prevention of heavy
metal pollution is one of the hot topics in environmental researches.
12
Several organizations have pointed out the need for monitoring heavy
metals concentrations in the environment (UNEP/FAO, 1996) because of
their toxicity, persistence and accumulation in the biota. Thus, there is a
need of scientific management in exploitation and conservation of the
precious natural resources. To achieve this goal there is an urgent need of
basic and applied research on various aspects of aquatic ecosystems for
their sustainable utilization through pollution control and conservation. A
regular monitoring of them not only prevent diseases and hazards but also
checks the water resources from going further polluted. It will be
necessary to review continuously, the quality standards of water in the
country‟s rivers as well as ground water aquifers.
Conventionally, heavy metal monitoring of water has been carried
out by analysis the concentration of heavy metals in water and sediments.
This technique does not show the relation between the metal levels in the
environment with availability of biological metal inside the body of
organism (Waldichuk, 1985). Furthermore, this technique does not give
any indication towards determining the harmful effect on the organism
and also the effect at the molecular level (Cajaraville et al., 2000). The
levels of toxic heavy metals when are low, are difficult to detect. As a
consequence, human populations are often at grave risk by Mandatory
Quality Control. In such a situation the development of biological assay
system to detect hazardous chemicals in water sources as well as actual
water supply is essential. It is proposed to develop some such systems
which would then serve to give what is described as Early Warning
Systems (EWS) for detection of hazardous pollution of water sources.
Biological organisms afford a simple and more meaningful means of
assessing risk of biological systems. Simple, effective, economically and
technically feasible systems of biological indication of pollution hazards
will be developed to use as Early Warning Systems (EWS‟s) for initiating
13
urgent remedial action. Thus there is need to use the biomonitoring
techniques to detect the very low metal pollution level in water and its
impact.
Biomonitoring of Heavy Metal Pollution
To reveal very low, undetectable heavy metals concentrations and
its effects for early warning, the idea has been planned to use
biomonitoring techniques, which includes study of bioaccumulation and
their responses (biomarkers) and is more capable and sensitive
(Cajaraville et al., 2000) as compare to conventional methods. According
to Van Der Oost et al., (2003) the biomonitoring, is a regular systematic
use of living organisms to assess changes in water quality, by assessing
either bioaccumulation, biological effect, health (occurrence of disease)
and/or ecosystem integrity. The typical method for biomonitoring is
based on bioindicators. Bioaccumulative indicators are commonly
regarded as biomonitors. Among the common approaches used to survey
environmental condition, the use of bioindicator species has been proven
to be important and informative tools (Philips, 1990). The advantages of
biomonitoring versus physical or chemical monitoring (Zhou et al., 2008)
are:
1. Biomonitoring reflects overall ecological integrity (i.e. physical,
chemical and biological);
2. it provides a holistic measure of environmental condition by
integrating stresses over time;
3. can tell us regarding the cumulative effect of different contaminants in
the ecosystem and about how long a problem may have been present;
4. has high sensitivity due to the rapid responses induced in the organisms
exposed to pollutants, which helps to the declare of the precaution;
5. realizes the monitoring of the pollutants at low levels which are below
the detection limits of the instrumental analytical techniques due to the
14
occurrence of the chronic toxicities of the pollutants in the organisms
under long-term exposure;
6. allows widely sampling even at remote areas;
7. avoids the limits of the convention chemical analysis such as
continuous sampling, needs of expensive instruments etc;
8. the public better understands living organisms as measures of a
"healthy" environment (Plafkin et al., 1989).
Bioindicators
Since the ultimate purpose of pollution monitoring is the protection
of ecosystems and human beings, the main interest of the use of
quantitative sentinel organisms with regard to water or sediment is their
capacity to give information on the bioavailability of pollutants (Cossa,
1989). Currently, the term "bioindicator" is a deeply ambiguous term
which has different meanings in different contexts (Heink and Kowarik,
2010). To prevent problems due to different interpretations of this term,
we use the definition of Blandin (1986) “a biological indicator (or
bioindicator) is an organism or a set of organisms that allows, by
reference to biochemical, cytological, physiological, ecological or
ethological variables, in a practical and safe way, to characterize the
status of an ecosystem or an eco-complex and to highlight as early as
possible their changes, natural or caused”. Bioindicators therefore allow
to accurately assessing the effects of anthropogenic activities on
ecosystems.
Salanki (1986) reported a good bioindicator is one which shows the
earliest responses to the pollutants allowing indicating the presence and
predicting the consequences of adverse anthropogenic effects. For the
biomonitoring of aquatic pollution including heavy metal, the organisms
in the given aquatic ecosystems are sampled for the analysis of various
biological responses to pollutant exposures. Suitable bioindicators
15
generally give great help to the biomonitoring. The evaluation of early
changes in indicator organisms will allow the prevention of long term
effects of the pollution at the population and community level (Bolognesi
et al., 1996). These organisms deliver information on alterations in the
environment or the quantity of environmental pollutants by changing in
one of the following ways: physiologically, chemically or behaviorally.
The information can be deduced through the study of, (i) their content of
certain elements or compounds, (ii) their morphological or cellular
structure, (iii) metabolic-biochemical processes, (iv) behaviour, or (v)
population structure(s).
An ideal bioindicator (biomonitor) should satisfy certain criteria
(Yan and Lv, 1989; Phillips and Rainbow, 1993; Langston and Spence,
1994: Zhoua et al., 2008). These include
(1) it can accumulate elevated levels of pollutants without death;
(2) it lives in a sessile style, thus definitely indicating the local pollution;
(3) it has sufficient abundance and wide distribution for the repetitious
sampling and comparison;
(4) its life is long sufficient to allow the sampling of more than one yearclass;
(5) be of reasonable size, giving adequate tissue for analysis,
(6) easy sampling and raising in the lab;
(7) it remains alive in water;
(8) it occupy the main position in food chain;
(9) well dose-effect relationship can be detected in it;
(10) it must be able to stand high concentrations of different toxic
substances so as to survive the pollutant studied;
(11) dose effect relationship can be observed in it;
(12) easy to handle and identify;
(13) hardy enough to survive in the laboratory and filed transplantations.
16
Therefore, a good biomonitor will indicate the presence of the
pollutant and also attempt to provide additional information about the
amount and intensity of the exposure. As it is too rigorous to find such
bioindicator for biomonitoring, the applicant bioindicator with numerous
characters is feasible according to the specific monitoring purpose.
Bivalves as bioindicator species
During the past few decades, many species have been studied to
determine their potential as biomonitoring organisms and molluscs have
become a popular choice for metal monitoring for several reasons as
follows (Hung et al., 2001). Many bivalves species have wide
geographical distribution and are dominant species in benthic ecosystem
(Burky, 1983), which facilitate comparison of result obtained in different
areas. Being sedentary organisms, bivalves reflect local condition and as
efficient filter feeders, which feed on algae, zooplankton and excreta of
all aquatic vertebrates mostly present in the bottom of aquatic ecosystem,
they
accumulate
measurable
contaminant
body
burdens
from
environmental condition that are near or below the limit of detection in
chemical analysis (Ullven, 1993; Huang et al., 2007). They have a long
life span (Farrington et al., 1983). They are sedentary organisms filtrating
large amounts of water allowing them to accumulate the substances from
the environment. In addition, their ability to biotransform accumulated
toxicants is generally lower than many other aquatic organisms (Borchert
et al., 1997). They are sturdy enough to survive in laboratory and field
studies in cages, provides a time integrated indication of environmental
contamination hence fulfilling the criteria as good bioindicators (Regoli,
1998; Olivier et al., 2002; Huang et al., 2007). In comparison to fish and
crustacean, bivalves have a very low level of activity of enzyme systems
capable of metabolizing persistent pollutants. Therefore contaminants
concentration in the tissues of bivalves more accurately reflects the
17
magnitude of environmental contamination (Phillips, 1977, Phillips,
1980, Phillips, 1990). For all these reasons, mussels are very widely used
in programs monitoring the chemical pollution of the aquatic
environment (Goldberg, 1975; Amiard et al., 1986; Kljakovic-Gaspic et
al., 2006).
To achieve the aim to monitor low level of heavy metal pollution
and its effect induced on organism/ecosystem, the biomonitoring
techniques can be categorized into two categories a) bioaccumulation
study b) biomarker study.
a) Bioaccumulation Study
Bioaccumulation is defined as the accumulation of chemicals in the
tissue of organisms by any route, including respiration, ingestion, or
direct contact with polluted water, sediment, and poor water in the
sediment (U.S.E.P.A., 2000). Aquatic organisms are exposed to both
dissolved and particulate metals and absorb essential and non essential
heavy metal from the surrounding environment with the potential to
accumulate
them
within
their
bodies
(Meyer
et
al.,
2005).
