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REVIEW
Biota as toxic metal indicators
Slavka Stankovic •Predrag Kalaba •
Ana R. Stankovic
Received: 25 May 2013 / Accepted: 27 May 2013 / Published online: 9 June 2013
ÓSpringer-Verlag Berlin Heidelberg 2013
Abstract Metal in the environment arises from both
natural sources and human activities. Toxic metals in air,
soil, and water have become a global problem. They are
potential hazards to aquatic, animal, and human life
because of their toxicity, bioaccumulative, and non-bio-
degradable nature. The major impacts of metal pollutants
can be stated as ecosystem contamination and health
problems of exposed human populations. Those problems
have been a cause of increasing public concern throughout
the world. Some trace metals are used by living organisms
to stabilize protein structures, facilitate electron transfer
reactions, and catalyze enzymatic reactions. But even
metals that are biologically essential can be harmful to
living organisms at high levels of exposure. An increasing
concentration of heavy metals in the environment can
modify mineral and enzyme functions of human beings.
During the last two decades, the interest in using bioindi-
cators as monitoring tools to assess environmental pollu-
tion with toxic metals has increased. Bioindicators are flora
and fauna members, which are collected and analyzed to
measure the levels of metal contaminants. Bioindicators
therefore identify health hazards. Various living organisms,
such as microbes, fungi, plants, animals, and humans, are
used to monitor toxic metals from air, water, sediment, soil,
and food chain. Here, we review recent bioindicators,
toxicity assessment, and ecological effects.
Keywords Heavy metals Air Water Soil
Bioindicators Flora Fauna Humans Toxic effects
Biomarkers
Introduction
In natural systems, potentially toxic heavy metals can orig-
inate from rocks, ore minerals, volcanoes, and weathering
releases of metals during soil formation transported to the
surface and/or aquifer waters (Szyczewski et al. 2009). In the
last few decades, the pressure from the activities of the urban
population has been intense and anthropogenic emissions of
potentially toxic trace metals have accelerated considerably.
Anthropogenic impacts of toxic metals are related mostly to
the mining, extraction, and refining stages and can be the
cause of substantial air, water, and soil pollution (Norgate
et al. 2007). Heavy metals once released into the environ-
ment—the air, water, and soil—do not disappear, but accu-
mulate in soils, sediments, and biota.
Metals serving as micronutrients in living organisms
usually occur in trace amounts that are precisely defined for
each species. Both, their deficiency and high excess, badly
affect living organisms. They, also directly or indirectly,
throughout air, water, and food (plants, animals) get into
human bodies. The excessive content of metals in the human
body may in many ways affect the body (Stankovic et al.
2011a,b; Jovic et al. 2012). Therefore, the content of heavy
metals needs to be known not only in water, air, soil, and
sediment, but also in biological samples: plants, animals, and
finally, in humans (Stankovic and Stankovic 2013).
Metals fall into one of two categories: essential and non-
essential. Essential metals or micronutrients are required
for the optimal functioning of biological and biochemical
processes in organisms, while non-essential elements have
S. Stankovic (&)A. R. Stankovic
Department of Analytical Chemistry, Faculty of Technology
and Metallurgy, University of Belgrade, Karnegijeva 4,
11000 Belgrade, Serbia
e-mail: slavka@tmf.bg.ac.rs
P. Kalaba
Department of Biochemistry, Faculty of Chemistry, University
of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia
123
Environ Chem Lett (2014) 12:63–84
DOI 10.1007/s10311-013-0430-6
no known biological functions and exert their toxicity by
competing with essential metals for active enzyme or
membrane protein sites (Torres et al. 2008). Metals that are
biologically essential have the potential to be harmful to
humans and other living organisms at high levels of
exposure (Stankovic et al. 2011a; Stankovic and Jovic
2012). Together with essential nutrients, living organisms
also take up heavy metals and can accumulate them.
Living organisms are commonly known as toxic metal
bioindicators and their exposure to metals can be measured
by either levels or effects. The widespread development
and application of plants and animals as bioindicators has
occurred primarily since the 1960s, using various animals
like birds, mollusks, and mammals (Holt and Miller 2011).
As shown in the review of Burger (2006), over 40 % of the
bioindicator papers were about metal pollution, wherein
fish, plants, invertebrates, and mammals were the domi-
nantly used bioindicator species. For aquatic metal pollu-
tion, the commonly used bioindicators mainly contained
organisms including plankton, insects, mollusks, fishes,
plants, and birds (Zhou et al. 2008; Lam and Wang 2008;
Jovic et al. 2011; Hargreaves et al. 2011; Joksimovic et al.
2011a,b; Markovic et al. 2012; Joksimovic and Stankovic
2012; Kitowski et al. 2012).
Higher plants, animals, algae, fungi, bacteria, and lichen
have been used as bioindicators in air, soil, and water
pollution surveys over the past few decades. Metal content
in bioindicators depends not only on the metal concentra-
tions in air, water, soil, sediment, and environmental con-
ditions, but also on the biological factors of the organisms
themselves. One of the largest problems associated with the
exposure to toxic metals is their potential for bioaccumu-
lation and biomagnification causing heavier exposure for
some organisms than toxic metals present in the environ-
ment alone. This article is an abridged version of the chapter
by Stankovic and Stankovic (2013) in the series Environ-
mental Chemistry for a Sustainable World (http://www.
springer.com/series/11480).
Toxic metals
Metals normally occur in nature and some are essential to
life, but can become toxic through accumulation in
organisms. Only a few of the numerous metals present in
the environment are essential and necessary in minute
amounts to all living organisms, the so-called micronutri-
ents. Micronutrients, such as Cu, Zn, Fe, Mn, Co, Mo, Cr,
and Se, are required by humans in small quantities, a few
milligram or microgram per day, and Ca, Mg, Na, P, and S,
are also required, but in larger quantities, 100 mg or more
per day, for the optimal functioning of biological and
biochemical processes in humans (Stankovic and Stankovic
2013). Micronutrients are involved in the functional
activities of living organisms.
Heavy metals occur naturally and from anthropogenic
sources in the ecosystems with large variations in con-
centrations. Pb, Cd, Cr, Cu, Zn, Ni, As, and Hg are the
most common heavy metal pollutants, and Hg, Pb, and Cd
are of the greatest concern. They can be bioaccumulated
through the food chain posing a toxic risk to species higher
in the food chain and to humans (Stankovic and Stankovic
2013). The classification of elements from the periodic
table, according to their toxicity and uptake, whether easily
exposed to organisms, is presented in Table 1.
Toxic metals in the environment
World mining activities are known to release significant
amounts of toxic metals into the surrounding environment,
Fig. 1. About 90 % of mine outputs of Cd, Cu, Zn, Ni, and
Pb were consumed in the last century (Nriagu 1996). These
elements are present at low levels in soil, rock, and water,
but the process of world metal mining may release quan-
tities harmful to the health of people and ecosystems, as
well as the electric power industry, the primary metal
industry, and world metal mining operations. Metals from
Table 1 Classifications of elements according to toxicity and their
uptake (Wood 1974)
Not critical Toxic, partially
dissolved
or easily exposed
Very toxic and
easily exposed
Na, C, F, K, S,
Sr, H, Cl
Ti, Ga, Hf, Rh,
Nb, Ir
Be, As, Au, Cu, Pd, Pb
P, Li, Mg, Al,
O, Br, Si
La, Zr, Os, Ta,
Ru, Re
Co, Se, Hg, Zn, Ag, Sb
Fe, Rb, Ca, N W Ni, Te, Tl, Pt, Sn, Cd, Bi
Fig. 1 a Value of world mining activities, bworld metals value at
mine (O
¨stensson 2006)
64 Environ Chem Lett (2014) 12:63–84
123
these sources are dispersed in the environment and they
contaminate air, water, and soil. They, directly or indi-
rectly, through air, water, plants, and animals, get into
human bodies too.
The metals classified as heavy metals are: Cu, Co, Cr, Cd,
Fe, Zn, Pb, Sn, Hg, Mn, Ni, Mo, V, and W (Szyczewski et al.
2009). Within the group of heavy metals, one can distinguish
both the elements essential for living organisms (micronu-
trients) and the elements whose physiological role is
unknown, and thus they are ‘‘inactive’’ toward plants, ani-
mals, and people. The metals serving as micronutrients in
living organisms usually occur in trace amounts that are
precisely defined for each species (Szyczewski et al. 2009).
Both their deficiency and excess affect the psychophysical
development of organisms as well as adults in a harmful way.
Metals are commonly considered as simulators or
inhibiting factors of life processes, due to which they may
appear toxic for living organisms. This depends on their
concentration, ability to form complexes, and degree of
oxidation. The strongest toxic properties are the charac-
teristics for inorganic metals compounds, which are easily
soluble and they can easily penetrate cell membranes and
get into organisms. The free-metal ionic activity may be
more important in producing metal toxicity than the total
concentration of a metal (Szyczewski et al. 2009).
Mercury (Hg)
The main natural sources of Hg are the emissions from the
geothermal and volcanic activity (WHO 2007). The largest
anthropogenic source of Hg on a global scale is the com-
bustion of coal and other fossil fuels, including metal and
cement production, forest fires, and waste disposal. Annu-
ally, anthropogenic pollution reaches approximately 2,000 t
of this element, most of which comes from burning coal,
milling of non-ferrous metals, and gold mining by amal-
gamation (Kalisinska et al. 2012). According to official
emission data, the total Hg anthropogenic emissions in
Europe was 413 t/year in 1990 and 195 t/year in 2003 (WHO
2007).
The total concentrations of Hg in natural waters are nor-
mally very low, below 1.0 ng/L; in drinking water, it is in the
average of 25 ng/L and the WHO guideline value is 1.0 lg/L
(WHO 2007). Dissolved total mercury values are: in an open
ocean, 0.5–3.0 ng/L; coastal seawater, 2.0–15 ng/L; fresh-
water lakes and rivers, 1.0–3.0 ng/L. In the European top
soils, Hg concentrations range from 10 to 160 lg/kg,
reaching a median value of 40 lg/kg (WHO 2007). An
inorganic-Hg form can be converted biologically to meth-
ylmercury (MeHg) in soil and water and enters the human
body readily via the dietary route (Stankovic and Stankovic
2013).
Arsenic (As)
The total annual anthropogenic As emissions were estimated
as 28,000–54,000 tones/year, but this range does not include
natural As emissions to the atmosphere, including volca-
nism, forest fires, and for example, significant amounts of As
are released through hydrothermal activities in Yellowstone
National Park (Reimann et al. 2009). Plants generally show
low As concentrations, much lower than the supporting soils;
the suggested value for the world reference plant of As is
0.1 mg/kg (Nagajyoti et al. 2010). The majority of plants
have mechanisms for avoiding As uptake, but mosses and
lichens consistently show higher As values than other ter-
restrial plants (Serbula et al. 2012).
