1. Introduction

Human activity generates increasing amounts of new compounds that are released into the environment without prior knowledge of their potential toxicity or impact in living organisms. Heavy metals (HM) and metalloid such as arsenic (As), cadmium (Cd), mercury (Hg), lead (Pb), and aluminum (Al) are major environmental pollutants, particularly in industrial areas. Heavy metals are generated as a result of anthropogenic activities such as metal-working industries, cement factories, mining industry, smelting plants, refineries, and traffic and heating systems [1]. HM and their ions are ubiquitous and by definition are metals having atomic weights between 63.5 and 200.6 g mol�<sup>1</sup> and specific gravity greater than 5 g cm�<sup>3</sup> [2]. Living organisms require small doses of some essential HM, including cobalt (Co), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo), vanadium (V), strontium (Sr), and zinc (Zn). However, in the case of essential metals and very toxic metals, excessive levels and, respectively, even small doses influence both the ecosystem and human health [3]. Nonessential HM which affect the surface water systems are Cd, chromium (Cr), Hg, Pb, As, and antimony (Sb).

and de-purination [15]. The use of DNA strand breaks as biomarker of genotoxicity is common in plants, for example, Bacopa monnieri (Plantaginaceae) exposed to Cd [16]; Nicotiana tabacum and Solanum tuberosum (Asteraceae) exposed to Cd, Cu, Pb, and Zn [17]; Pisum sativum (Fabaceae) exposed to Cr (VI) [18]; Lycopersicum esculentum (Solanaceae) and Cucumis sativus (Cucurbitaceae) exposed to Cu [19]; and Prosopis laevigata (Fabaceae [20]), Vachellia farnesiana (Fabaceae [21]), Pithecellobium dulce [22], and Wigandia urens exposed to Pb, Cu, and Zn [23]. These alterations at the DNA level can trigger changes at the biochemical level that lead to diverse effects at the cellular, physiological, or morphological level, as early effects

The Use of Biosensors for Biomonitoring Environmental Metal Pollution

Among the responses at the cellular level that occur in plants as a result of exposure to HM, we can mention oxidative damage, the production of chelating agents, and alterations in cell division [25]. In the case of oxidative damage, reactive oxygen species (ROS) production indirectly influences the production of antioxidant enzymes [26]. For example, the hydrogen peroxide (H2O2) acts as a signaling molecule in response to HM and other stresses [27]. Under a pollution scenario by HM, H2O2 levels increase in response to Cu and Cd treatment as it has been reported in Arabidopsis thaliana [28], in Hg exposure in tomato (Lycopersicon esculentum [29]), and in response to Mn toxicity in barley (Hordeum vulgare [29]). As a consequence of oxidative stress, the plants experience cellular damage and accumulate metal ions that disturb cellular ionic homeostasis [30]. To minimize these detrimental effects, plants have evolved detoxification mechanisms based on chelation and subcellular compartmentalization. Chelation of HM is a detoxification strategy and the best characterized classes of HM chelator in plants are phytochelatins

PCs are capable of chelating HM, thereby reducing the concentration of cytotoxic free metal ions [32]. In particular, the synthesis of this type of chelant proteins is quickly active with the presence of Cd, Cu, Zn, Ag, Au, Hg, and Pb [33]. For example, Cd is not essential for plant growth, but it is readily taken up by many plant species. Higher plants react to excess Cd by stimulating sulfate absorption [34] and production of PCs involved in Cd chelation and transport into vacuoles [35, 36]. Increased availability of Cd for root uptake may cause considerable alterations in mineral nutrition [37, 38], lipid biosynthesis [39], photosynthetic rate [40], and nitrogen metabolism [41] in plants. Consequently, this led to a severe growth inhibition [42, 43] and finally death [44]. PC induction has been reported in copper-tolerant plants of Mimulus guttatus (Phrymaceae [45]), Brassica juncea (Brassicaceae) following the intracellular accumulation of Cd [46], Rauwolfia serpentina (Apocynaceae), Arabidopsis thaliana (Brassicaceae), and Silene vulgaris (Caryophyllaceae) exposed to As [47] and Lotus japonicus (Fabaceae) exposed to