Bioaccumulation results from a dynamic equilibrium between exposure
from the outside environment and uptake, excretion, storage and
degradation within an organism (Gobas et al., 1988). In aquatic
environment metals are not removed rapidly, are not readily detoxified, as
a result they accumulate in the vital organs of the animals (Rainbow and
Moore, 1986). An increase in the concentration of a chemical in a
biological organism over time may occur compared to the concentrations
of chemicals in the environment.
Bivalves are known to accumulate and tolerate high concentration
of heavy metals (Okazaki, 1976 and Goldberg, 1980). This content results
from a balance between the concentration in the organism and its
environment, dependent on the process of absorption, excretion and
18
accumulation (Cossa, 1989). High concentrations of trace metals in soft
tissues have been detected in several species of bivalves in many parts of
the world (Ikuta, 1991). Bivalve accumulates higher level of contaminant
body burdens from environmental condition that are near or below the
limit of detection in chemical analysis (Ullven, 1993). After a stay of
several months in water, the content of contaminants in the tissues of
mussels reflects the concentration of bioavailable contaminants in the
water (Casas and Bacher 2006). Molluscs are capable of achieving tissue
concentrations of metals that are 100 to 1000 times higher than those in
water concentrations (Hartwig, 1995). The accumulation of several
metals is due to the low capacity of these molluscs for discriminating
among metals, which are similar in some characteristics such as ionic
radius (Jeffree et al., 1993; Metcalfe-Smith, 1994). Gundacker (1999)
reported that Zebra mussel accumulates high amounts of potentially toxic
heavy metals and is widely used as a bio-monitoring organism. One
fundamental assumption and basis of biomonitoring programmes is that
the concentration in the bivalves reflects the available levels of metals in
the ambient environment.
Bioaccumulation of heavy metals in tissues of aquatic organisms
has been identified as an indirect measure of the abundance and
availability of metals in the marine environment (Chapman, 1997;
Kucuksegin et al., 2006). The biomonitoring of chemical contaminants in
waters is mostly carried out by direct quantification of the accumulated
pollutants within individuals (Blackmore and Wang 2003, Andral et al.,
2004, Andral et al., 2011, Galgani et al., 2011). For this reason,
monitoring tissue contamination serves an important function as an early
warning indicator of sediment contamination or related water quality
problems (Mansour and Sidky, 2002; Barak and Mason, 1990) and
19
enables us to take appropriate action to protect public health and the
environment.
The use of biomonitoring tool is widespread in environments
because the measuring of the pollutant content in the organism is the only
way of evaluating the bioavailability of a pollutant present in the
environment. This technique makes it possible to measure concentration
of trace element even when their amounts in the natural environment are
lower than the detection limits of the methods commonly used. The
bioaccumulation of heavy metals in the bivalve‟s tissues can be helpful to
detect the polluted area and can be used as environmental bio-indicator
(Langston and Spence, 1995). The analysis of heavy metal content in
whole soft body tissues of bivalves as bio-indicator gives some
information about the degree of metal pollution in the environment. It
was found that metals concentration in benthic organisms is directly
correlated with concentrations of metals present in sediments and water.
Metal concentration in sediments and water decreased after a period of
time, but concentration in benthic animals remained relatively unchanged
(David, 2003). Tissue metal concentrations can reveal contamination and
mollusk in particular may therefore be sensitive biomonitors of
anthropogenic metal contributions (Hendozko et al., 2010). In addition,
the pollutant concentrations in the organism are the result of the past as
well as the recent pollution level of the environment in which the
organism lives, while the pollutant concentrations in the water only
indicate the situation at the time of sampling (Ravera et al., 2003).
Based on the use these molluscs for monitoring purposes, two
types of strategies have been adopted: some scientists use of the native
populations of wild or cultivated mussels (passive biomonitoring e.g.: the
Mussel Watch Program in the USA, Goldberg 1975, the RNO program in
France, Chiffoleau et al., 2005) while others resort to transplants of
20
individuals (active biomonitoring e.g.: RINBIO and MYTILOS programs
in the Mediterranean, Andral et al., 2004; Benedicto et al., 2011). In the
latter case, caged mussels are placed several months during their sexual
dormancy in the water body of interest in order to accumulate
contaminants up to achieve a balance with their transplantation
environment.
„Mussel Watch‟ and „Status and Trend Monitoring Programs‟ were
two suitable examples of biomonitoring programs in which the
concentrations of various pollutants in bivalve molluscs were measured
and used for explaining the differences between locations and time trend
(Goldberg, 1975; Phillips, 1980, 1985; Farrington et al., 1987;Monirith et
al., 2003). Many systematic „„Mussel Watch‟‟ monitoring programs in
estuarine and coastal waters have been carried out by California Mussel
Watch Program (Stephenson et al., 1995), NOAA‟s National Status and
Trend Program (O‟Connor, 2002), International Mussel Watch Program
Initial Phase (South America, Central America, Caribbean, and Mexico)
(Tripp et al., 1992) and Asia-Pacific Phase (Monirith et al., 2003; Fung et
al., 2004), and other studies (Gunther et al., 1999; O‟Connor, 2002).
Numerous monitoring studies have employed mussels as bioindicator to
evaluate the spatial and temporal trends of contaminants in the
environment (Fang et al., 2001; Salanki et al., 2003; Smolders et al.,
2003; Mubiana et al., 2005).
Factors affecting bioaccumulation of heavy metals
In aquatic environment multiple factors including season, physical
and chemical properties of water and biological parameters can play a
significant role in metal accumulation in organism (Hayat et al., 2007;
Romeo et al., 1999). Physicochemical parameters that influence on metal
concentration and accumulation are temperature, pH, dissolved oxygen,
hardness, salinity, and organic components (Ademoroti, 1996; Cusimano
21
et al., 1986; Heath, 1987; Allen, 1995; Ingersoll et al., 1997; Chapman et
al., 1998; Karthikeyan et al., 2007).
The
important
biological
factors,
that
affecting
metal
bioaccumulation in organisms are organism‟s feeding behavior, mobility,
respiration, and where and how it lives (Langston and Spence, 1995;
Ingersoll et al., 1997). Additionally, many factors influencing the metal
accumulation are age (Phillips and Rainbow, 1994; Cossa, 1989), body
size (Riget et al., 1996), internal cavity volume (Mubiana et al., 2006),
and physiological condition of the organism (Fischer, 1983; Soto et al.,
2000). Williamson (1980) reported the higher metabolic rate of small
organisms may partially account for the higher concentration of the
elements. Metal concentrations in organism may differ according to the
species. Within a single species metal accumulation can be function as
age, size, weight, sex and genotype, phenotype, feeding activity and
reproductive state (Boening, 1997). Seasonal variation in the tissue
weight of bioindicator organisms can significantly affect metal
concentrations by simply diluting or concentrating the animal's total
metal body burden (Lobel and Wright, 1982; Popham and D‟Auria, 1983;
Joiris et al., 2000). Birungi et al., (2007), studies from the field and
laboratory experiments showed that accumulation of heavy metals in a
tissue is mainly dependent upon concentrations of metals in water and
exposure period; besides other environmental factors such as salinity, pH,
hardness and temperature.
Body burden of heavy metal
The history of an organism‟s metal exposure determines its body
burden (i.e., whole-body chemical residues) and may influence its
physiological or behavioral response to metals (Widdows et al., 1984).
The body burden of a substance may be the result of exposures from
more than one source. The tissue body burden and the detoxificatory fate
22
of metals in animals seem to be more important in affecting metal
accumulation than the nature of the exposure routes (aqueous vs. dietary)
or of the exposure regimes (Gupta and Singh, 2011).
The actual body burden attained by an organism may depend on a
number of factors, including the physicochemical properties of the
contaminant, exposure route and subsequent internal fate and
detoxification. In cases where the body burden is directly related to body
weight, as has been found for a variety of elements in several species
(Boyden, 1974), a function of body weight such as binding of specific
compounds within the tissue may play some role in determining the total
burden. The body burden can increase with element availability until it
reaches a threshold above which no further increase is possible in
response to environmental increases (Ravera et al., 2003).
Bivalves are often used to evaluate the level of environmental
pollution (Philips, 1976). Generally these indicators are capable to
evaluate the level of metal pollutants but the problem of individual
variability within and between sample batches still remains and this
causes problems on the interpretation of the results (White, 1982). For
this instance, body size or weight of the organism is one of the parameters
that may affect the bioaccumulation of the elements in the body.
According to Boyden (1974) if the metal concentrations are expressed as
concentration per unit body weight (µg/g), we would then predicted the
highest value to be recorded amongst the smallest organisms and could
therefore render a misleading interpretation. The total metal content is
directly proportional to the body weight (Ibrahim and Mat, (1995).
According to Davies and Pirie, (1980) the trace metals body
burdens in most bivalves have been used to 1) identify and map areas
with extremely high levels of trace metals and organic pollutants, 2)
identifying
source/magnitude
and
23
calculating
concentrations
of
“bioavailable” metals, 3) assessing comparative levels of stress affecting
biota of different habitat, trophic or behavioral characteristics and 4)
quantifying ecological impact traceable to pollution.