The toxicity of As has been well known at least since
Roman time (Stankovic and Stankovic 2013). The most
carcinogenic of all substances named in current drinking
water regulations is As. The drinking water action limit for
As was quite recently lowered to 10 lg/L (from 50 lg/L).
The As concentration in air is generally very low and ranges
from 0.4 to 30 ng/m
3
; in seawater from 1.0 to 8.0 lg/L, while
in marine sediments it ranges from 1.0 to 60 mg/kg. The
world-average value for As in soil is 5.0 mg/kg, but varies
considerably across geographic regions (Reimann et al.
2009).
Lead (Pb)
Pb is released into the atmosphere from natural and anthro-
pogenic sources. Natural Pb emissions originate from
volcanoes, forest fires, and biogenic sources. Major anthro-
pogenic emission sources of Pb include the combustion of
fossil fuels from traffic, non-ferrous metal production, and
iron and steel production (WHO 2007). The levels of Pb in
the environment vary between 4 and 20 mg/g of dust;
uncontaminated waters contain Pb in concentrations ranging
from 0.001 to 0.06 mg/L, while seawater contains up to
0.03 lg/L (Bardi 2010). In top soils, Pb concentrations are
spatially heterogeneous and vary from below 10 mg/kg up to
[70 mg/kg. The median value is estimated to be 22.6 mg/kg
in the European soil.
In general, ingestion of Pb through food and water is the
major exposure pathway for Pb in humans. Individuals will
absorb more Pb in their food if their diets are deficient in Ca,
Fe, or Zn (Hu 2002). The EU ambient air quality guideline
for Pb is 0.5 lg/m
3
and in the immediate vicinity of specific
industrial sources, the value was 1 lg/m
3
until 2010 (WHO
2007). The limit value for Pb in drinking water in the EU
is currently 25 lg/L and will be reduced to 10 lg/L by
2013 (WHO 2007). Levels of Pb in soils range from 5.0 to
30 mg/kg. In addition to atmospheric deposition, agricul-
tural practices are a source of Pb input to soils from mineral
and organic fertilizers (WHO 2007). According to Krystofova
Environ Chem Lett (2014) 12:63–84 65
123
et al. (2009), levels of Pb in the environment are not stable and
vary according to industrialproduction, urbanization, climate
changes, and many other factors.
Cadmium (Cd)
Cd is released to the biosphere from both natural and
anthropogenic sources too. The total Cd emission to air
from the natural sources is estimated at about 150–2,600 t
(WHO 2007). The global Cd production increased with a
factor of four from 1950 to 1990 and in the recent decade
the production has slightly decreased. Global emission of
Cd into air until the year 2000 was closed to 3,000 t/year
and in Europe 257 t in 2003. Emissions of Cd in EU
countries have decreased by 50 % and the dominant
sources of Cd are atmospheric deposition and commercial
phosphate fertilizers (OECD 1994).
On the basis of the Cd contents in surface soils from
many parts of the world, the average value lies between
0.07 and 1.1 mg/kg. Values above 0.5 mg/kg usually
reflect anthropogenic Cd inputs (WHO 2007). There are
three main anthropogenic sources of terrestrial Cd: atmo-
spheric deposition, agricultural application of phosphate
fertilizers, and use of municipal sewage sludge as a fer-
tilizer on agricultural soils. It has been reported that 90 %
of the Cd in soil remains in the top 15 cm (WHO 2007). Cd
levels of up to 5.0 mg/kg have been reported in sediments
from river and lakes, and from 0.03 to 1.0 mg/kg in marine
sediments. The average Cd content is about 5.0–20 ng/L in
seawater, in European rivers roughly varies from 10 to
100 ng/L (OSPAR 2002) and in drinking waters Cd con-
centrations usually vary between 0.01 and 1.0 lg/L (WHO
2007).
Chromium (Cr)
Cr(VI) exists in soils naturally and is the sixth most
abundant element in the Earth’s crust. Cr is found in all
phases of the environment, including air, water, and soil.
Cr occurs naturally in Irish agricultural soils in concen-
trations between 5.0 and 250 mg/kg and in various soil
types ranging from 1.04 to 3,015 mg/kg worldwide (Boyle
and Kakouli-Duarte 2008). It is also present as a result of
human practices, and mostly associated with industry.
Naturally occurring in soil, Cr ranges from 10 to 50 mg/kg
depending on the parental material (Shanker et al. 2005). In
freshwater, Cr concentrations generally range from 0.1 to
117 mg/L, whereas values for seawater range from 0.2 to
50 lg/L. In the atmosphere, Cr concentrations vary widely
from the background concentration of 0.0012 lg/m
3
(Shanker et al. 2005). Cr has been extensively shown to
induce general environmental toxicity, as well as more
specific effects of an acute and chronic toxicity nature, such
as neurotoxicity, dermatoxicity, genotoxicity, carcinoge-
nicity, and immunotoxicity. It is believed that Cr inflicts
more damage during Cr(VI) reduction to Cr(III), a process
considered to be initiated in the cell by glutathione (Boyle
and Kakouli-Duarte 2008).
Air, water, and soil metal pollution
Air
Atmospheric pollution causes serious damage to human
health and to all natural ecosystems. Among the many
inorganic air pollutants originating from anthropogenic
activities, heavy metals such as As, Cd, Cr, Hg, and Pb are of
a major concern due to their toxicity. One of the most
important sources of Cd, Cr, and Pb in the urban environment
is road traffic (Melaku et al. 2008). Other anthropogenic air
pollutant sources are constructions and agricultural activi-
ties, mining and mineral processing, wind-blown dust, and
power plants. Coal-fired power plants are responsible for
99 % of Hg emissions. The Hg concentration in air is, in most
areas, close to the mean global background value, which is
1.5–2.0 ng/m
3
(WHO 2007). In recent years, emissions of
Hg into the air in Europe have been declining.
According to Harmens et al. (2008), the main sources of
Pb emissions come from manufacturing industries (41 %)
and road traffic (17 %). Pb levels in the ambient air in
Europe have decreased in recent decades, that is, between
1990 and 2003 they fell by 50–70 %. In the air of rural
areas, Pb concentrations are between 0.05 and 0.10 lg/m
3
.
Natural Pb content is estimated to be 6.0 ng/m
3
in the
atmosphere and the air quality guideline recommended the
Cd level of 5.0 ng/m
3
to prevent any further increases in
Cd level in agricultural soils (WHO 2007).
Water
Heavy metals in aquatic systems can be naturally produced
by the slow leaching from soil/rock to water, which are
usually at low levels, causing no serious effects on human
health. Nowadays, the industrial and agricultural develop-
ment promotes the rapid increase in water metal pollution.
Metals can accumulate in aquatic organisms and persist in
water and sediments (Sevcikova et al. 2011).
Aquatic heavy metal pollutions usually represent high
levels of Hg, Cr, Pb, Cd, Cu, Zn, and Ni in water systems
(Zhou et al. 2008). Cu, Ni, Cr, and Zn are essential trace
metals to living organisms, but become toxic at higher con-
centrations. Heavy metals including Hg, Cr, Cd, Ni, Cu, As,
and Pb introduced into environmental water system may
pose high toxicities on the aquatic organisms (Stankovic and
Stankovic 2013). Concentrations of inorganic Hg in surface
and ground waters are generally below concentrations
66 Environ Chem Lett (2014) 12:63–84
123
known to cause adverse health effects, but MeHg in fresh-
water fish originates from the soil inorganic Hg and direct
atmospheric deposition (Chen et al. 2008). Although the
anthropogenic Hg emission in Europe decreased approxi-
mately by 50 % after 1990, the MeHg concentration
remained the same in freshwater fish (WHO 2007).
Soil
Soil is a fundamental natural resource for agriculture.
Initially, heavy metals are naturally present in soils. The
presence of heavy metals in the environment has accel-
erated due to human activities. The contamination of soil
mainly occurs through air pollution, wastewater intake,
and use of fertilizers in agriculture. The soil environment
is a major sink of heavy metals. An extensive literature
review of the urban soils trace metals contaminations
worldwide is given by Wong et al. (2006). Purely theo-
retically, every 1,000 kg of ‘‘normal’’ soil contains 200 g
Cr, 80 g Ni, 16 g Pb, 0.5 g Hg, and 0.2 g Cd (CAOBI-
SCO 1996). In areas of agricultural and industrial activity,
higher concentrations of heavy metals, in comparison to
background levels, can be detected (Babula et al. 2008)
and may disturb the soil ecosystem, plant productivity,
and also pose threat to human health and ecosystems
(Musarrat et al. 2011).
Trace metal assessments of urban soils frequently
examined to detect metals that were traditionally signifi-
cant for the environment and health, particularly Cu, Zn,
Pb, and Cd (Wong et al. 2006). In the EU, the limit value
for Cu in soils is 50–140 mg/kg and the mean levels vary
between 13 and 24 mg/kg; the Zn soil level usually falls in
the range of 10–300 mg/kg, and in the EU, the limit Zn
value for soil is between 150 and 300 mg/kg dw. The limit
values in the EU for Pb, Cd, and Hg are between 50 and
300, 1.0 and 3.0, and 1.0 and 1.5 mg/kg dw, respectively
(Council Directive, 86/278) (Serbula et al. 2012).
The mean Pb concentration is estimated at 25 mg/kg on
the world scale for surface soils. The average contents of
Cd in soils are between 0.07 and 1.1 mg/kg, and for var-
ious soils, the mean concentrations of Hg do not exceed
0.4 mg/kg on the world scale for surface soils (Serbula
et al. 2012). The world-average value for As in soils is
5 mg/kg (Reimann et al. 2009) and the EU limit for As in
agricultural soil is 20 mg/kg (Bhattacharya et al. 2010).
As(III) is more soluble and more mobile, and is much more
toxic than As(V) in soils (Lai et al. 2010), but in well-
aerated soils, arsenate [As(V)] is the predominant form.
Currently, about half of the world’s land and 70 % of total
water are used for agriculture (Reijnders and Huijbregts
2009) and consequently soil pollution problems arise from
agrochemicals being used.
Biota
Biota growth and development are essential processes of
life and propagation of the species. They are mainly
depending on external resources present in water, soil, and
air. The presence of toxic metals in the external environ-
ment leads to changes in the growth and development
pattern of the biota. Metals are commonly considered as
simulators or inhibitors of life processes, due to which they
may appear toxic for living organisms (Szyczewski et al.
2009). Various metals play a key role to maintain and
control various vital functions of living organisms. The
essential metals have biochemical and physiological
functions in living organisms. Two major functions of
essential metals are: (a) participation in redox reaction and
(b) direct participation as an integral part of several
enzymes (Nagajyoti et al. 2010). The availability of metals
in medium varies, and metals such as Cu, Zn, Fe, Mn, Mo,
Cr, Ni, and Co are essential micronutrients whose uptake in
excess results in toxic effects (Nagajyoti et al. 2010) and
can damage living organisms, or in some cases, the
organism can be injured or die (Szyczewski et al. 2009).