Metallothioneins (MTs) are proteins that play a key role in the binding and transport of various metals [49]. The structure of these highly conserved proteins is linked to their role in the homeostasis of essential metals such as Zn and Cu and detoxification of toxic elements such as Cd and Hg [50]. In wheat (Triticum) and in rice (Oryza sativa), MTs are induced by metal ions, such as Cu and Cd; in A. thaliana MT gene expression is activated in response to Cu and Cd [51]. MTs bind metal ions in Cicer arietinum (Fabaceae), Quercus suber (Fagaceae), and Triticum

Also, HM mixtures affect cell division [54]. In general, Pb, Cd, Fe, and Zn reduce the synthesis of the cell wall components, causing damage to the Golgi apparatus and other cell organelles. The inhibition of mitosis is also limited by links between HM and cell wall pectin, which becomes more rigid and limits both the expansion and size of the intracellular space [55]. For example, Lerda found that Pb

of exposure to HM in plants [18, 24].

DOI: http://dx.doi.org/10.5772/intechopen.84309

(PCs) and metallothioneins (MTs) [25, 30, 31].

aestivum (Poaceae) exposed to Zn and Cd [52, 53].

Cd [48].

99

Some metals have been classified as toxic, persistent, and accumulative elements. According to the Agency for Toxic Substances and Disease Registry [4], among the 10 most hazardous substances to human health, four are toxic metals: Pb, Hg, As, and Cd. Interestingly, Pb and Hg rank first among the most harmful metals to plants, followed by Cu, Cd/As, Co/Ni, and Mn [5]. Pb and Hg have been reported as mutagenic agents in plants [6]. Due to its chemical similarity to phosphorous, arsenic may interfere with several physiological and biochemical processes [6]. Cd does not appear to have any physiological function, with the exception of the marine diatom Thalassiosira weissflogii that possesses a carbonic anhydrase with Cd as its metal center [7]. Cu, Ni, Co, Zn, and Mn are all plant micronutrients. They participate in prosthetic groups and as cofactors of many proteins and are therefore essential for growth and development and, however, at high concentrations cause oxidative stress [8]. Al is another toxic element with significant implications for agriculture, because 30% of the world's land areas consist of acid soils [9].

Exposure to toxic metals can result in inhibition of seed germination, photosynthesis, and plant growth and consequently causes yield losses. These symptoms are normally related with overproduction or reactive oxygen species (ROS), changes in the permeability and structure of cell membranes, imbalance of mineral nutrients, incorporation of the metal into S-containing molecules, and cell cycle disruption [10]. Also, environmental metal exposure can affect all levels of biological organization. HM bioaccumulation in plants, lichens, small mammals, and bacteria might respond to this chemical stress on the molecular, cellular, or morphological scale and, at population, community and even ecosystem levels [11]. These different types of responses to toxic stress induced by HM (biomarkers) offer a powerful tool for documenting the extent of exposure and the effects of environmental metal contamination [12], revealing the potential use of plant, lichens, small mammals, and bacterial species as biosensors.

### 2. The use of plants as biosensors of heavy metal pollution

The use of plants as biosensors has a long history. For decades, they were used as a part of ecological risk assessment of agricultural and industrial chemicals, solid wastes, food additives, and chemically and radioactively polluted soil and water. However, the use of plants as environmental biomonitors has some drawbacks. Despite being higher eukaryotes, plants have completely different mechanisms of uptake, distribution, storage, compartmentalization, and metabolism of various pollutants [13].

#### 2.1 Plants as biosensors

Vegetal species have the ability to absorb metals, particularly those essential for their development and growth through their root system from the soil, water, and overground vegetative organs from the atmosphere [14]. Also, these chemicals may be transported, transformed, stored, and accumulated in different cells and plant tissues. Genotoxicity or DNA damage is an early effect biomarker at the molecular level that has been used in several plant species exposed to HM. Metal binding to the cell nucleus causes damage including DNA base modifications, inter- and intramolecular cross-linkage of DNA and proteins, DNA strand breaks, rearrangements,

### The Use of Biosensors for Biomonitoring Environmental Metal Pollution DOI: http://dx.doi.org/10.5772/intechopen.84309

and de-purination [15]. The use of DNA strand breaks as biomarker of genotoxicity is common in plants, for example, Bacopa monnieri (Plantaginaceae) exposed to Cd [16]; Nicotiana tabacum and Solanum tuberosum (Asteraceae) exposed to Cd, Cu, Pb, and Zn [17]; Pisum sativum (Fabaceae) exposed to Cr (VI) [18]; Lycopersicum esculentum (Solanaceae) and Cucumis sativus (Cucurbitaceae) exposed to Cu [19]; and Prosopis laevigata (Fabaceae [20]), Vachellia farnesiana (Fabaceae [21]), Pithecellobium dulce [22], and Wigandia urens exposed to Pb, Cu, and Zn [23]. These alterations at the DNA level can trigger changes at the biochemical level that lead to diverse effects at the cellular, physiological, or morphological level, as early effects of exposure to HM in plants [18, 24].