If the goal of a monitoring program is to detect elevated levels of
contaminants being introduced to a water body, then body burden
measurement seems to be a reliable and cost-effective tool.
In fact
mussels are considered to be best indicator for reflecting bioavailable
concentrations of environmental contaminants (Tessier et al., 1993;
Deshmukh, 2013).
Biowater
Accumulation
Factor
(BWAF)
and
Biosediment
Accumulation Factor (BSAF)
The accumulation of contaminants from the water column by
bivalves is referred to as „bioconcentration‟, a property that makes
bivalves potentially useful as „biomonitors‟ for water quality monitoring
programmes, and also for bioremediation to improve the quality of
polluted waters. The efficiency of metal accumulation in different species
of bivalve from a medium (water/soil) into its tissues can be evaluated by
calculating the Biowater Accumulation Factor (BWAF) and Biosediment
Accumulation Factor (BSAF). BWAF is defined as the ratio between the
metal concentration in the organism and that in the water (Lau et al.,
1998; Szefer et al., 1999). BSAF is defined as the ratio between the metal
concentration in the organism and that in the sediments. It is commonly
used to estimate propensity to accumulate metals in the organisms. By
comparing BWAF/BSAF values, one can compare the potential of
different bivalve species to accumulate metals from water and sediments.
Liao et al., (2003) stated that BWAF/BSAF is a requisite component for
both aquaculture ecosystems and human health risk assessment. Many
investigators have calculated BWAF/BSAF values to determine the
potential of animals to accumulate metal from water/sediment in to body
24
of aquatic animals. Usero et al., (2005) were calculated biosediment
accumulation factor (BSAF) for Cr, Ni, Cu, Cd, Pb, Zn, As and Hg in
Donax trunculus and Chamelea gallina. BSAF and BWAF values were
calculated by Adeniyi et al., (2008) to compare the bioaccumulation
capacity of Tilapia sp. and Chrysichthys sp. Biosediment accumulation
factor (BSAF) in D. trunculus and P. textile for Cd, Co, Cu, Zn, Mn and
Fe were calculated by Maha et al., (2008). Deshmukh (2013) and Waykar
and Petare (2014) calculated BWAF and BSAF in mollusk to find put
sentinel animal species to monitor heavy metals pollution.
Literature review
Monitoring of heavy metals in fresh water system, using freshwater
bivalves was carried out by many authors. According to Goldberg et al.,
(1983) and Phillips (1990), among the common approaches used to
survey environmental pollution, the use of bioindicator species has
proven to be an important and informative tool. Luoma et al., (1985)
studied temporal variation of Cu, Ag and Zn concentration measured in
the soft tissues of the estuarine bivalve, Macoma balthica in South San
Francisco Bay. Borcharnt (1989) studied heavy metal concentration in
Mytilus edulis from the central North Sea. Francesco and Enzo (1994)
studied accumulation and subcellular distribution of metals (Cu, Fe, Mn,
Pb and Zn) during a field transplant experiment in the Mediterranean
mussel, Mytilus galloprovincialis. Regoli and Orlando (1994) studied
seasonal variation of metal concentrations (Cu, Fe, Mn, Pb and Zn) in the
digestive glands of the Mediterranean mussel, Mytilus galloprovincialis:
comparison between a polluted and a non polluted site. Paster et al.,
(1994) studied the levels of heavy metals (mercury, cadmium and lead) in
some marine organisms (fish, molluscs and crustaceans) from the
Western Mediterranean areas. Hickey et al., (1995) studied the metal
concentrations in resident and transplanted freshwater mussels, Hyridella
25
menziesi and sediments in the Waikato River, New Zealand. Pip (1995)
studied bioaccumulation and biomonitoring potential of the White
Heelsplitter (Lasmigona complanata) and numerous other species in the
Assiniboine River, Manitoba (Canada) .Amanulla Hameed (1995) used
freshwater mussel as a bio indicator organism in freshwater pond
(Chediyan pond). Fish tissue metal content has also been successfully
utilized in assessment of metal pollution into large European and
American rivers (Chevreuil et al., 1995; Saiki et al., 1995; Carru et al.,
1996). The Mussel Watch project in the USA has been developed using
bivalves and oysters to monitor spatial and temporal trends of pollutants
concentrations in coastal and estuarine areas (Goldberg et al., 1978;
O‟Conner, 1996). Amiard et al., (1986) studied the ecotoxicological
impact of cadmium, copper and zinc in the mussel Mytilus edulis. Wang
and Yin (1987) studied the accumulation and the distribution of
cadmium, lead and copper in the clam Arca granosa by analyzing the
metal content in blood and soft parts. Szefer et al., (1997) studied
the
distribution and association of trace metals in soft tissues and byssus of
Mytilus edulis from the east coast of Kyushu Island, Japan. The
distribution of heavy metals in the different soft tissues such as mantle,
muscle, ligament, foot, remainder, gill and digestive gland of mussels was
studied by Sato et al., (1997). The distribution of trace metals in green
mussels, Perna viridis from Thailand coastal waters was studied
Boonchalermkit et al., (1998). Senthilnathan et al., (1998) studied
concentration of metals in mussels, Perna viridis and oyster Crassostrea
madrasensis from some parts in southeast coast of India. Szefer (1999)
studied the distribution and relationships of metals in molluscs and
associated sediments from the Gulf of Aden, Yemen. Bat (1999) used the
Mediterranean mussel, Mytilus galloprovincialis to monitor the metal
pollution in coastal region. Villar et al., (1999) studied Cd, Cu and Zn
26
content in bivalves, L. fortunei and C. fluminea to assess their utility as
indicators of heavy metal pollution. Ismail et al., (2000) studied greenlipped mussel, Perna viridis as a biomonitoring agent of heavy metal
pollution in the west coast of Peninsular Malaysia. Gundacker (2000)
used gastropods, Radix ovata and Viviparus sp. to monitor the heavy
metal pollution. Wong et al., (2000) studied the concentration of heavy
metals in green-lipped mussels collected from Tolo Harbour and markets
in Hong Kong and Shenzhen. The effect of pH and temperature on the
cadmium uptake by Corbicula fluminea and Dreissena polymorpha was
studied by Fraysse et al., (2000). Chase et al., (2001) have been
successfully used Mytilus sps. as tools in Mussel watch environmental
monitoring programmes to reveal geographical patterns and to identify
temporal trends of coastal pollution. Bivalves have been used to assess
the bioavailability of heavy metals in the waters of different parts of the
world (Blackmore, 2001; Cohen et al., 2001; Hung et al., 2001; Sunlu,
2002). Szefer et al., (2002) studied distribution of trace metals in soft
tissue, byssus and shells of Mytilus edulis trossuls from southern Baltic.
In Malaysia, Yap et al., (2003b, 2004b, 2006), used different parts of
Perna viridis to monitor the metal pollution in the coastal water of
Peninsular Malaysia. Chou et al., (2003) used the digestive glands,
residuals and the adductor muscles of the horse mussel, Modiolus
modiolus to monitor metal pollution. Salanki et al., (2003) used bivalves
and snails to monitor the level of pollution, Lake Balaton. The study on
assessment of different soft tissues of the green lipped mussel, Perna
viridis as biomonitoring agents of Pb in field and laboratory studies was
carried out by Yap et al., (2003). A large body of field studies has since
indicated that the metal concentrations in bivalves have the potentiality of
using bivalve as indicator for monitoring of heavy metal pollution in
aquatic system (Gagnaire et al., 2004). Sokolova et al., (2005) reported
27
high Cd levels in the different soft tissues of mussels from Gelang Patah,
Senggarang and Kampung Masai. Mubiana (2005) studied temporal and
spatial variation of metal concentrations in mussels to determine the
influence of season on metal concentrations in mussels from Western
Scheldt estuary (The Netherlands). Yap et al., (2006) studied the different
soft tissues of Perna viridis as biomonitors of bioavailability and heavy
metals (Cd, Cu, Fe, Pb, Ni, and Zn) contamination in semi-enclosed
intertidal water, the Johore Straits. Determination of heavy metal
distributions in the green-lipped mussel, Perna viridis as bioindicators of
heavy metal contamination in the Johore Straits and Senggarang,
Peninsular Malaysia has been studied by Yap et al., (2007). MariaCervantes et al., (2009) studied arsenic content in the mollusks, Hexaplex
trunculus and Tapes decussatus from coastal Zones of a mediterranean
lagoon affected by mining wastes. Mokhtar et al., (2009) calculated
Metal Pollution Index for tiger prawns (Prnaeus monodon) and tilapia
fish (Oreochromis spp) collected from aquaculture ponds of high
densities development area. Different tissues of Perna viridis as
biomonitors of polycyclic aromatic hydrocarbons in the coastal water of
Peninsular Malaysia was studied by Shahbazi et al., (2010). Yap and
Edward (2010) studied the distribution of Cu, Cd, Zn, Pb, Ni and Fe in
the different soft tissues of Cerithidea obtusa collected form Bako,
Sematan and Deralik. Studies have been conducted to investigate heavy
metal levels in environmental samples, as well as heavy metal
accumulation and their effects on organism, and factors affecting heavy
metal accumulation by various organisms (Murugan and Arunkumar,
2010). Adedji et al., (2011) determined heavy metals in snail and water
samples to assess the pollution of Alaro River at Oluyole Industrial
Estate, Ibadan southwestern Nigeria. Kamaruzzaman et al., (2011)
studied the bioaccumulation of some metals by green mussel Perna
28
viridis. Eugene et al., (2013) studied the assessment of heavy metal
contamination by using transplanted caged mussel, Perna viridis in the
straits of Johore.