Fauna
Metals uptake by fauna occurs through diffusion as well as
ingestion of food. Metals can be stored in the skeletal
structure and intracellular matrices of fauna organisms, and
excreted in feces and eggs. Fauna organisms have addi-
tional defense mechanisms, such as heavy metal-binding
proteins (Stankovic and Stankovic 2013). In comparison to
plants, non-sessile animals can avoid a certain number of
environmental or anthropogenic stressors by their mobility.
Mollusks have been successfully used as bioindicators
in monitoring aquatic programs (Stankovic and Jovic 2013;
Stankovic and Stankovic 2013). Terrestrial ecosystems
were much less considered than the aquatic environment. It
can be stated that terrestrial bivalves, gastropods, and
especially the other mollusks classes have not yet received
the attention they probably deserve, in terms of their eco-
logical importance. A number of invertebrate species are
known to be efficient accumulators of trace elements
(Stankovic and Stankovic 2013).
Terrestrial vertebrates, including wild animals, absorb
toxic metals via food, water, air, and through the skin and
accumulate them mainly in the liver, kidney, and also in
the brain (Kalisinska et al. 2012). Prey, predatory birds and
mammals, are accumulators of significant amounts of
organic Hg by feeding on beans and small warm-blooded
vertebrate herbivores contaminated with Hg (Basu and
Head 2010). Toxicity of methylmercury (MeHg) and Cd in
mammals has been well documented (Basu and Head
2010).
Environ Chem Lett (2014) 12:63–84 67
123
Birds are primary and secondary consumers feeding on
plants, invertebrates and vertebrates. Colonial waterbirds
(herons, storks, ibises, pelicans, cormorants, gulls, terns,
and marine birds) often have been used or proposed as
indicators of Pb, Cd, As, Se, and Hg. Toxic metals such as
Cr, Mn, Cu, Zn, As, Se, Rb, Sr, Mo, Ag, Cd, and Hg have
been studied in several terrestrial, aquatic, and seabirds, as
birds are ordinarily at the top of the food web (Horai et al.
2007; Hargreaves et al. 2011).
Flora
Uptake of elements into plants can happen via different
routes: elements can be taken up via roots from soil and
sediment and transported to the leaves; also, they may be
taken up from the air or by precipitation directly via the
leaves. Trace elements may also be taken up via both
aforesaid ways, from water if they are aquatic. Many fac-
tors influence the plants’ metal uptake and include the
growing parameters (T, pH, soil aeration, Eh condition,
light), the root system, the type of leaves, the type of plant
size, fertilization, competition between the plant species,
the availability of the elements in the soil/sediment/water
or foliar deposits, soil moisture, and plant energy supply to
roots and leaves (Nagajyoti et al. 2010).
Absorbed substances may be transported, converted,
stored, and accumulated in the different cells and tissues of
the plant. Accumulation and distribution of metals in the
plant depend on the plant species, the metal level in the
soil, water and air, the element species, pH, cation
exchange capacity, bioavailability, vegetation period, and
other factors (Filipovic-Trajkovic et al. 2012). Plants might
react to environmental stress on the cellular biochemical,
or morphological scale, and at species or population level
(Johnson et al. 2011).
Plants need macronutrients (N, P, K, S, Ca, and Mg) for
their development, and essential micronutrients such as Fe,
Zn, Mn, Ni, Cu, and Mo for their metabolic needs (Na-
gajyoti et al. 2010). These metals in high concentrations in
plant tissues might have phytotoxic effects, sometimes
resulting in plant death (Szyczewski et al. 2009). Heavy
metals such as As, Cd, Cr, Hg, and Pb are non-essential
elements and toxic to plants. The levels of heavy metals in
plants, both terrestrial and aquatic, vary widely because of
the influence of environmental factors and the type of plant
itself. The metal ranges observed in plants are presented in
the review paper of Nagajyoti et al. (2010).
Babula et al. (2008) claim that essential elements are
important constituents of pigments and enzymes, mainly
Cu, Ni, Zn, Co, Fe, and Mo for algae and higher plants, but
all metals/metalloids, especially Cd, Pb, Hg, and Cu are
toxic in higher concentrations because of disrupting
enzyme functions, replacing essential metals in pigments or
producing reactive oxygen species (ROS). The similarity of
certain heavy metals to essential heavy metals, for exam-
ple, couples Cd–Zn, Se–S or As–P, predestinates their high
toxicity due to the possibility to replace essential metals in
enzymatic systems (Babula et al. 2008).
Plants contribute to the circulation of heavy metals in
the food chain through active and passive absorption
thereof, accumulation in tissues as well as subsequent
being grazed by animals or consumed by humans. Plants
have the ability to absorb all metals, especially those
essential for their growth and development through the root
from the soil, water, and through over-ground vegetative
organs from the atmosphere. Levels of heavy metals in
various species of plants growing in the same habitats may
vary considerably (Wisłocka et al. 2006).
Human-health implications
Humans can be exposed to metals through inhalation of
dust or gaseous particles, or ingestion through food and
drink. The 11 elements of highest concern within the
European Community are: As, Cd, Co, Cr, Cu, Hg, Mn, Fe,
Zn, Ni, and Pb. Some of these elements are actually nec-
essary for humans in trace amounts (Co, Cu, Cr, Mn, Ni),
while others are carcinogenic or toxic, affecting the central
nervous system (Hg, Pb, As, Mn), the kidneys or liver (Hg,
Pb, Cd, Cu) or skin, bones, or teeth (Ni, Cd, Cu, Cr) (Rai
and Pal 2002; Chen et al. 2008; Lavery et al. 2009).
Exposure to heavy metals continues, although adverse
health effects of heavy metals have been known for a long
time (Rai and Pal 2002). Different levels of human expo-
sure to heavy metals can lead to three different effects:
acute effects in which symptoms appear immediately after
exposure to heavy metals during a short exposure period;
chronic effects are a result of low-level exposure over a
long period of time, and finally, lethal effects can be
defined as responses that occur when physical or chemical
agents interfere with cellular and subcellular processes in
the organism at the high level thus causing death (Kakkar
and Jaffery 2005). Figure 2depicts the deposition of metals
in humans.
Metals are significant to humans because some of them
are most important trace elements in various metabolic
enzymes and constituents of cells: Zn, Cu, and Fe form
important component of cell and they are the co-factors in
several enzymes, while organically chelated Cr
3?
ion acts
as a co-factor in insulin hormone response controlling
carbohydrate metabolism in humans (Rai and Pal 2002).
The daily need for Cr in humans should be around 50 lg.
International Agency for Research on Cancer (IARC) has
assessed that Cr(VI) compounds are carcinogenic to
humans (Rai and Pal 2002). Other heavy metals such as
68 Environ Chem Lett (2014) 12:63–84
123
Hg, Cd, As, and Pb are toxic and have no known vital or
beneficial effects on humans (Stankovic et al. 2011a) and
their accumulation in the body over time can cause serious
illness (Stankovic and Stankovic 2013).
The main source of Cd exposure in the general popu-
lation is food. Chronic Cd poisoning induces disturbances
in Ca metabolism accompanied by softening of bones,
fractures, and skeletal deformations, and it became known
as ‘‘itai-itai’’ disease (Stankovic et al. 2011a). Liver and
kidney tissues are the two main sites of Cd storage and
these organs accumulate considerable amounts of Cd,
about 40–80 % of the body burden. Important health end-
points include kidney and bone damage and cancer (WHO
2007).
Hg is considered to be a highly toxic metal for living
organisms. Human poisoning with MeHg is observed in
various parts of the world (Stankovic and Stankovic 2013).
Even at very low concentrations, Hg and its compounds
present potential hazards due to enrichment in food chain.
Human exposure to MeHg occurs mainly through the diet,
more specifically, the consumption of fish. The importance
of As as a health hazard, also known as a ‘‘slow killer,’’ is
now well recognized. As is the most common cause of
acute heavy metal poisoning in humans, and does not leave
the body once it has entered. There is no medicament
available for chronic As toxicity. The poison also attacks
internal organs, notably the lungs and kidneys, which can
result in illnesses including cancer. Long-term ingestion of
As contaminated drinking water produces gastrointestinal,
skin, liver, and nerve tissue injuries and cancer (Stankovic
and Stankovic 2013).
Even the Romans were aware that Pb could cause seri-
ous health problems (Rai and Pal 2002). Pb causes serious
health hazards to humans, especially to young children,
affecting the membrane permeability of kidney, liver, and
brain cells, resulting in either reduced functioning or a
complete breakdown of these tissues since Pb is a cumu-
lative poison. The full impact of Pb poisoning on the health
of children and adults is becoming clearer to most coun-
tries, and many governments have begun to take action in
this respect (Stankovic and Stankovic 2013).
With regard to essential and non-essential elements, the
Joint Food and Agricultural Organization and World
Health Organization (FAO/WHO) have established the
provisional tolerable weekly intake (PTWI) level, which is
defined as an upper intake limit above which adverse health
effects might be expected in humans (FAO/WHO 2004,
2007,2010, Table 2).
Regulations
Today, compelled by the growing environmental and
health awareness of the public, assessments of an array of
trace metals in soils, sediments, water, air, as well as foods
are demanded by regulatory guidelines. Those routinely
regulated trace metals include As, Ba, Cd, Co, Cr, Cu, Hg,
Pb, Mo, Ni, V, and Zn (Stankovic and Stankovic 2013).
The significance of heavy metals for the environment
quality has been reported in several European Union
Directives: Directive 2004/107/EC for As, Cd, Hg, Pb, and
Ni monitoring in an air, Directive 2008/1/EC for integrated
pollution prevention and control in Europe, Directive
2008/50/EC on ambient air quality.
The aim of a directive is to protect the environment by
monitoring water, air, sediment, soil, biota, and to establish
common quality rules for their chemical analysis. Within
the EU countries, free access to environmental information
is guaranteed according to the information directive
90/313/EEC (EEC 1990). The first EU directive in the field
Fig. 2 Deposition of metals in humans (Kakkar and Jaffery 2005)
Table 2 Tolerable intake levels for trace elements appointed by the
FAO/WHO (http://www.inchem.org/ November 6, 2009)
Element Provisional tolerable
weekly intake (PTWI)
(mg/kg body weight)
Provisional maximum
tolerable daily intake
(PMTDI) (mg/kg
body weight)
Arsenic 0.015 –
Cadmium 0.007 –
Copper – 0.50
Iron – 0.80
Lead 0.025 –
Mercury 0.005 –
Methylmercury 0.0016 –
Zinc – 1.00
Environ Chem Lett (2014) 12:63–84 69
123
of nature protection was the Bird Directive in 1978 (79/
409/EEC) (EEC 1979). Intensive Monitoring of Forest
Ecosystems was established in 1994 within the framework
of the UN-ECE ICP Forest, UN/ECE and EC (2000).
Monitoring of heavy metals and other air toxicants is
planned according to the directive of the CAFE (Clean Air
for Europe), Directive 2004/107/EC (EC 2004).