Recently in India, considerable attention has been placed on
monitoring of pollution in water bodies; and bio-assay studies of the flora
and fauna are carried out to find out level of heavy metal concentration.
Seasonality‟s in bioaccumulation of trace metals such as copper,
cadmium, iron, manganese, zinc and mercury in the oyster, Crassostrea
madrasensis in Cochin backwaters in the west coast was studied by Nair
and Nair (1986). Pillai et al., (1986) observed significant variations in the
concentration of metals like iron, zinc, copper, lead, cadmium, nickel and
cobalt in clams, Vellorita cyprinoides, Meretrix costa, Crassostrea
madrasensis, Perna viridis and P. indica. Bioaccumulation of mercury,
arsenic and lead in freshwater gastropod, Bellamya (Viviparous)
bengalensis was studied by Mahajan (2005). Bioaccumulation of arsenic
and lead in freshwater bivalve, Lamellidens corrianus was studied by
Nawale (2008). Waykar and Shinde (2011) and Waykar et al., (2011)
studied the bioaccumulation of metals in freshwater pelecypod molluscs
under controlled experimental condition. Yasmeen (2011) investigated
the bioaccumulation patterns of Cd in whole body and different body
parts of freshwater bivalve Lamellidens marginalis. Waykar and
Deshmukh (2012) studied the comparative bioaccumulation of As, Cd,
Cu, Pb, Hg and Zn in different freshwater bivalve species. Waykar and
Shinde (2013) studied bioaccumulation of As, Cu, Cd, Pb and Zn in
mantle, gills, digestive glands and whole soft body tissues in freshwater
bivalve, Parreysia corrugata. Deshmukh (2013) studied bioaccumulation
in different bivalve species from different study sites of Jayakwadi
reservoir. Waykar and Petare (2014) studied bioaccumulation in snail
species sampled from Latipada dam.
29
Therefore the aim of this study is to determine the concentrations
of heavy metals zinc, copper, cadmium and lead in water, sediment and
native freshwater bivalve species, Lamellidens corrianus, Lamellidens
marginalis and Parreysia cylindrica inhabiting the four reservoirs of
Nasik district and to determine BWAF/BSAF values to find out suitable
bivalves species as sentinel animal for monitoring of metal pollution in
the fresh water ecosystem. These freshwater bivalve species were
selected for present study, because they are native and abundantly found
in the study area, data of application of these bivalve species as
bioindicator is lacking and there is no any published report on metal
accumulation in these species.
30
b) Oxidative Stress as Biomarkers
Bioaccumulation study only gives information regarding the levels
of contaminants in aquatic organisms (Andral et al., 2004, Andral et al.,
2011, Galgani et al., 2011). Scientists have suggested recently that there
is a need for new environmental monitoring procedures that focus mostly
on the effects of contaminants rather than the levels of contaminants
(Lam and Gray, 2003). The use of biomarkers is a new concept that gives
the information on the biological effects of contamination (Narbonne,
2000; Clements, 2000 Bocchetti et al., 2008, Serafim et al., 2011).
Biomarkers are more significant than bioaccumulation because biomarker
study deals with xenobiotically induced variations in cellular or
biochemical components or processes, physiological or behavioral
variations in the tissue or body fluids, structure or function that is
measurable in a biological system or sample that provide evidence of
exposure to chemical pollutants and may also indicate a toxic effect
(NRC, 1987; de Lafontaine et al., 2000, Allen and Moore, 2004; Long et
al., 2004).These xenobiotically induced changes are grouped under the
term biomarkers, and
can
be determined
through
variety of
observations/measurements (Amiard et al., 2000). The biological
responses to environmental stressors can often detect alterations at an
early stage, before other disturbances, such as disease, mortality, or
population changes, and thus may present early warnings of pollution
impact (Depledge and Fossi, 1994; Moore, 1985). Biomarkers may help
to predict changes at higher levels of biological organization, so that
undesirable and irreversible effects at higher levels of biological
organization can be avoided (Lam and Gray, 2001; Perceval et al., 2004;
Gagne et al., 2005). Biomarkers can provide a spatial and temporal
integrated measure of minute amounts of bioavailable pollutant levels
(Chapman, 1992). Specific responses can be used to attribute exposure to
31
pollutants, biomarkers can provide information on the relative toxicities
of specific chemicals and biomarkers are applicable in both the laboratory
and the field (Amiard et al., 2000; Moolman, 2004). Therefore,
biomarkers that are prognostic of advanced toxicity at higher biological
levels have historically been heralded as potentially dominant tools for
use in biomonitoring because of their sensitivity and specificity to
xenobiotic exposures and also for pragmatic reasons such as the cost and
time associated with measuring a stress response. Such scenarios have
triggered the research to establish early-warning signals or molecular
biomarkers, reflecting the adverse biological responses towards
anthropogenic environmental toxicants (Bucheli and Fent, 1995). The use
of biomarkers in environmental monitoring gives significant advantages
over conventional chemical measurements because measured biological
effects can be meaningfully related to environmental consequences so
that environmental concerns can be directly addressed (Wu et al., 2005).
The use of biomarkers does not replace chemical monitoring, but it
integrates them providing a unique contribution in determining the
toxicity of pollutants, even when they are present at low, sub-lethal
concentration (Shugart, 1996). It is suggested that biomarkers can be used
to develop rapid, effective screening assays, which can complement other
testing techniques by significantly reducing the number of samples that
may require a more elaborate, specific evaluation (STAP, 2003).
Consequently, in ecological quality monitoring programs, the
integration of chemical data with biological responses (biomarkers) is
strongly recommended to characterize effects of contaminants on
organisms (Clements, 2000; Manns et al., 2003; Hagger et al., 2008). In
the last three decades, however, recommended biomarkers in aquatic
organisms have become a major tool for environmental quality evaluation
and risk assessment (Regoli et al., 2004; Amiard et al., 2006).
32
Biomarkers are increasingly worldwide-recognized method for the
assessment of pollution impacts in the aquatic environment, and some are
already included in environmental monitoring programs (Cajaraville et
al., 2000; ICES, 2004; Viarengo et al., 2007). These are modifications in
some parameters of blood composition, oxidative stress and DNAadducts appearance, cytotoxicological responses such as genotoxicity,
lysosomal alterations, cytochrome P450, metallothioneins (MTs), cellular
energy allocation (CEA), acetylcholine esterase (AChE) and appearance
of specific antibodies against a xenobiotic or a particular cellular fraction.
Some special protein can be used as biomarker for metal exposure as
well.
It is well understood that no one biomarker has been validated as a
unique tool
for detecting specific pollutant exposure and effects
(McCarthy, 1990; Lagadic et al., 2000) and cannot provide an adequate
base for environmental health assessment, management and sustainability
planning. The suite/battery of biomarkers at different levels of biological
organization is more realistic assessment, effectively applicable, helpful
and offers an effective early warning system in biomonitoring of aquatic
environments (Parolini et al., 2010; Jebali et al., 2011). Many authors
have stressed the importance of various biomarkers and have further
advocated the utilization of different endpoints at different levels of
biological organization (Lagadic et al., 1997; Smolders et al., 2003). The
multiparametric approach using different biomarkers will enable an
assessment of the effects of the different pollutants present in the aquatic
environment (Serafim et al., 2011). Oxidative stress as main biomarkers
in assessment of the pollution-related toxicity should be members of the
battery.
In the present investigation the biomarkers of oxidative stress were
selected for monitoring of pollution because variety of different toxicants
33
can cause damage in aquatic organisms by this mechanism of toxicity and
it is multi-parameter approach. Oxidative stress indicator biomarkers
have become a major tool for environmental quality evaluation and risk
assessment (Regoli et al., 2004; Amiard et al., 2006).
Overview of biomarkers for measuring oxidative stress
Among the numerous parameters of oxidative stress indicator
biomarkers, one area of interest has been the study of antioxidants system
involved in the detoxification of xenobiotics (Sheehan and Power, 1999).
Aquatic organisms have evolved a suite of enzymatic and non-enzymatic
defenses to cope with the production of reactive oxygen species (ROS).
The antioxidant defense enzyme system comprises several enzymes such
as superoxide dismutase (SOD), catalase (CAT), and glutathione
peroxidase (GPx), while non-enzymatic antioxidants or scavengers
include fat soluble compounds like tocopherol (Vit. E), β-carotene (pro
Vit. A) and water soluble compounds like glutathione and L-ascorbic acid
(Vit. C). Many of these antioxidants interact in a concerted manner to
eliminate reactive oxygen species and prevent damage to cellular
components. Superoxide radicals generated are converted to H2O2 by the
action of SOD, and the accumulation of H2O2 is prevented in the cell by
CAT and GPx.
Relationships between xenobiotic exposures and biomarker responses
All biological systems generate endogenous reactive oxygen
species (ROS) and other oxidants during their aerobic metabolism and
energy production in the mitochondria. Natural antioxidant enzymes
manufactured in the body provide an important defense against free
radicals. Under normal physiological condition, animals maintain a
precise balance between generation, neutralization of reactive oxygen
species (ROS) and the level of antioxidant molecules.
34
A common pathway of toxicity induced by a wide range of
environmental toxicants is the enhancement of intracellular production of
reactive oxygen species (ROS), „„oxygen-derived species‟‟ or oxyradicals
and comprises both radical and non-radical species (Reist et al., 1998;
Livingstone, 2001) and reduce the antioxidant protection (Hausladen and
Stambler, 1999; Halliwell and Gutteridge, 2001). Heavy metals are
known to disturb redox homeostasis in mollusk by stimulating the
formation of free radicals and reactive oxygen species (ROS), which
exceeds their scavenging capacity (Livingstone, 2001; Company et al.,
2004; Chandran et al., 2005). Excessive ROS production can overwhelm
natural defense mechanisms leading to oxidative stress through the
Fenton reaction (Sheehan and Power, 1999; Regoli, 2000), with direct
influence on the activity of antioxidant enzyme and level of lipid
peroxidation (Runnalls et al., 2007; Faria et al., 2010). Organisms are
able to react with these radicals by several mechanisms, including the
generation of antioxidant enzymes such as superoxide dismutase (SOD),
catalase (CAT), glutathione peroxidases (GPx). Damage can occur to the
biological membranes resulting in lipid peroxidation, when antioxidant
systems are in sufficient to neutralize the oxyradicals in the cells
(Halliwell and Gutteridge, 1999; Livingstone et al., 2000).
Therefore biomarkers indicating oxidative stress (antioxidant
enzyme system and LPO) in bivalve are proposed for the biomonitoring
of aquatic metal pollution. The modulation of enzymatic antioxidants
includes superoxide dismutase (SOD), catalase (CAT), glutathione
peroxidase (GPx), glutathione S-transferase (GST) and level of reduced
glutathione (GSH), and malondialdehyde (MDA) formation to investigate
the oxidative stress generated by metals. These enzymes can be induced
by reactive oxygen species (ROS) and therefore they may represent
indicators of oxidative stress (Pavlovic et al., 2004; Barata et al., 2005).
35
These enzymes can give an evaluation of the physiological state of an
organism. Measuring activities of these enzymes provides indicators of
the effects of contaminants in the aquatic environment and on biota.
These antioxidant enzymes have been proposed to use as biomarkers for
exposure to ROS-mediating contaminants, such as heavy metals (Chevre
et al., 2003). Oxidative stress is more often used as a biomarker of the
effects of exposure to environmental pollutants in aquatic environments
(van der Oost et al., 2003; Valavanidis et al., 2006). In recent years, there
has been considerable interest in the use of antioxidant defense system of
bivalves in environmental monitoring system because of its potential
utility to provide biochemical biomarkers (Oliveira et al., 2010).
The two kinds of responses induction and/or inhibition of the
enzyme activities and lipid peroxidation (LPO) depend on the intensity
and duration of the stress applied on one hand and on the susceptibility of
the exposed species on the other (Cossu et al., 2000). Measurement of
antioxidant enzymes and lipid peroxidation in mussels can be used as
sensitive biomarkers for the biomonitoring (Lionetto et al., 2003;
Vlachogianni et al., 2007).
The results of numerous worldwide studies carried out so far, have
indicated that the oxidative stress indicator (antioxidant enzyme system
and LPO) biomarkers are a powerful and cost-effective tool to get
information on the state of the environment and the effect of
contaminants on living biological resources. Therefore, many of these
have been accepted in national and international monitoring programmes
in Europe and USA (Collier et al., 1995).
Factors affecting antioxidant enzyme activity
Factors other than contaminants may affect measures of
antioxidants and oxidative damage in bivalves, and confound their use in
biomonitoring. Temperature may affect antioxidants in bivalves by
36
changing the catalytic rate of antioxidant enzymes. Dissolved oxygen
may affect biomarkers of oxidative stress in bivalve molluscs. Past
studies have confirmed that invertebrates adapted to environments with
high dissolved oxygen often have high levels of antioxidants to protect
them from related increases in oxyradical production (Shick and Dykens,
1985; Regoli et al., 2000). Conversely, antioxidant inhibition may occur
in molluscs during anoxic or hypoxic conditions (Viarengo et al., 1989;
Pannunzio and Storey, 1998). Food availability could affect oxidative
stress responses in bivalves by a variety of mechanisms, including
changing the amount of amino acids available for antioxidant production
and altering the composition of polyunsaturated fatty acids in membranes
there by affecting lipid peroxidation rates.
Many abiotic and biotic factors changes with season and influence
metabolic activities of aquatic organisms. Seasonal changes in metabolic
activities of an organism can put stress on the organism; which in turn
may lead to an increase in the production of ROS. Oxidative stress levels
may also change throughout the year due to seasonal changes in
reproductive status, growth, water temperature and nutrient availability
(Sheehan and Power, 1999). Biomarker levels in natural populations of
organisms can be predictable to change due to physiological adaptations
caused by seasonality (Verlecar et al., 2007). Preferably, the ideal
biomarker would not change seasonally in response to seasonal changes,
and would only respond to changes in exposure to contaminants.
Therefore Sheehan and Power (1999) reported that it is important to
define the natural seasonal changes in biomarkers to confirm their use as
a biomarker of exposure to contaminants. To confirm the use of
antioxidant enzymes as biomarkers of heavy metal contamination and it is
essential to define the natural range of biomarker activities by taking into
account their seasonal and natural variations (Sole and Albaiges, 1995;
37
Cajaraville et al., 2000) before applying the use of biomarkers in field
studies and in environmental monitoring programmes (Cajaraville et al.,
2000). Manduzio et al., (2004) reported that there was a correlation
between antioxidant defences and seasonality and this can be associated
to food consumption and reproductive status. To check the applicability
of antioxidant enzymes as biomarkers of diminished health, an
appropriately designed study of animals living in their natural
environment needs to be carried out to evaluate alterations occurring in
field-typical conditions (De Coen et al., 2006).
Literature review
The vast amounts of literature are available on the establishment
and utilization of some biomarkers in field to identify polluted sites and
to assess the impact of pollution in aquatic environment and laboratory
based approaches (Lagadic et al., 2000; Chevre et al., 2003; Viarengo et
al., 2007). Viarengo et al., (1991) studied seasonal variations in the
antioxidant defense systems and lipid peroxidation in the digestive gland
of mussels. Biomarkers are considered useful devices and are
increasingly included into environmental monitoring programs such as
the United Nations Environment Programme has funded a biomonitoring
programme in the Mediterranean Sea that contains variety of biomarkers
(UNEP, 1997). Cossu et al., (1997) studied glutathione reductase,
selenium dependent glutathione peroxidase, glutathione levels and lipid
peroxidation in freshwater bivalve, Unio tumidus, in field studies as
biomarkers of aquatic contamination. Doyotte et al., (1997) studied
antioxydants enzymes, glutathione and lipid peroxidation as applicable
biomarkers of experimental or field exposure in gills and digestive gland
of the fresh water bivalve, Unio tumidus. Da Ros et al., (2000) studied
biomarkers and trace metals in the digestive gland of indigenous and
transplanted mussels, Mytilus galloprovincialis, in Venice Lagoon, Italy.
38
Cajaraville et al., (2000) studied the use of biomarkers to assess the effect
of pollutants in coastal environments of the Iberian Peninsula. Several
scientific programmes in different Mediterranean countries are taking
approach to the biological effects of pollutants with the aim of promoting
a common and integrated strategy of utilizing marine biomarkers in
suggested sentinel species (Cajaraville et al., 2000; ICES, 2004). Lipid
peroxidation and catalase activities were found to be modulated by metals
or organic contaminants under both laboratory exposure (Livingstone,
1991) and field conditions (Hoareau et al., 2001; Durou et al., 2007).
Porte et al., (2001) studied the relation of antioxidant defense system
enzymes as a marker of chemical pollution by using M. galloprovincialis
in the Venice Lake, Italy and Galician coast in NW Spain. Cheung et al.,
(2002) studied the relationships between tissue concentrations of
chlorinated hydrocarbons (polychlorinated biphenyls and chlorinated
pesticides) and antioxidative responses of marine mussels, Perna viridis.