The increasing bioindicator importance is also encour-
aged within the European Union’s water framework direc-
tive (WFD) (EC 2000). The directive aims to achieve a good
ecological and chemical status in all European water bodies,
such as rivers, lakes, and coastal waters, and requires that the
assessment of the ecological status of a system be accom-
plished primarily utilizing biological indicators. Among the
wide range of bioindicators, five biological elements as
bioindicators are listed within the WFD: phytoplankton,
macroalgae, angiosperms, benthic invertebrates, and fish
(Frontalini and Coccioni 2011). The Marine Strategy
Framework Directive 2008/56/EC (MSFD) is formulated as
follows: ‘‘concentrations of contaminants are at levels not
giving rise to pollution effects.’’ The recent adoption of
human biomonitoring (HBM) as Action 3 in the Environ-
ment and Health Action Plan 2004–2010 of the European
Commission COM (2004) has motivated the implementa-
tion and application of HBM in the European environment
and health research.
Bioindicators
A bioindicator is an organism or a part of an organism or a
community of organisms, which contains information on
the quantitative aspects of the quality of the environment;
exposure of organisms can be measured by either levels or
effects. The importance of metals to ecosystems can be
evaluated by measuring metal levels in air, water, and soil,
and potential effects on organisms. In practice, bioindica-
tors can be any animal, plant, or microbial systems that can
be used to formulate conclusions about the environmental
conditions they are continuously exposed to (Stankovic and
Stankovic 2013). According to Hodkinson and Jackson
(2005), a bioindicator is a species or a group of species that
reflects biotic and/or abiotic levels of contamination of an
environment. Organisms used as metal pollution bioindi-
cators must meet certain criteria: the body must constantly
accumulate and tolerate large amounts of toxic metals, it
must be tied to a single place to make it a true ‘‘repre-
sentative’’ for the soil, air, and water environmental area; it
must be available for collection, identification, and han-
dling; it must have sufficient tissue for chemical analysis
and a long life span to ensure sampling over a longer period
of time.
Animals as bioindicators
The animal species mostly used as bioindicators are zoo-
plankton, invertebrates, and vertebrates. Animal species
have been commonly used as indicators of aquatic eco-
systems. Generally, metal accumulation by animals is
favored by their limited ability to excrete these contami-
nants directly after their uptake because of metal inacti-
vation by binding to MTs (Zhou et al. 2008).
Zooplankton
Plankton is composed of phytoplankton and zooplankton
microscopic organisms that float freely within oceanic
currents and in other bodies of water. Primary source of
food in the aquatic food chain is phytoplankton that use
chlorophyll to convert energy (from sunlight), inorganic
chemicals (like nitrogen), and dissolved carbon dioxide gas
into carbohydrates. Phyto- and other plankton are zoo-
plankton’s food. The zooplankton genus Daphnia is an
important link in freshwater trophic chain, as it both con-
sumes phytoplankton and represents a food source to
invertebrate and vertebrate predators. The freshwater
Daphnia magna is one of the oldest and most widely used
zooplankton species as a test organism in aquatic toxicol-
ogy (Ratte et al. 2003). D. magna is the most commonly
tested freshwater species in acute and chronic tests on toxic
metals, such as Cu, Cd, Zn, and Se in water (Lam and
Wang 2008).
Invertebrates
Mollusks play important ecological roles in the different
aquatic and terrestrial ecosystems of the world due to their
ubiquitous distribution and enormous species number.
Although mollusks are basically a marine group of animals,
terrestrial and freshwater mollusks have also been suc-
cessfully used to obtain information on the quality of ter-
restrial and freshwater ecosystems and to quantify
contaminants in their environment. This is particularly the
case with the two most diverse classes of mollusks, gas-
tropods, and bivalves (Moloukhia and Sleem 2011).
Gastropods represent the only mollusks class in terres-
trial ecosystems and consequently, snails are the only
mollusks which can be used for bioindication and bio-
monitoring purposes in these environments. Particularly
terrestrial snails can be utilized as accumulation indicators
of metal pollution. Fritsch et al. (2011) compared the
concentrations of Cd, Cu, Pb, and Zn in the grove snail and
the glass snail, a herbivorous and a carnivorous species,
respectively. Based on their results, toxic metal accumu-
lation in snails and small mammals is governed by eco-
logical (diet, habitat, and mobility) and physiological
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123
(assimilation and excretion of toxic metals) characteristics
of animals (Fritsch et al. 2011).
Since 1976, bivalves have been used to assess the levels of
contamination in marine ecosystems, and certain systematic
groups, notably mussels and oysters, have been extensively
studied worldwide. Within a single species, accumulation
can be a function of age, size, sex and genotype, nutritional,
and reproductive status. Factors that influence metal bioac-
cumulation are physical and chemical water properties:
temperature, pH, dissolved oxygen, salinity, sediment grain
size, and hydrological features of the system (Stankovic et al.
2011a; Stankovic and Jovic 2013).
Marine mussels, oysters, and clams are the most com-
monly used bivalve groups for toxic metals bioindicators
(Jovic et al. 2011; Markovic et al. 2012; Stankovic and
Stankovic 2013). Bellotto and Miekeley (2007) confirmed
that the mussel Perna perna is an efficient toxic metal indi-
cator in Asia–Pacific, Brazil and in tropical areas. Freshwater
mollusks have also been used as toxic metal indicators and
the most frequently used freshwater bivalve as a toxic metal
indicator is the zebra mussel Dreissena polymorpha. The
zebra mussel, a widespread invasive species in Europe and
North America, is an important toxic metal bioindicator of
freshwaters (Stankovic and Jovic 2013).
Vertebrates
The diet appears to be the most important factor that affects
the levels of metals in the tissues of animals. Usually, the
higher levels of metals in plants and animals from lower
trophic levels mean the greater concentrations of metals in
the tissues of higher level of animals, such as fish, birds,
and mammals.
Fish
Fish are used as test organisms in aquatic toxicology
because of their top position in the trophic chain and their
role as food for humans. In fishes, trace metals, such as Mn,
Co, Fe, V, Cu, Zn, and Se, are necessary in small amounts
for metabolic processes, but Ni, Pb, Cr, Cd, Hg as non-
essential elements perform no biological role for fish and
become toxic above certain concentrations. Fish are well
recognized bioindicators of environmental changes and are
adequate for water monitoring programs (Hauser-Davis
et al. 2012). At present, the main database of fish toxicity
data exists for freshwater species. Comparative studies of
toxicity data of freshwater fish and marine fish species
show that marine species are more sensitive than fresh-
water species for the majority of substances tested
(Chovanec et al. 2003). Gills, liver, kidney, and muscle fish
are the organs mostly used for toxic metals investigations.
The mobility of many fish species makes it difficult to
identify not only the exact source of pollution, but also the
time and duration of pollution exposure.
Birds
Birds can play an important role as heavy metal bioindicators.
The general biology and ecology of birds are well known and
birds are easy to identify, but birds are used as often as they
could be as metal bioindicators. One disadvantage is that
many species are migratory, making it difficult to determine
where exposure occurred (Burger and Gochfeld 2004).
Birds have been successfully used to indicate temporal
and spatial trends in toxic metal pollutions in terrestrial and
aquatic ecosystems (Hargreaves et al. 2011; Zhang and Ma
2011; Kitowski et al. 2012). Birds have been proposed as
useful biomonitoring species of pollutants from the year
1993. In many European areas, and also in other continents,
birds have proven to be very useful bioindicators of Hg,
especially sea- and waterbirds (Burger and Gochfeld 2004;
Kitowski et al. 2012). Burger and Gochfeld (2004) analyzed
methyl and inorganic Hg separately. MeHg makes up more
than 90 % of the total mercury in liver, kidney, muscle, and
feathers of birds. Feathers are good indicators of metal pol-
lution (Zhang and Ma 2011). There is usually a significant
correlation between concentrations of Pb in feathers and
those in internal tissues, including blood (Burger and
Gochfeld 2004). The quantities of a metal incorporated into
the feather represent the body level at the time of feather
growth. Waterbirds’ eggs were used as an indicator to detect
heavy metals concentrations or their temporal-spatial trends,
as they are easier to obtain and can be saved for a longer
period as compared to soft tissues (Zhang and Ma 2011).
Hargreaves et al.(2011) investigated As, Be, Cd, Co, Cr,
Cu, Fe, Hg, Mn, Mo, Ni, Pb, Sb, Se, Tl, V, Zn, and Hg
concentrations in the tissues, food, and abiotic environment
of Arctic shorebirds and showed significant elements bi-
odilution from soil to invertebrates to shorebirds. The
highest levels of metals were recorded in the tissues of
carnivore seabirds (Jakimska et al. 2011). The level of Hg
concentration was found to be significantly lower in young
birds than in adults. It can be either stored in internal tis-
sues such as kidneys and liver or it can be excreted in
feathers and eggs (Kitowski et al. 2012). Over the last
decade, the international council for the exploration of the
sea (ICES) has established a working group on ‘‘Seabird
Ecology,’’ dealing with the question of the usefulness of
seabirds as indicators of the marine environment state.
Mammals
Mammals represent useful organisms for biomonitoring pur-
poses and can be used when both temporal and spatial infor-
mation is required. Among the numerous members of the class
Environ Chem Lett (2014) 12:63–84 71
123
of mammals, free-ranging animals or ‘‘wildlife’’ fit best the
requirements of a biomonitor. This is because they depend
exclusively on the quality of food, water, and air in their
habitat. They consume flora or fauna that reflect the local soil,
water, and air contamination. Any contamination present will
influence the animal and can have an effect on its health. The
metal toxic levels in mammals depend on their diet compo-
sition and are often influenced by food-chain effects: whether
they eat other animals or/and plants.
An omnivore is a kind of animal that eats other animals as
well as plants (generally only the fruits and vegetables).
Some omnivores will hunt and eat carnivores, herbivores,
and other omnivores. A carnivore is an animal that gets food
from killing and eating other animals. The most widespread
predator in the order Carnivora is the red fox Vulpes vulpes.It
lives in almost the entire northern hemisphere, from the
Arctic Circle to North Africa, Central America, and the
Asian steppes, with the exception of Iceland, the Arctic
islands and parts of Siberia (Kalisinska et al. 2012). A her-
bivore is an animal that gets its energy from eating only
plants, including grasses. Therefore, potentially toxic trace
of Pb, Cd, Hg, As accumulates in mammals (Pokorny 2006;
Rudy 2010 and Kalisinska et al. 2012).
Although many wildlife species could be used for toxic
metals biomonitoring, the available literature concentrates
on only a few species: wild boar, red deer, brown hare, and
red fox (Pokorny 2006; Rudy 2010 and Kalisinska et al.
2012). Several taxes of small mammals can also be used
for toxic metals biomonitoring of terrestrial (Sanchez-
Chardi et al. 2007) and for aquatic ecosystems (Basu et al.
2007). The population of the European brown hare (Lepus
europaeus) in Central Europe represents one of the best
biomonitors for agriculturally used land (Rudy 2010). A
very good bioindicator is the roe deer (Capreolus capreo-
lus), the species abundant in nearly all parts of Europe in
agricultural and forest areas, which is one of the most
suitable species for bioindication of toxic metals pollution
in terrestrial ecosystems (Pokorny 2006).
In the past decade, marine mammals, especially seals and
dolphins, have been accepted as bioindicators due to their long
life span and their position at the very top of the marine food
chain (Agusa et al. 2011; Bellante et al. 2012; Kakuschke et al.
2012). Marine mammals are extremely susceptible to Hg
contamination from both natural and anthropogenic inputs
(Wintle et al. 2011; Kakuschke et al. 2012). Metal concen-
trations were measured in their liver, kidney, and muscle tis-
sue, as well as in blood and plasma (Kakuschke et al. 2012).
Absorption and distribution of toxic metals
among mammalian organs
Usually liver and kidney samples are analyzed in order to
monitor the Pb, As, Hg, and Cd exposure of an animal
(Kakuschke et al. 2012; Bellante et al. 2012). The dis-
tribution pattern of Pb and As within these organs is not
as uniform as it is found with Cd (e.g., Bellante et al.
(2012)). Cd is a non-essential element for mammals. The
retention rate of Cd increases in mammals when proteins,
Cu, Zn, Fe, or vitamin D, are low in their diet. In addition
to dietary uptake, another exposure pathway for Cd is the
inhalation of contaminated air. Inhalation of particles
containing Cd is the most important exposure pathway in
areas with elevated atmospheric Cd levels (Tataruch and
Kierdorf 2003).
After absorption in the lung and gut, Cd is transported
via the bloodstream to body stores, particularly the liver. In
blood, more than 95 % of Cd is bound to protein in the
blood cells. In the liver, Cd is bound to MT (Zhou et al.
2008), and the formed complex is transported to the kid-
neys; approximately 50 % of the total Cd burden of the
body is found in the liver and kidneys (Jakimska et al.
2011). In the kidneys, Cd has a very long retention time.
Since the kidneys are the main target organ for Cd, they are
the best tissue samples for the analysis of Cd in mammals
(Tataruch and Kierdorf 2003; Bellante et al. 2012). In
muscles, Cd concentrations are low.
The level of gastrointestinal absorption of Hg in animals
depends on its chemical form. In its inorganic form, Hg is
absorbed up to about 7 % from food, but on the other hand,
the absorption of MeHg can be as high as 95 %. The organ
distribution of Hg in mammals follows the sequence: kid-
neys [liver [spleen [brain, in descending order. Con-
centrations in blood and muscle are low (Tataruch and
Kierdorf 2003).
The literature on Hg and Cd in mammals indicates that
concentration levels are influenced by feeding patterns.
Among terrestrial mammals, Hg concentrations increased
from herbivores to omnivores and carnivores. For example,
herbivores such as mule deer and various species of rabbits
usually contained \1.0 mg Hg/kg fresh weight in liver and
kidney, but carnivores such as red fox (Vulpes vulpes)
contained 10 mg/kg ww (Kalisinska et al. 2012).
Animals as accumulative monitors of heavy metal pol-
lution have some advantages over plants, such as area-
related results and comparability to man (Pokorny 2006).
Mammalian wildlife has physiological systems that are
similar to those of humans in mediating the uptake, dis-
tribution, metabolism, and elimination of toxicants (Basu
et al. 2007). Humans and many species of mammalian
wildlife inhabit similar ecosystems and are exposed to
common climates, food sources, and pollutants.
Plants as bioindicators
Algae, fungi (micro and macro), and other plant commu-
nities play a fundamental role for nutrition and life on
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earth. The main sources of trace elements in them are their
growth media. As non-mobile organisms, they are always
exposed to the environmental conditions: air, soil, and
water pollutants at their sites of growth. Various physio-
logical and biochemical processes in them are affected by
metals. Algae and plants growing in metal-polluted sites
exhibit altered metabolism, growth reduction, lower bio-
mass production, and metal accumulation.
Accumulation and distribution of heavy metals in the
plant depend on the plant species, the levels of the metals in
the soil/sediment, water and air, the element species and
bioavailability, pH, vegetation period, and multiple other
factors (Nagajyoti et al. 2010). The quantity or level of heavy
metal absorption in a plant not only depends on the con-
centration levels of the metals in the physical and chemical
composition of the soil/sediment and water, but also varies in
different parts of the plant. Plants also have the ability to
accumulate heavy metals which have no known biological
function (Filipovic-Trajkovic et al. 2012).
Elements are often classified as macronutrients or
micronutrients, and as either essential or non-essential for
the plant, whereas their concentrations are generally indi-
cated as deficient, sufficient, or toxic. Plant nutrients yet
identified and best known as essential are C, H, O, N, P, K,
S, Ca, Mg (as macronutrients) and B, Cl, Co, Cu, Fe, Mn,
Mo, Ni, Si, Na, and Zn (as micronutrients). When these
metals are present in bioavailable forms and at excessive
levels, they have the potential to become toxic to plants
(Nagajyoti et al. 2010).
Biomaterials such as micro- and macroalgae, fungi,
lichens, mosses, tree bark, and leaves of higher plants have
been used to detect the deposition, accumulation, and dis-
tribution of metal pollution in soil, water, and air. Accu-
mulation and distribution of heavy metals in algae and
plants depend on the species, the levels of the metals in the
soil, water and air, the element species and bioavailability,
pH, cation exchange capacity, vegetation period, and
multiple other factors. Plants are able to minimize the
adverse effects of excess heavy metals by regulating the
distribution and translocation thereof within their organs or
cells (Hossain et al. 2012).
Lower plants
Algae,Fungi,Lichen,and Moss In terrestrial environments,
bacteria, fungi, algae, and other lower plants play the
dominant role in the biochemical cycling of heavy metals.
In aquatic environments, algae play a key role in biogeo-
chemical cycling of metals and their accumulation in
sediments. For example, the uptake of toxic metals by
phytoplankton is the first step in the bioaccumulation in
aquatic food webs. Micro- and macroalgae also play an
important role in the removal of toxic metals (Azizi et al.
2012; Rybak et al. 2012). Metals sequestered by microal-
gae are a major contributor to the metal load of the water
column as well as to the metal content of sediments (Torres
et al. 2008).
Some green algae or phytoplankton like Scenedesmus
subspicatus,Chlorella vulgaris,orPseudokirchneriella
subcapitata are in use as standard bioindicators representing
primary producers (Ratte et al. 2003). For example, Klebso-
rmidium-dominated algalmats are good indicators of high Fe
concentration in water, whereas the presence of Fucus vesi-
culosus suggests heavy metal pollution in marine environ-
ment. Algal and fungal biomasses are reported to show
efficient metal removal from wastewater. The adsorption of
heavy metals by algae is highly variable, depending on the
metal, the taxon, and other conditions (Das et al. 2009).
Mosses and lichens, due to their bioaccumulative
properties, are probably the most frequently used organ-
isms as bioindicators of an aerial heavy metal contamina-
tion (Backor and Loppi 2009; Blagnyt _
e and Paliulis 2010;
Serbula et al. 2012). Mosses have been applied to measure
heavy metal levels and trends within and around urban and
industrial areas (Suchara et al. 2011). The use of fungi in
the monitoring of heavy metal pollution is limited but some
fungal groups are better bioaccumulators than others
(Stankovic and Stankovic 2013).
Fresh- and seawater macrophytes are probably the main
source of metals for many animals feeding on them, like
invertebrates and fish, which are commonly consumed as
human food. Therefore, the investigation of metal con-
centrations in the macroalgae species may provide useful
information on the transfer of potentially toxic elements
from abiotic compartments, water and sediment, to higher
consumers including man. The use of marine macroalgae
as bioindicators for trace metal pollution is currently very
common (Joksimovic et al. 2011b; Joksimovic and
Stankovic 2012). Freshwater and sea macroalgae are able
to accumulate trace metals, reaching concentration values
that are thousands of times higher than the corresponding
concentration in water (Vardanyan et al. 2008; Akcali and
Kucuksezgin 2011; Wolff et al. 2012).
Higher plants
Higher plants have been used as bioindicators in areas with
significant air pollution in the absence of mosses, but the
use of plants in the bioindication of heavy metal levels is
not routinely practiced yet. Different plant organs (leaves,
flowers, bark, or roots) from naturally occurring wild plants
and trees, and cultivated plants (vegetables and fruits) were
evaluated as possible bioindicators of heavy metal pollu-
tion (Filipovic-Trajkovic et al. 2012). The most often used
parts of higher plants are leaves/needles and a bark of
different trees (Serbula et al. 2012).
Environ Chem Lett (2014) 12:63–84 73
123
Trace elements taken up by leaves can be translocated to
other plant organs according to Filipovic-Trajkovic et al.
(2012) and Serbula et al. (2012). Unlike Pb, Cd contami-
nation cannot be removed from plants by washing/rinsing;
it is already distributed throughout the organism. Gener-
ally, it is accepted that the normal Cd concentrations in
plants are between 0.2 and 0.8 mg/kg and toxic concen-
trations of Cd are defined as ranging from 5.0 to 30 mg/kg,
while Zn is not considered to be highly phytotoxic and the
toxicity limit for Zn (300–400 mg/kg) depends on the plant
species as well as on the growth stage. According to Ka-
bata-Pendias and Pendias (1992), normal concentrations of
Pb in plants are 0.1–10 mg/kg dw; element uptake and
release depend on plant species, growth stage, and com-
position of the soil/sediment, especially Ca. Generally,
toxic concentrations of Pb are defined as range of
30–300 mg/kg (Filipovic-Trajkovic et al. 2012).
Distribution of heavy metals in plants is unequal and the
largest is in the tree bark. After the tree bark, heavy metals
are mostly accumulated in the roots, then in the leaves, and
as well as in the plant fruits. In over-ground organs, the
highest amounts of Pb are found in the leaves and then in
the fruits and vegetables (Filipovic-Trajkovic et al. 2012).
Hg that presents in the soil has very low availability to
plants as the roots function as a barrier. When organ-
mercuric compounds were still in use as fungicides, it was
shown that in wheat, barley, oats, and corn, Hg was
transferred from the seed dressing into the new seedling.
During growth, dilution of the mercury occurred and the
concentration in the grain was reduced (Tataruch and Ki-
erdorf 2003).
Toxic metal bioindicators
The oldest and widely used species for toxic metal bioindi-
cators are fish, bird, and mosses species, while species rep-
resenting soil organisms are currently becoming more
important in toxic metals bioindication. Representative spe-
cies traditionally used for risk assessment of chemicals are
various freshwater species representing bacteria, microalgae,
invertebrates, and fishes and among the terrestrial species
mainly used are the earthworm Eisenia fetida and some
higher plants. The standardized bioindicators of the different
trophic levels for the most commonly studied toxic metals in
the environment compartments (air, soil, and water) are
shown in Fig. 3. Up to now, toxic metals distributions were
considered more homogeneous in aquatic systems than in
terrestrial ones (Stankovic and Stankovic 2013).