Mytilus spp. and other marine bivalves are commonly used in both,
laboratory and field studies to assess pre-pathological changes as
responses to increased pollution stress at the cellular and organism levels
(Lionetto et al., 2003; Regoli et al., 2004; Gravato et al., 2005). Romeo et
al., (2003) studied the activities of glutathione-S-transferase (GST),
catalase (CAT), acetylcholinesterase (AchE) and lipid peroxidation
(LPO) in transplanted mussels to evaluate the water quality. Lionetto et
al.,
(2003)
measured
the
integrated
use
of
biomarkers,
acetylcholinesterase (AchE) and antioxidant enzymes (CAT, GPx)
activities for identifying the possible effect of chemical pollutants in
Mytilus galloprovincialis and Mullus barbatus in an Italian coastal
marine area. Company et al., (2004) studied the impact of cadmium,
copper and mercury on activity of antioxidant enzymes and lipid
peroxidation in the gills of the hydrothermal vent mussel, Bathymodiolus
39
azoricus. The study on seasonal variations in battery of biomarkers and
physiological
indices
for
the
mussel,
Mytilus
galloprovincialis
transplanted into the northwest Mediterranean Sea was conducted by
Bodin et al., (2004). Manduzio et al., (2004) studied the seasonal
variation in the activity levels of GST, GPx, GR, and three isoforms of
Cu/Zn-superoxide dismutase in gills and digestive glands in the blue
mussel, Mytilus edulis. Bebianno et al., (2004) studied the role of several
biomarkers: metallothioneins (MT), superoxide dismutase (SOD),
catalase (CAT), glutathione peroxidases (GPx), lipid peroxidation (LPO),
glutathione S-transferase (GST) and acetylcholinesterase (AChE), in
different tissues of the clam R. decussatus, in laboratory conditions and
under
various
environmental
stresses,
in
a
perspective
of
a
multibiomarker approach to assess environmental changes. Antioxidant
systems and lipid peroxidation in Bathymodiolus azoricus from MidAtlantic Ridge vent fields was studied by Bebianno et al., (2005). Leinio
and Lehtonen (2005) studied the seasonal variation in biomarkers in the
bivalve‟s Mytilus edulis and Macoma balthica from the northern Baltic
Sea. Several studies have used a group of enzymes (including GR, GST,
MT, GPx, CAT, GSSG, GSH and SOD) to measure alterations in enzyme
activity as a result of environmental pollution. Box et al., (2007) and
Osman et al., (2007) showed that antioxidant enzyme activities decreased
with increase in pollutant levels. Perendija (2007) studied activities of
glutathione peroxidase (GSH-Px), glutathione reductase (GR), and
glutathione-S-transferase (GST) in the foot of three species of freshwater
mussels, Unio pictorum, Unio tumidus, and Sinanodonta woodiana from
the Sava River. Biomarkers are used in Joint Monitoring Program of the
OSPAR convention; MED POL, to distinguish anthropogenic pollution
(Viarengo et al., 2007). Vlachogianni et al., (2007) use of biomarkers
(superoxide dismuatse, catalase and lipid peroxidation) in mussel, Mytilus
40
galloprovincialis for evaluating heavy metals pollution in coastal areas
from the Saronikos Gulf of Greece.
Morales-Caselles et al., (2008)
highlighted the in identifying cause-effect relationships between
organisms, biomarker responses, and contaminants and its importance of
field studies. Seasonality is a typical characteristic of aquatic
environments. Antioxidant biochemical responses to long-term copper
exposure in Bathymodiolus azoricus from Menez-Gwen hydrothermal
vent was studied by Company et al., (2008). The study on sub-lethal
effects of cadmium on the antioxidant defence system of the
hydrothermal vent mussel, Bathymodiolus azoricus was conducted by
Company et al., (2010). Khebbeb et al., (2010) studied the effect of
cadmium exposure on malonedialdehyde and reduced glutathione
concentrations in various tissues of a bivalve, Ruditapes decussatus
fished from Mellah lagoon (North East of Algeria). Antioxidant and lipid
peroxidation responses in Mytilus galloprovincialis exposed to mixtures
of benzo (a) pyrene and copper studied by Maria and Bebianno (2011).
Wang et al., (2011) studied effect of cadmium exposure on enzyme
responses and lipid peroxidation in gills and hepatopancreas of clam,
Mactra vereformis. Netpae et al., (2012) studied the antioxidant enzyme
activities and DNA damage as biomarker of copper effect on Corbicula
fluminea.
In the present study the oxidative stress indicator biomarkers that
were selected to indicate the pollution of four reservoirs were
malondialdehyde (MDA) formation, reduced glutathione (GSH) level and
activity of antioxidant enzymes, superoxide dismutase (SOD), catalase
(CAT), glutathione peroxidase (GPx) and glutathione-S-transferase
(GST). This group of multiple biomarker assays using bivalves was
subjected to multivariate analysis to provide an integrated biomarker
response assessment of the ecosystem. As such oxidative stress
41
measurement indicators has been used as sensitive, early warning
indicators of adverse effects in polluted aquatic ecosystem.
There is apparently lack of information on bioaccumulation of
contaminants and its relation to biomarker responses in the freshwater
organisms. Hence greatest need is felt to study the concentration of
different metals and its response to biochemical levels in sedentary
organisms such as bivalves. This provides not only early warning
regarding degradation in environmental quality, but also specific
measures of the toxic, mutagenic and carcinogenic compounds in the
biological materials.
Therefore aim of present study is to measure the level of oxidative
stress indicator parameters like lipid peroxidation and reduced glutathione
(GSH) and activity of antioxidant enzyme superoxide dismutase (SOD),
catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase
(GST) in digestive glands of fresh water bivalve, Lamellidens corrianus,
Lamellidens marginalis and Parreysia cylindrica inhabiting at four
reservoirs of Nasik district, to indicate the pollution status of the
reservoirs.
However, the study of antioxidant enzymes in conjunction with
trace metal body burden contribute to a more comprehensive picture of
environmental pollution and biological responses in bivalves representing
useful reference value for future heavy metal pollution assessment.
Hence there is utmost need to examine the concentration of various heavy
metals and their responses to different oxidative stress indicator
parameters in sedentary organisms such as bivalves. Furthermore, since
these biomarkers are a part of the detoxification mechanism, these
enzymes are secreted at sub lethal levels of toxic components. This
provides not only early warning about degradation in environmental
42
quality, but also specific measures of the toxic, carcinogenic and
mutagenic compounds in the biological materials.
43
c) Biochemical Study
In aquatic ecosystem, heavy metals are considered as the most
important pollutants due to their highly bioaccumulative nature, persistent
behavior and potential of higher toxicity (Niyogi and Wood, 2004; Censi
et al., 2006). When animals are exposed to metals in a polluted aquatic
ecosystem, metal accumulate in various tissues significantly (Framobi et
al., 2007; Mohamed, 2008; Fernandes et al., 2008).
Heavy metal
bioaccumulation in the organisms of food chain is highly dangerous to
human health. Accumulated heavy metals induce generation of reactive
oxygen species (ROS). The ROS attack unsaturated fatty acids of the cell
membrane that lead to the formation of LPO (Viarengo, 1989). Increased
formation of lipid peroxidation causes alterations in the levels of GSH
and activities of the antioxidant enzymes which leads to oxidative stress
(Regoli, 2000) and results into oxidative damage to cellular
macromolecules such as proteins, lipids, nucleic acids and carbohydrates
(Manning et al., 2005; Araujo et al., 2006; Kaoud and El-Dahshan,
2010).
In any tissues, toxicants exert their effect first at the molecular and
biochemical level (Robbins and Angel, 1976). It has been observed that
metals can cause inhibition of enzymes, genetic damage, metabolic
disorder, and affecting physiological and biochemical mechanisms of
animals (Lucky and Venugopal, 1977; Radhakrishnan et al., 1991). The
biochemical composition varies according to the situation like seasonal
changes, environmental factors (temperature, salinity), starvation and
toxicants in the water (Verlecar et al., 2007; Nandurkar and Zambare,
2010; Salaskar and Nayak 2011). Hence changes in normal biochemical
parameters serve as the initial sensitive indicators of toxic effect on
tissues (Thaker and Haritos, 1989).
44
The convenience of biochemical approaches in environmental
pollution monitoring and characterization of effect/exposure to stressor
for the use in environmental risk assessment is based on the assumption
that low concentrations of a toxicant causes biochemical responses within
individual organisms before these effects are observed at higher levels of
biological organization (Bayne, 1985; Sarkar et al., 2006). Such
biochemical responses are considered to be rapidly responding endpoints
(Adams, 2002). This often provides sensitive and specific response to
particular toxicant. Hence, this kind of biomarkers gives an opportunity to
measure and diagnose the type of contaminant or toxicant that particular
organism is exposed (McCarthy and Shugart, 1990). Biochemical
responses in aquatic organisms have been employed in several
monitoring programs to characterize anthropogenic pollution (Cajaraville
et al., 2000). To assess the overall quality of the aquatic environment,
different biological responses can be examined to estimate the impact of
pollutant. In fact the changes in biological functions, structures and
proteins in response to metal pollution may be used to assess the health of
aquatic animals as early warning signals of various environmental risks
(Venier and Zampieron, 2005) and to access the fitness of the animal.