Toxic metal biomonitoring in the air using mosses and
lichens has been established for many years in Europe and
North America. The terrestrial plants are promising indi-
cators for the soil metal pollution. Biomonitoring based on
plants as heavy metal air pollution indicators has been
widespread all over the world since the 1970s. Foliage and
bark of trees are widely used in this regard (Dmuchowski
and Bytnerowicz 2009).
Bioindicators for air metal pollution
Numerous different bioindicators are used in monitoring
air pollution: bacteria, fungi, mosses, lichens, grasses,
agricultural crops, and plants (Stankovic and Stankovic
2013). Which plant species will be used as bioindicators
also depends on how widely they are distributed throughout
the region. The usage of air pollution bioindicators usually
covers metal toxicity in traffics, smelters, mining indus-
tries, industrial pollution, coal-burning power plants, and
agriculture (Harmens et al. 2008; Blagnyt _
e and Paliulis
2010; Suchara et al. 2011; Paoli et al. 2012).
Typical examples of biological indicators of air pollution
are lichens and mosses (Conti and Cecchetti 2001; Blagnyt _
e
and Paliulis 2010). Lichens and mosses are mostly used for
atmospheric trace elements bioindication due to their
capacity to accumulate and store heavy metals and other
toxins. How lichens and mosses do not have roots, they are
able to be direct bioindicators of the air pollution (Suchara
et al. 2011). Lichens and mosses may be considered as the
most commonly applied organisms as bioindicators. The
most commonly used lichen and moss species for toxic
metals biomonitoring were Parmelia sulcata,Hypnum cu-
pressiforme,Hylocomium splendens, and Pleurozium
schreberi (Paoli et al. 2012; Blagnyt _
e and Paliulis 2010).
Lichens are symbiotic organisms of fungi and algae, and
have been widely used in biomonitoring of air pollution by
Fig. 3 The most analyzed heavy metals in the biota of environmental
compartments from reviewed literature (Stankovic and Stankovic
2013)
74 Environ Chem Lett (2014) 12:63–84
123
trace elements and can be used to monitor air quality changes
in urban areas over intervals of several years (Paoli et al.
2012). Naturally growing mosses have been widely used as
effective bioindicators of metal air pollution. Moss appears
to be an excellent bioindicator for the next monitoring ele-
ments and their uptake by mosses decreases in the order:
Cu [Pb [Ni [Co [Cd [Zn, Mn (Harmens et al.
2008). Moss still appears to be one of the best air pollution
bioindicators; however, based on literature search of Aboal
et al. (2010), it could be concluded that mosses can be bio-
indicators for certain metals, such as Pb or Cd, probably
because these elements are of almost exclusively atmo-
spheric origin.
According to Harmens et al. (2008), many European
countries have used mosses in national and multinational
surveys of atmospheric-metal deposition. Different moss
types are currently widely used as bioindicators since they
obtain most of their nutrients directly from the air and by
dry deposition. The procedures for the moss as a bioindi-
cator, the methods of collection, processing and analyses of
moss samples are outlined in the International Moss
Monitoring Manual (Markert et al. 2011). But it is
important to note that a unique species that can be a suit-
able indicator for biomonitoring of toxic metal pollution all
over the world has not been found yet. For this reason,
different species of mosses are useful as bioindicators in
different parts of the world (Blagnyt _
e and Paliulis 2010).
In the last decades of the twentieth century, a rapid
increase in bioindication studies of pollutant loads in higher
plants is observed (Stankovic and Stankovic 2013).
Depending upon the type of the tree, the deployment of
heavy metal content and manners of accumulation show a
great variety: in some trees heavy metals are filtered out by
the leaves from the air, while in others they are taken up by
their crown or by their roots. Different plant organs (leaves,
flowers, stems, or roots) from naturally occurring wild plants
and trees, and cultivated plants (vegetables and fruits) were
evaluated as possible bioindicators of heavy metal pollution
too (Filipovic-Trajkovic et al. 2012). Filipovic-Trajkovic
et al. (2012) found the highest amounts of heavy metals in the
leaves, especially Pb, followed by the fruits and vegetables.
They concluded that fruits and vegetables were metal
avoiders. Tree barks of different tree species are used as
metal bioindicators for longer term of air pollution (Baslar
et al. 2009). Tree bark and higher plants leaves started to be
used to detect the deposition, accumulation, and distribution
of air metal pollution on large-scale air pollution (Kord et al.
2010; Serbula et al. 2012).
Bioindicators for aquatic metal pollution
Specific programs for monitoring toxic metals in aquatic
systems were undertaken as early as the 1960s using
various animals like invertebrates, mollusks, and verte-
brates, like mammals and birds (Stankovic and Stankovic
2013). Heavy metals play an important role as substances
affecting aquatic organisms. Aquatic heavy metal pollution
usually represents high levels of Hg, Cr, Pb, Cd, Cu, Zn,
As, etc., which, introduced into environmental water sys-
tem, may pose high toxicities on the aquatic organisms
(Zhou et al. 2008). Many pollutants are associated with
sediments in aquatic systems. Living benthic foraminiferal
taxa were studied in surface sediment samples (Frontalini
and Coccioni 2011). Foraminifera have been proven to be
successful candidates as part of an integrated monitoring
aquatic pollution program. The statistical analysis reveals a
strong relationship between trace elements, in particular
Hg, Mn, Ni, Pb, and Zn, and the occurrence of abnormal-
ities in foraminiferal taxa (Frontalini and Coccioni 2011).
Microorganisms, such as protozoa, green algae, or bac-
teria, reflect the water quality only 1 week or 2 weeks prior
to their sampling and analysis, whereas insect larvae,
worms, snails, and other macroinvertebrate organisms reflect
the condition from more than a month, and possibly several
years prior to sampling. Microorganisms have proven
capability to take up heavy metals from aqueous solutions,
especially when the metal concentrations range from\
1.0 to
about 20 mg/L (Ahmad 2006). Microbes such as bacteria
exist at the lowest trophic level, so bacteria have the ability
to detect toxic compound before other organisms. Thus,
bioindicators using bacteria have been commercialized, such
as the Lux-Fluoro, the Polytox
TM
, the Deltatox
TM
, and the
Microtox
TM
(Ahmad 2006). The environmental monitoring
of toxic metals by bioindicators like bioluminescent bacteria
(BLB) in marine environment is reported by Ahmad (2006).
Phytoplankton, as the important elementary producer in
marine and inland waters, plays the key role to the whole
ecosystem. Some phytoplanktons are bacteria, some are
protists, and most are single-celled plants. Among them, the
common kinds are cyanobacteria, green algae, and fungi
(Das et al. 2009). The aquatic alga species and amounts can
directly reflect the water quality (Zhou et al. 2008). As an
example, the green alga Chlorella ellipsoidea was reported
to exhibit growth inhibition due to Cu, Zn, Ni, and Cd
exposure. Zooplankton species D. magna is the most com-
monly tested freshwater species in acute as well as in
chronic tests (Zhou et al. 2008). Daphniidae toxicity test is
the essential assay for worldwide water quality assessment.
The usefulness of river plankton for the toxicity of metals
is measured as their inhibitory effect on the photosynthesis
of natural algal assemblages, such as Aulacoseira granulata,
Actinocyclus normanii,Stephanodiscus neoastrea, and Cy-
clotella meneghiniana, among many others. Several algal
species accumulate considerable amounts of metals and can
thus be used as indicators for Cd, Cu, or Pb. Most metals are
slightly to highly toxic to algae, with As, Cu, Hg, and Zn
Environ Chem Lett (2014) 12:63–84 75
123
having the greatest toxic effects. Both cyanobacteria and
green algae exhibit concentrations of various metals pro-
portionally to ambient concentrations (Azizi et al. 2012).
Most of what is stated for freshwater indicators equally
applies to bioindicators in the marine environment, such as
bacteria and phytoplankton, but macroalgae in coastal marine
waters are far more important as bioindicators than macro-
phytic algae in freshwaters. Accumulation of heavy metals
from the surrounding seawater makes them ideal bioindica-
tors. Red algae (Gracilariasp.), often dominant macroalgae of
sea communities, are frequently deployed for Cd, Cu, and Zn
coastal monitoring (Jakimska et al. 2011). Among the green
macroalgae, the genera Ulva, Enteromorpha,andPosidonia,
have attracted considerable attention as toxic metal bioindi-
cators (Stankovic and Stankovic 2013). Aquatic macroalgae
have been used for monitoring the contamination level of
various heavy metals in aquatic environments, such as Zn, Pb,
Cd, and Hg, and many others, as these plants have the ability to
accumulate metallic ions (Vardanyan et al. 2008; Joksimovic
et al. 2011b; Luy et al. 2012.
Fish are used as test organisms in aquatic toxicology
because of their top position in the trophic chain and their
role as food for humans. During their life cycle, fish feed
on algae, rotifers, microcrustaceans, macroinvertebrates,
higher plants, and other small fish. Fish are one of the most
frequently used groups of bioindicators in water bodies. In
the review by Sevcikova et al. (2011), the most important
and most studied metals in fish are Fe, Cu, Cr, Hg, and Pb.
Freshwater mussels are used as bioindicators of Pb, Cd,
Hg, and Zn (Stankovic and Jovic 2013). It appears that
freshwater gastropods and freshwater species, in general,
may be less sensitive than their marine relatives, although
there are insufficient freshwater data to ascertain whether
there is a real difference in sensitivity.
Various aquatic organisms occurring in rivers, lakes, seas,
and marines are potentially useful as metal pollution bioin-
dicators for sediments and waters, including fish, shellfish,
oyster, mussels, clams, aquatic plankton, and macroalgae
(Joksimovic and Stankovic 2012; Stankovic and Stankovic
2013). To achieve adequate geographical and temporal toxi-
cant bioindication, mussels (the blue mussel—Mytilus edulis,
the Mediterranean mussel–Mytilus galloprovincialis) and fish
(Atlantic cod—Gadus morhua,herring—Clupea harengus
and flounder—Platichthys flesus) were selected as state indi-
cators (EEA 2003). Other marine species, like marine mam-
mals (seal, sea lion) and seabirds can also be used for the
biomonitoring of metal pollution (Jakimska et al. 2011).
Bioindicators for soil metal pollution
Various abiotic and biotic soil characteristics can be used
as indicators for evaluating soil health. With increasing
heavy metal concentrations, the activities of soil microbes,
soil enzymes, and nitrogen fixation are inhibited, and
growth of microfloral communities such as fungi, algae,
and photosynthetic bacteria is reduced. Plants and soil
inhabiting organisms such as soil microflora, and micro-
fauna (protozoa), fungi, nematodes, earthworms, mites, and
insects have been used as biotic indicators of soil toxicity
(Park et al. 2011).
There are many ways in which bacteria, microfungi, and
algae can take up toxic metal ions from a soil. The envi-
ronmental monitoring of toxic pollutants by biolumines-
cent bacteria (BLB) in terrestrial environment is reported;
the presence of toxicants in the soil sample reduced light
emission of the bioluminescent microorganisms. General
reductions in microfungal numbers have often been noted
in soils polluted with Cu, Cd, Pb, As, and Zn. The soil
protozoa Tetrahymena pyriformis are used for Cu and Zn
determination in soil (Ahmad 2006). Mosses as well as
higher fungi have developed accumulation mechanisms
with regard to heavy metals from the soil (Kalac 2010).
The types of soil invertebrates used in monitoring pollu-
tant effects include: nematodes, oligochaetes (earthworms),
gastropods, springtails, isopods, arachnids (Stankovic and
Stankovic 2013). The soil nematode community has been
suggested to be a useful indicator of the status of soil pollution
and soil ecological status because of their influence on soil
food webs and plant–soil interactions (Sochova et al. 2006).
The concentrations of heavy metals such as Cr, Cd, Pb, Zn,
and Ni influence the soil nematode community structure
(Park et al. 2011). In the United States, a guide was accepted
and described the use of nematode Caenorhabditis elegans in
soil toxicity tests (Boyle and Kakouli-Duarte 2008).
By far, the most common invertebrate soil bioindicators
used to assess soil metal contaminations are members of
the Family Lubricidae and Eisenia spp. earthworms
(Annelida, Oligochaeta) (Hirano and Tamae 2010). Earth-
worms (E. foetida) are capable of accumulating Hg and Cd,
Cu, Pb, and Zn; significant positive correlations have been
found between metal concentrations in the earthworm and
in the soil (Hirano and Tamae 2010; Olayinka et al. 2011),
impacted by the substrate they consume and the length of
exposure. Earthworms may be available alternative to tra-
ditionally applied organisms in aquatic ecosystems, such as
fish, because they are simple and they can provide indi-
cations of metals bioavailability in a short time at relatively
low cost. They have been extensively studied, and are
approved for use in toxicity testing by the US EPA and the
European Economic Community and the Organization for
Economic Cooperation and Development.
Besides aquatic and marine gastropods, terrestrial gas-
tropods (snails) are recognized as adequate bioindicators,
because of their ability to accumulate Pb, Zn, Cu, and Cd
(Madoz-Escande and Simon 2006). They also exhibit a
very wide distribution of a limited number of species, for
76 Environ Chem Lett (2014) 12:63–84
123
example, Deroceras reticulatum snails are found across
much of North America, Europe, North Africa, and
Atlantic islands (Hall et al. 2008). Snails are known to play
an important role in the diet of many species, including
snakes, toads, beetles, and birds.
In the case of vertebrates, in general, only a few verte-
brate species spend most of their time throughout the year
in close contact with the soil ecosystem. Examples would
include some small mammals, such as ground squirrels,
and some larger mammals, such as fox (Stankovic and
Stankovic 2013). Red fox is a species who lives in a wide
geographical range (Europe and North America), but
occurring in a small home range for the entire year. It has a
high position in the trophic pyramid and accumulates
various toxic metals, including Hg (Kalisinska et al. 2012).
Exposure among terrestrial vertebrates occurs through
ingestion of contaminated biotic or abiotic matter, contami-
nant absorption through skin, or via inhalation of volatile,
aerosolized, or particle-bound contaminants. In most cases,
exposure of wild mammals to contaminants is likely through
oral consumption, either by ingesting contaminants incorpo-
rated into dietary food and water. Dietary exposure in mam-
mals is a function of age, sex, and season, with mammalian
dietary range from pure herbivorous to exclusively carnivo-
rous diets, with virtually all gradients in between, which also
impacts their contaminant exposure (Smith et al. 2007).
The use of physiological and biochemical plant param-
eters in the bioindication of heavy metal soil contamination
is not routinely practiced yet. Some responses of higher
plants to soil heavy metal contamination have a certain
potential. The symptoms of reduced root growth, reduced
seed sprouting, necrosis, and chlorosis appear in suscepti-
ble plants grown in soils contaminated with heavy metals
(Park et al. 2011). Plants, besides macronutrients, require
essential micronutrients for their development: Fe, Zn, Mn,
Ni, Cu, and Mo. Most of these micronutrients accumulate
in the plant tissues for their metabolic needs, but they never
exceed 10 mg/kg. Yildiz et al. (2010) have reported the
normal natural concentration intervals for toxic metals in
terrestrial plants such as the following: Cd: 0.2–2.4 lg/g,
Ni: 1–5 lg/g, Zn: 20–400 lg/g, Fe: 70–700 lg/g, Pb:
1–13 lg/g, Mn: 20–700 lg/g; they found that the level of
accumulation in the high plant sample was soil-oriented.
Higher levels of these metals and other heavy metals in
plant tissue might have phytotoxic effects, sometimes
resulting in death (Winkelmann 2005). But high levels of
heavy metals in the soil do not always indicate similar high
concentrations in plants (Brej 1998).
Metal toxicity bioindicators for humans
For human contaminants, the approach firstly involves
examining the contaminants in all the media, which form
pathways for human exposure—food, drinking water, air,
and soil, and then setting out data on contaminants in dif-
ferent human tissues: blood, urine, hair, nails, etc. The use of
hair as an indicator is not new. Over 200 years ago, hair was
analyzed to measure As levels in the body (Hubbart 2012).
Hair can provide a more permanent record of trace elements
associated with normal and abnormal metabolism, as well as
trace elements assimilated from the environment (Hashem
and Abed 2007). Furthermore, hair is easily collected, and
may better reflect the total body pool of certain elements
than either blood or urine as short-term indicators (Hubbart
2012). For example, hair is a long-term exposure bioindi-
cator to MeHg: once Hg is incorporated into the hair, it
remains unchanged (WHO 2007).
To detect the presence of As, Hg, or Pb, placenta, urine,
finger/toenails, and human milk have been repeatedly used
as indicators of humans’ toxic metals exposure (Smolders
et al. 2009). Heavy metals such as Hg, Pb, and Cd are the
well-known toxicants to cross the placenta and to accu-
mulate in fetal tissues (Gundacker and Hengstschla
¨ger
2012). Unlike urine, blood, placenta, and human milk, hair
and fingernails can record the level and changes of ele-
ments in the body over a long period of time (Ayodele and
Bayero 2010).
Humans can be affected directly by air and water metal
pollutions, as well as indirectly through contaminated food
supplies. Metals that are biologically essential have the
potential to be harmful to humans and other living organ-
isms at high levels of exposure (Freisinger 2010), because
they irreversibly bind to active sites of enzymes; destroying
normal metabolism by producing high-level toxicity and
excessive content of heavy metals in human body may affect
the body and psychophysical development (Szyczewski
et al. 2009).
Biomarkers of metal toxicity in living organisms
A conventional monitoring system of environmental metal
pollution includes measuring the level of selected metals in
the whole organism or in respective organs. However,
measuring only the metal content in particular organs does
not give information about its effect at the subcellular
level. Therefore, the evaluation of biochemical biomarkers,
metallothioneins (MTs), phytochelatins (PCs), and antiox-
idant enzymes (catalase—CAT, superoxide dismutase—
SOD, glutathione S-transferases—GST and glutathione
peroxidases—GPXs, lipid peroxidation—LP), may be
useful in assessing metal exposure and the prediction of
potential detrimental effects induced by environmental
metal contaminants (Stankovic and Stankovic 2013).
Toxicity does not depend on total accumulated metal
concentration. Accumulated metal concentrations should
Environ Chem Lett (2014) 12:63–84 77
123
be interpreted in terms of different trace metal accumula-
tion patterns dividing accumulated metals into two com-
ponents: metabolically available metals and stored
detoxified metals. The relationship between metal accu-
mulation and toxicity is influenced by physiological
activity of living organisms (Shariati and Shariati 2011).
After penetrating the plasma membrane, the incoming
metal is bound immediately by ligands and distributed
between sites of storage, efflux, or toxic action.
A number of trace metals are used by living organisms
to stabilize protein structures, facilitate electron transfer
reactions, and catalyze enzymatic reactions (Nagajyoti
et al. 2010). For example, Cu, Zn, and Fe are essential as
constituents of the catalytic sites of several enzymes.
Although some heavy metals are essential micronutrients
for animals, plants, and many microorganisms, depending
on the route and dose, all heavy metals demonstrate toxic
effects on living organisms via metabolic interference and
mutagenesis. The mechanisms by which metals exert their
toxicity in living organisms are very diverse, especially
their involvement in oxidative biochemical reactions
through the formation of reactive oxygen species (ROS)
(Torres et al. 2008; Nagajyoti et al. 2010). Heavy metals
are involved in toxic redox mechanisms through the gen-
eration reactive oxygen species, associated with oxidative
damage to important biomolecules, and molecular mecha-
nisms of metal toxicity and carcinogenicity (Vlahogianni
and Valavanidis 2007).
Metal ions can penetrate inside the cell interrupting cel-
lular metabolism and in some cases can enter the nucleus.
The entrance of the metal into the cell can mobilize several
metabolic pathways and genetic processes to neutralize the
source of toxicity (Azevedo and Rodriguez 2012). The
entrance of certain metals into the nucleus can enhance the
synthesis of RNA that codes from metallothioneins (MTs).
MTs are low-molecular-weight peptides found mainly in the
cytosol, lysosomes, and nucleus, high in the amino acid
cysteine that contains a thiol group (-SH), which enables
MTs to bind heavy metals (Nordberg and Nordberg 2009).
MT in the physiological system has several roles,
especially in the metabolism and kinetics of metals: trans-
port and detoxification of metal ions and protection from
metal toxicity, free radical scavenging, storage of metal
ions, metabolism of essential metal ions, immune response,
genotoxicity and carcinogenicity, chemoresistance, and
radiotherapy resistance (Shariati and Shariati 2011). Con-
sidering the heavy metal detoxification significance of MTs,
these proteins seem to be more specifically involved in
responses to heavy metals and can serve as the environment
heavy metal pollution biomarkers (Valavanidis et al. 2006;
Nordberg and Nordberg 2009; Shariati and Shariati 2011;
Hauser-Davis et al. 2012). MTs and other selectively metal-
binding proteins have found a comparable attention and
application for aquatic studies, in aquatic mollusks and fish,
especially snails and mussels, gastropods, insect, crusta-
ceans, mussels, fishes, as well as in terrestrial surveys
(Dallinger et al. 2000).
MTs are low-molecular-weight proteins with many
sulfhydryl groups binding a variety of metals, showing a
strong affinity toward certain essential and non-essential
trace elements, such as Cu, Zn, Cd, and Hg. So far, MTs
have been identified in a large number of species
throughout the animal kingdom, and although a variety of
biochemical data prove MTs to be structurally well defined,
but their biological function is still under discussion
(Freisinger 2010). A number of studies demonstrated that
the synthesis of MTs can be induced by certain trace ele-
ments, but also by organic chemicals and other non-
chemical stress factors, like infections, starvation, and
injuries. Nevertheless, it has been shown that Zn, Cu, Cd,
Hg, and other trace elements are the most potent inducers
of MT synthesis (Nordberg and Nordberg 2009).