In past few years monitoring programs, carried out to evaluate
water quality, generally include chemical and common biological
parameters, the use of biochemical markers is less frequent, but recently
more efforts have been given to propose these biomarkers of exposure
and effect, in toxicity testing aiming an application in pollution
monitoring (Goulet et al., 2005).
1. Protein:
Proteins are important biomolecules involved in a wide spectrum
on cellular functions. They interplay between enzymatic and nonenzymatic proteins to govern the metabolic harmony (Lehninger, 1984).
45
They are also involved in major physiological events to maintain the
homeostasis of the cell. Protein acts as growth material for organism and
as a source of energy. Harper et al., (1978) stated that, the proteins are
among the most abundant biological macromolecules and are extremely
versatile in their function.
A wide range of metal pollution or stresses are responsible for the
secretion or suppression of the proteins (Iwana et al., 1998 and Kohler et
al., 2001) in the body of organism. Proteins are one of the main targets of
free radicals attack. Over produced radicals can react with protein and
amino acids to oxidize and cross-link them. Radical-protein reactions can
impair the function of important cellular and extracellular proteins like
enzymes and connective tissue proteins permanently. Any alteration in
the environment affects the level of protein content by changing the
physiology of organism (Young, 1970). Several studies reported that
accumulated heavy metal stress causes biochemical alterations in
organism (Verlecar et al., 2008; Zhang et al., 2010; Rajkumar and
Milton, 2011).
Combining “Sentinel species” with specific biomarkers provides
important biological information on the potential impact of xenobiotics
on the health of organisms and ecosystems (Van der oost et al., 1997).
Various biomarkers have been measured in molluscs, such as vitelline like proteins, metallothionin like proteins (MT), lipid peroxidation
protein, free DNA strands and glutathione-s-transferase (GST) (Gagne et
al., 2001 a, 2004, Hoarau et al., 2004). Therefore, the estimation of
protein is considered to be important parameter to indicate the stress
condition in the organism.
2. Ascorbic acid
Ascorbic acid is important micronutrient, which functions as a
factor in several metabolic reactions (Halliwell, 2001; Kaya, 2003). It is
46
a low molecular weight antioxidant and in vitro it inhibit the lipid
peroxidation (Serbecic and Beutelspacher, 2005; Verma et al., 2007).
Ascorbic acid is well known to inhibit oxidative damage against metal
toxicity (Houston and Johnson, 2000; Rao et al., 2001; Nandi et al.,
2005). Ascorbic acid helps to maintain the oxidation-reduction potential
of the cell at the stabilized level. Ascorbic acid acts against the toxic,
mutagenic and carcinogenic effects of environmental pollutants by
stimulating liver detoxifying enzymes (Sweetman et al., 1997;
Kronhausen et al., 1989). Antioxidant property of ascorbic acid helps to
prevent free radical formation from toxic water soluble molecules which
may cause cellular injuries and diseases. It can act as a hydrogen carrier
and may have an essential role in the metabolism of carbohydrate or
protein or both.
Ascorbic acid, being important constituent in cellular metabolism,
the interactions of the biomolecules gives proper idea of toxicant stress
and its effect. During acute and chronic response to different stressors
such as metals, ascorbic acid levels were depleted (Parihar and Dubey,
1995; Lackner, 1998). The study regarding the change in ascorbic acid
content in bivalves exposed to heavy metals can be useful as an indicator
of stress situation.
3. Deoxyribonucleic acid (DNA)
Deoxyribose nucleic acid contents can be the index of capacity of
an organism for protein synthesis in different stress conditions affected by
heavy metals or any toxic metals or pesticides. Different toxic levels and
stressed conditions may alter or damage the nucleic acid. Number of
researchers reported changes in DNA content due to heavy metal stress
(Black et al., 1996; Bolognesi et al., 1999; Gulbhile, 2006; Nawale, 2008;
Patil, 2010; Andhale and Zambare, 2011; Ali and Shakoori, 2011). DNA
damage results from exposure to many contaminants, and is widely used
47
as an indicator or biomarker of biological effects (van der Oost et al.,
2003). DNA damage is an important mechanism of toxicity for a variety
of pollutants, and therefore, is often used as an indicator of pollutant
effects in eco-toxicological studies. RNA: DNA ratio is a measure of
protein synthesis that has been used as a biochemical biomarker of
growth reflecting a general response to environmental stressors
(Humphrey et al., 2007).
4. Ribonucleic acid (RNA)
RNA is an important molecule with long chains of nucleotides. Just
like DNA, RNA is vital for living beings. The main job of RNA is to
transfer the genetic code need for the creation of proteins from the
nucleus to the ribosome. This process prevents the DNA from having to
leave the nucleus. This keeps the DNA and genetic code protected from
damage. Without RNA, proteins could never be made. The RNA/DNA
ratio has been used as an alternative measure of the physiological
conditions of ecologically relevant organisms (Amaral et al., 2009) and
has furthermore been described as being well correlated with growth and
nutritional condition of such organisms. Alterations in RNA content due
to heavy metal exposure was reported by several researchers (Gulbhile,
2006; Nawale, 2008; Srivastava and Verma, 2009; Andhale and Zambare,
2011).
Literature review
Changes in the protein, ascorbic acid, DNA and RNA contents due
toxicant stress were studied by the several researchers in different
mollusc species. Siddiqui (1967) observed the ascorbic acid levels during
summer season in liver, gonad and serum of Ophiocephalus punctatus.
Chitra and Ramana Rao (1977) reported variety of changes in ascorbic
acid levels at low temperature. Rao et al., (1987), studied Bio-chemical
composition in respect to pH and fluoride in the bivalve Indonaia
48
caeruleus.
Kulkarni et al., (1988) observed the effect of pH and
temperature on ascorbic acid content of Indonaia caeruleus. Vedpathak
(1989) reported enhanced ascorbic acid levels with increase in
temperature in Indonaia caeruleus. The alteration in ascorbic acid level
due to the pollutants in Corbicula striatella was studied by Zambare
(1991). Muley and Mane (1995) found increase in ascorbic acid content
in hepatopancreas, mantle and gonads of fresh water bivalve, Lamellidens
marginalis exposed to pH. Deshmukh and Lomte, (1998) studied the
protein content in mantle, foot, gill, digestive gland and whole soft body
tissue of fresh water bivalve, Parreysia corrugata after acute and chronic
exposure to copper sulphate. Seasonal changes in biochemical
composition in two unionid mussels, Actinonaias ligamentina and
Amblema plicata was observed by Baker and Hornbach (2001). Waykar
et al., (2001) studied the changes in the ascorbic acid content during acute
and chronic exposure to pollutant in mantle, gill, foot, digestive gland and
whole body tissues of the bivalves, Parreysia cylindrica. Mahajan and
Zambare (2001) studied the protein profile in different tissues of
freshwater bivalve, Corbicula striatella after exposure to chronic dose of
copper sulphate and mercuric chloride. Waykar and Lomte (2001a)
studied total protein alteration in different tissues of the fresh water
bivalve, Parreysia cylindrica after exposure to pollutants. David et al.,
(2003)
studied
some
aspects
of
protein
metabolism in
foot,
hepatopancreas and mantle tissues of snail, Pila globosa on exposure to
heavy metal. Waykar and Lomte (2004) studied carbaryl induced changes
in the ascorbic acid content in different tissues of freshwater bivalve
Parreysia cylindrica. Mahajan (2005) studied the biochemical changes
induced by heavy metals mercury, lead and arsenic in the gastropod,
Bellamya (Viviparous) bengalensis. Pardeshi and Zambare (2005) studied
the ascorbic acid contents of different organs in relation to reproduction
49
of the freshwater bivalve, Parreysia cylindrica. Gulbhile (2006) studied
the alterations in biochemical constituents in different tissues of
freshwater bivalve, L. corrianus after acute exposure to arsenic and
mercury. Mahajan and Zambare (2006) studied the changes in ascorbic
acid content in freshwater bivalve, Laemellidens marginalis after chronic
exposure to arsenic. The seasonal variation in RNA content in
Austrovenus stutchburyi at different sites was studied by Norkko and
Thrush (2006). Mahajan (2007) studied the changes in the biochemical
composition of the gill, foot, digestive gland and whole soft body tissues
of freshwater bivalve, Lamellidens marginalis exposed to chronic
concentration of arsenic trioxide, cadmium chloride and lead chloride.
Nawale (2008) studied changes in biochemical content in different tissues
of freshwater bivalve, L. corrianus after chronic exposure to lead nitrate
and sodium arsenate. Injal and Raut, (2009) reported lead induced
alterations in protein levels of gills and mantle of freshwater bivalve,
Lamellidens marginalis. Singh et al., (2010) studied the effect of
sublethal treatment of deltamethrin on protein, amino acid and nucleic
acids levels in gonadal, nervous and foot tissue of Lymnaea acuminata.