It has been concluded that the detoxification of metals is
the primary biological function of these proteins. The
involvement of MTs in Cd detoxification of terrestrial gas-
tropods has been proven in detail for a number of species
(Dallinger et al. 2000). MTs are found in all tissues of fish,
particularly in the liver and kidney, and play an important
role in the intracellular regulation of the essential metals Zn
and Cu. MT concentrations in fish tissues increase by fish
exposure to Cu, Zn, Cd, or Hg and the affinity of Cd and Hg to
MTs is even higher so that they may displace essential metals
(Zhou et al. 2008). Increased levels of stress proteins reflect
the gradual impact on the fish metabolism ranging from
adaptive to degenerative responses with severe conse-
quences for fish survival.
MT can be used as an indicator in both environmental and
biological monitoring reflecting human exposure to metals.
Metal-binding proteins have special functions in the detox-
ification of toxic metals and also play a role in the metabo-
lism of essential metals. MTs are involved in the regulation
of the essential metals Cu and Zn and in the detoxification of
Cd and Hg (Zhou et al. 2008; Freisinger 2010; Sevcikova
et al. 2011). The synthesis of this protein probably represents
the body’s defense mechanism against the toxic Cd and Hg
ion. MT in urine can be used as a sensitive biomarker for
metal-induced nephrotoxicity and MT is an established
biomarker in biomonitoring of human Cd exposure. MT
mRNA in lymphocytes in humans has been suggested as an
indicator of susceptible groups in relation to metal exposure
(Nordberg and Nordberg 2009).
Both animal and plant cells are capable of generating—
via multiple sources—a number of different reactive oxy-
gen species (ROS), including the superoxide anion (O
2-
),
hydrogen peroxide (H
2
O
2
), and the hydroxyl radical (OH);
all ROS are harmful to organisms at high concentrations
78 Environ Chem Lett (2014) 12:63–84
123
(Torres et al. 2008). The potential of oxygen-free radicals
and other reactive oxygen species that able to damage
tissues and cellular components in biological systems,
called oxidative stress, have become a topic of significant
interest for environmental toxicology studies (Valavanidis
et al. 2006). Metal ions possess the ability to produce
reactive radicals, resulting in DNA damage, LP, and
depletion of protein sulfhydryls. When ROS levels exceed
antioxidant defenses, the cells go into oxidative stress
which causes membrane LP and changes in the activity of
the antioxidant defense enzymes (Vlahogianni et al. 2007),
like superoxide dismutase (SOD), catalase (CAT), and
glutathione peroxidase (GPX), and no enzymatic antioxi-
dants, such as glutathione, vitamin E, ascorbate, B-caro-
tene, and urate.
Antioxidant defense enzymes play an important role in
cellular antioxidant defense systems and protection from
oxidative damage by ROS. Heavy metals are involved in
toxic redox mechanisms through the generation of ROS,
associated with oxidative damage to important biomole-
cules and molecular mechanisms of metal toxicity and
carcinogenicity. Although there are considerable gaps in
the knowledge of cellular damage, response mechanisms,
repair processes, and disease etiology in biological sys-
tems, free radical reactions and the production of toxic
ROS are known to be responsible for a variety of oxidative
damages leading to adverse health effects and diseases
(Valavanidis et al. 2006).
Many enzymes need cofactors to work properly, such as
Fe
2?
,Mg
2?
,Cu
2?
, and Ca
2?
. The substitution of one heavy
metal ion by another leads to the inhibition or/and loss of
enzymatic activity. Antioxidant enzyme activities, oxidative
damages such as lipid peroxidation—LP, and metal content
in the marine species have manly been studied (Stankovic
and Stankovic 2013). LP by ROS is considered to be a major
mechanism by which heavy metals can cause tissue damage.
The negative correlations found for LP with SOD and mainly
GST activity highlighted the importance of these enzymes in
preventing oxidative damage in mussels. CAT activity was
also positively correlated with SOD and GST activities,
which emphasizes that the three enzymes respond in a
coordinated way to metal-induced oxidative stress (Semedo
et al. 2012). Correlations between metal accumulation and
biomarkers of oxidative stress, such as LP, CAT, and SOD in
marine mollusks were found (Vlahogianni and Valavanidis
2007; Duarte et al. 2011; Giarratano et al. 2011). The study of
Vlahogianni et al. (2007) showed that seasonal variations of
the antioxidant defense enzymes and LP concentrations in
mussels can be used as potential biomarkers of metal toxicity
for long-term monitoring in marine coastal ecosystems.
Heavy metal toxicity is one of the major abiotic stresses
also leading to hazardous effects in plants. A common
consequence of heavy metal toxicity is the excessive
accumulation of reactive oxygen species (ROS) and
methylglyoxal (MG). Both can cause lipids peroxidation,
protein oxidation, inactivation of enzymes, DNA damage,
and/or interact with other vital constituents of plant cells.
Higher plants have evolved a sophisticated antioxidant
defense system to scavenge ROS and MG (Hossain et al.
2012).
Heavy metal toxicity results in the accumulation of
excessive ROS inside the plant cell. For example, Cu can
directly generate ROS, whereas Cd is a redox-inactive
heavy metal and can only generate ROS indirectly by
enzyme inactivation. Potentially a very important mecha-
nism of heavy metal detoxification and tolerance in plants
under heavy metal stress is chelation of heavy metals in the
cytosol or intracellular fluids. Plants make two types of
peptide metal-binding ligands: phytochelatins (PCs) and
metallothioneins (MTs) (Hossain et al. 2012).
MTs and PCs have been identified in a wide variety of
plant species and in some microorganisms (Hegelund et al.
2012; Hossain et al. 2012). PCs and MTs are different
classes of cysteine-rich heavy metal-binding protein mol-
ecules. MTs are cysteine-rich polypeptides encoded by a
family of genes. In contrast, PCs are a family of enzy-
matically synthesized cysteine-rich peptides (Hossain et al.
2012). PCs are a family of Cys-rich polypeptides, although
the most common PC forms have 2–4 peptides (Hossain
et al. 2012).
PCs are structurally related to glutathione (GSH), and
numerous physiological, biochemical, and genetic studies
have confirmed that GSH is the substrate for PC biosynthesis.
GSH is a tripeptide with thiol group (-SH) of cysteine. It can
be synthesized in the human body from the amino acids.
While all cells in the human body are capable of synthesizing
glutathione, liver glutathione synthesis has been shown to be
essential. In animal cells, GSH is catalyzed by glutathione
S-transferase enzymes. It is an antioxidant, preventing
damage to important cellular components caused by reactive
oxygen species such as free radicals and peroxides. GHS has
a vital function in Fe metabolism (Kumar et al. 2011) and it is
the major free radical scavenger in the brain (Gawryluk et al.
2011).
Recent plant molecular studies have shown that GSH by
itself and its metabolizing enzymes act additively and
coordinately for efficient protection against heavy metal
damage in plants. PC synthase is primarily regulated by the
activation of the enzyme in the presence of heavy metals
(Hossain et al. 2012). The biosynthesis of PCs is induced
by many heavy metals, including Cd, Hg, Ag, Cu, Ni, Au,
Pb, As, and Zn; however, Cd is by far the strongest inducer.
Plants are not able to metabolize or eliminate Cd
(Hossain et al. 2012). They adopt the strategy of making
Cd-GSH and Cd-PCs complexes. Zhang and Ge (2008)
found a close relationship between Cd level and GSH
Environ Chem Lett (2014) 12:63–84 79
123
content as well as enzyme glutathione S-transferase (GST)
activity in rice, suggesting that these two parameters of
antioxidant defense system may be used as biomarkers of
Cd-induced stress in plants. Glutathione peroxidases
(GPXs) are key enzymes of the antioxidant network in
plants. Cuypers et al. (2002) suggested that peroxidase
activity can be used as a potential biomarker for heavy
metal toxicity in plants. Similarly, a significant increase in
GPX activity was also observed in red onion exposed to a
variety of Hg, Pb, Cr, Cu, Zn, or Cd concentrations sug-
gesting that the elevated activity of GPX was a result of
heavy metal-induced free radical generation (Fatima and
Ahmad 2005).
Research on plant MTs lags behind what is known about
the vertebrate forms (Freisinger 2010). The large diversity
in the metal-binding regions of plant MTs suggests that
they have the ability to bind a greater range of metals than
their animal counterparts and, consequently, a greater
range of function (Cobbett and Goldsbrough 2002). In
plants, MTs are extremely diverse (Hossain et al. 2012) and
their role in the detoxification process has not been con-
clusively shown (Freisinger 2010). The high metal ion
binding capacity of MTs suggests a role in metal ion
storage, metabolism, and trafficking of essential Cu
?
and
Zn
2?
ions, as well as the detoxification of non-essential
metal ions such as Cd
2?
and Hg
2?
in living organisms. The
precise MTs role in living organism remains elusive
(Freisinger 2010).
PCs have been shown to play an important role in the
detoxification of certain heavy metals in both plants and
animals. PCs play a wider role in heavy metal detoxifica-
tion in biology than previously expected, but it appears that
some organisms probably do not express a PCs synthesis.
There is, for example, no evidence for PC synthase in
mouse and human genomes. Organisms with an aquatic or
soil habitats are more likely to express PCs (Cobbett and
Goldsbrough 2002).
Conclusion
Without question, economic growth and social develop-
ment are critical for improving environment, human health,
and well-being. The toxic metals are widely distributed in
the environment and their early identification is funda-
mental to prevention or control of the damages to humans
and ecosystems. A wide range of legislation now exists in
Europe and other countries to address the release of metals
into the environment, including water, soil, and air, as well
as toxic metal control of food.
In the last few decades, investigations have focused on
searching for bioindicators such as microorganisms, plants,
and animals that accumulate toxic metals, even man.
Bioindication is the use of an organism, a part of an organ-
ism, or a community of organisms, in order to obtain infor-
mation on the quality of its/their environment. Thus, the use
of bioindicators should help to describe the natural envi-
ronment and to detect and assess human impacts. Consid-
ering all the results presented in Stankovic and Stankovic
(2013), it can be concluded that Zn, Cu, Pb, Cd, and Hg are
the most intensively investigated elements in air, water, and
soil bioindicators. Their concentrations depend on the
investigated species and environmental compartment.
The largest number of bacteria, microorganisms, plant,
and animal species are used as bioindicators of heavy
metals in the aquatic ecosystems. The lowest number is
used for the soil contamination. Mosses are particularly
effective bioindicators of aerial heavy metal levels and
trends within and around urban and industrial areas. Higher
plants have appeal as indicators in air pollution monitoring
in highly polluted areas where mosses are often absent. The
use of plants in the bioindication of heavy metal contam-
ination is not routinely practiced. Some responses of higher
plants as bioindicators of soil contamination to heavy
metals have the potential.
Acknowledgments This research was financed by the Ministry of
Science and Technological Development of the Republic of Serbia,
Contract No. III43009.
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