Kamble et al., (2011) studied the impact of pollutants on ascorbic acid
content in freshwater Lamellibranch mollusc, Lamellidens corrianus.
Jagtap et al., (2011) studied the changes in protein composition of gonad,
gills and digestive gland of freshwater bivalve, Lamellidens marginalis
after exposure to acute concentrations of organotin tri-butyltin chloride.
Rajkumar and Milton (2011) studied the alterations in total protein levels
in Perna viridis after chronic exposure to different concentrations of
cadmium, copper, lead and zinc. Mahajan (2012) studied the changes in
protein content in Bellamya dissimilis after chronic exposure to copper
sulphate. Pardeshi and Gapat (2012) studied the effect of nickel
intoxication on the protein content in freshwater bivalve, Lamellidens
50
corrianus. Salaskar and Nayak (2011) reported seasonal changes in
protein content of bivalves, Crassostrea madrasensis and Perna viridis in
the Kali estuary, Karnataka, India. Soumady and Asokan (2011) studied
seasonal changes in protein content in various organs of Perna viridis at
Tranquebar Coastal Waters, Tamilnadu, India. Babu et al., (2012) studied
protein content in different body parts of bivalve, Gafrarium tumidum.
Wenne and Styczyfiska-Jurewicz (1987) studied seasonal changes in
protein content in Macoma bahhica from five sampling sites in the
Gulf of Gdafisk. Waykar and Pulate (2012) studied the ameliorating
effect of L-ascorbic acid on profenofos induced alterations in the protein
contents of the freshwater bivalve, Lamellidens marginalis (L.).
Under certain unfavorable conditions, the organism develops
certain adaptive methods such as mobilization of energy from reserves to
tide over the crisis and to protect themselves (Bryan et al., 1986). Hence,
the study of biochemical components would be much meaningful to
estimate the nutritive value of the organism, and its further analysis with
the metal effect would provide an intricate relation between the metal
pollutants and the metabolism of the basic biochemical constituents.
Therefore, it is an attempt to investigate the effect of metal stress on the
biochemical components.
Pollution Sources at Study Area:
Girna, Ozarkhed, Chankapur and Gangapur reservoirs of Nasik
district is under permanent pressure of anthropogenic pollutants
originating from various sources located at the catchment areas and
details are given below
Girna reservoir:
The Girna reservoir is constructed on the Girna river. Girna river
originate from the hilly ranges of Sahyadries and flows from mountain to
plan, and weathering soil and rock have become sources of heavy metal.
51
The Girna river is mainly polluted by their rivulets namely Tamdi,
Punand, Aram, Mosam, Masa Nadi, Baindki and markhandi etc. These
rivulets are surrounded by the agricultural land, which is polluted by
fertilizers and pesticides. Four sugar factories and one cotton mill is
located in its basin, which drain untreated waste water into the Girna
river. The large numbers of small scale bricks industries are on the bank
of Girna river and its rivulets. The large quantity of ash gets released
from bricks industries into river water. The ash released in the water gets
settled and act as source of heavy metal pollution. The Kalwan, Satana,
Deola and Malegaon towns and number of villages are settled on the bank
of the river, releases untreated domestic waste and sewage water in river
which is also one of the important sources of heavy metal pollution. The
Mosam river, brings the industrial waste and domestic waste of Malegaon
town. Malegaon town is well known as most population density and city
of power looms in Maharashtra state. In all there are 1.1 lakh large,
medium and small scale power looms unit. Mainly the power loom
industries are engaged in grey cloths, synthetic and cotton fabrics, dyes
cloths, printed/dyed sarees, lungis, processing of raw clothing. The
printing is mainly carried out by the synthetic dyes. Dyes industries
required lot of water during the process. This untreated water is released
in to the open drainage system and finely to the Mosam river. In
Malegaon city apart from power looms industries, the other important
industries are agro-industries, plastic pipe manufacturing, cotton
spinning, ginning, oil mills, fabrications, tile manufacturing‟s and Lie
factories releases the waste in the drainage and finely come to the Mosam
river and Mosam river join to the Girna river. The waste water effluents
from textile dyeing and printing industries contains dyes, total dissolved
solids, bleaching agents, salts, acids, heavy metals like Cr, Cu, Pb, Zn,
and Fe are discharged continuously without treatment into Mosam river.
52
The river also receives traffic runoff, National highway-3 (BombayAgra). The vehicles release large quantity of smoke, oil droplets and
other particulate matter which get settled and dissolved in the river water
and acts as source of metal pollution.
Ozarkhed reservoir:
Ozarkhed reservoir is constructed at Dindori and receiving fresh
water from Unanda river. Unanda river originate hilly ranges of Satmala
and flows from mountain to plain, and weathering soil and rock have
become sources of heavy metals. Satmals hills are made up of basalt rock.
Unanda river receives freshwater from several small rivers like Dev nadi,
Parasheri river, Natravati nala etc and from other small streams. The
catchment area of Unanda river is surrounded by the highly productive
agricultural filed, which is polluted by fertilizers and pesticides. In some
catchment area of river is also know for cultivation of paddy crops and
vegetables. The large numbers of small scale and large scale bricks
industries are on the bank of river and its rivulets. The large quantity of
ash gets released from bricks industries into river water. The ash released
in the water gets settled and act as source of heavy metal pollution. The
cement asbestos sheet making factories are located in its basin, which
drain untreated waste water into the Unanda river. The high boating
activity due to ecotourism and fishing in dam, which leads to changes in
the water quality of dam. Sawmills and toys making units are on the bank
of river, discharges waste water into the river. The numbers of villages
are settled on the bank of the river, releases domestic waste and sewage
water in river which is also one of the important sources of heavy metal
pollution. Vehicular run-off also contributes to the pollution of river.
Chankapur reservoir:
Chankapur dam is constructed on Girna river at Kalwan in Nasik
district. The Girna river originate from hilly ranges, which are build from
53
basalt rock and flows from mountain to plain, and weathering soil and
rock have become sources of heavy metals. Girna river receives
freshwater from Tambdi river and from other small streams. Catchment
area of reservoir is agricultural land, which is polluted by fertilizers and
pesticides. In some catchment area of river is also know for cultivation of
paddy crops and vegetables. Villages settled on bank of river, releases
domestic waste, sewage water, traffic run-off in to rivers. The large
numbers of small scale bricks industries, sawmills and toys making units
are on the bank of river, discharges waste material into river. The
atmospheric fall out and vehicular run-off also contributes to the pollution
of river
Gangapur reservoir:
Gangapur dam, 10km away from Nisik city is constructed on
Godavari river. The anthropogenic activities in the study area have
affected the wetland. Godavari river originate from hilly ranges and flows
from mountain to plain, and weathering soil and rock have become
sources of heavy metals. Catchment area of reservoir is agricultural land,
which is polluted by fertilizers and pesticides. Villages settled on bank of
river, releases domestic waste, sewage water in to rivers. The large
numbers of small scale bricks industries are on the bank of river. The
high boating activity due to ecotourism and fishing in dam, which leads
to changes in the water quality of dam.
The reservoirs of Nasik district provides drinking water to nearby
towns and villages of Nasik district and Jalgaon district and its water also
used for irrigation, industrial supply, and for fishing purpose, therefore it
is imperative to monitor the metal pollution of the reservoir.
Therefore there is urgent need to monitor the heavy metal pollution
of the reservoir. A literature surveys revealed that, most of researchers
have studied physico-chemical parameters of the reservoir and never
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carried out biomonitoring study to detect heavy metal pollution level in
water, sediment and biota, hence present study was undertaken.
Objectives of the present study
1) To determine the level of heavy metals Cd, Zn, Cu and Pb in surface
water, soil sediments and whole soft body tissues of three bivalve
species, Lamellidens corrianus, Lamellidens marginalis and Parreysia
cylindrica inhabiting at the four reservoirs of Nasik district during
different seasons.
2) To find out most appropriate bivalve species as sentinel animal for
monitoring of metal pollution in the freshwater ecosystem.
3) To study the effect of heavy metals on oxidative stress indicator
biomarker parameters like activity of antioxidant enzymes, superoxide
dismutase (SOD), catalase(CAT), glutathione peroxidase (GPx), and
Glutathione-S-transferase and level of reduced glutathione (GSH) and
Lipid peroxidation in digestive glands of three bivalve species,
Lamellidens
corrianus, Lamellidens marginalis and
Parreysia
cylindrica inhabiting at the four reservoirs of Nasik district during
different seasons.
4) To study the effect of heavy metals on biochemical composition
(Protein, Ascorbic acid, DNA and RNA contents) in soft body tissues
of
three
bivalve
species
Lamellidens
corrianus,
Lamellidens
marginalis and Parreysia cylindrica inhabiting at the four reservoirs
of Nasik district during different seasons.
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