Among the responses at the cellular level that occur in plants as a result of exposure to HM, we can mention oxidative damage, the production of chelating agents, and alterations in cell division [25]. In the case of oxidative damage, reactive oxygen species (ROS) production indirectly influences the production of antioxidant enzymes [26]. For example, the hydrogen peroxide (H2O2) acts as a signaling molecule in response to HM and other stresses [27]. Under a pollution scenario by HM, H2O2 levels increase in response to Cu and Cd treatment as it has been reported in Arabidopsis thaliana [28], in Hg exposure in tomato (Lycopersicon esculentum [29]), and in response to Mn toxicity in barley (Hordeum vulgare [29]). As a consequence of oxidative stress, the plants experience cellular damage and accumulate metal ions that disturb cellular ionic homeostasis [30]. To minimize these detrimental effects, plants have evolved detoxification mechanisms based on chelation and subcellular compartmentalization. Chelation of HM is a detoxification strategy and the best characterized classes of HM chelator in plants are phytochelatins (PCs) and metallothioneins (MTs) [25, 30, 31].

PCs are capable of chelating HM, thereby reducing the concentration of cytotoxic free metal ions [32]. In particular, the synthesis of this type of chelant proteins is quickly active with the presence of Cd, Cu, Zn, Ag, Au, Hg, and Pb [33]. For example, Cd is not essential for plant growth, but it is readily taken up by many plant species. Higher plants react to excess Cd by stimulating sulfate absorption [34] and production of PCs involved in Cd chelation and transport into vacuoles [35, 36]. Increased availability of Cd for root uptake may cause considerable alterations in mineral nutrition [37, 38], lipid biosynthesis [39], photosynthetic rate [40], and nitrogen metabolism [41] in plants. Consequently, this led to a severe growth inhibition [42, 43] and finally death [44]. PC induction has been reported in copper-tolerant plants of Mimulus guttatus (Phrymaceae [45]), Brassica juncea (Brassicaceae) following the intracellular accumulation of Cd [46], Rauwolfia serpentina (Apocynaceae), Arabidopsis thaliana (Brassicaceae), and Silene vulgaris (Caryophyllaceae) exposed to As [47] and Lotus japonicus (Fabaceae) exposed to Cd [48].

Metallothioneins (MTs) are proteins that play a key role in the binding and transport of various metals [49]. The structure of these highly conserved proteins is linked to their role in the homeostasis of essential metals such as Zn and Cu and detoxification of toxic elements such as Cd and Hg [50]. In wheat (Triticum) and in rice (Oryza sativa), MTs are induced by metal ions, such as Cu and Cd; in A. thaliana MT gene expression is activated in response to Cu and Cd [51]. MTs bind metal ions in Cicer arietinum (Fabaceae), Quercus suber (Fagaceae), and Triticum aestivum (Poaceae) exposed to Zn and Cd [52, 53].

Also, HM mixtures affect cell division [54]. In general, Pb, Cd, Fe, and Zn reduce the synthesis of the cell wall components, causing damage to the Golgi apparatus and other cell organelles. The inhibition of mitosis is also limited by links between HM and cell wall pectin, which becomes more rigid and limits both the expansion and size of the intracellular space [55]. For example, Lerda found that Pb

strontium (Sr), and zinc (Zn). However, in the case of essential metals and very toxic metals, excessive levels and, respectively, even small doses influence both the ecosystem and human health [3]. Nonessential HM which affect the surface water

Some metals have been classified as toxic, persistent, and accumulative elements. According to the Agency for Toxic Substances and Disease Registry [4], among the 10 most hazardous substances to human health, four are toxic metals: Pb, Hg, As, and Cd. Interestingly, Pb and Hg rank first among the most harmful metals to plants, followed by Cu, Cd/As, Co/Ni, and Mn [5]. Pb and Hg have been reported as mutagenic agents in plants [6]. Due to its chemical similarity to phosphorous, arsenic may interfere with several physiological and biochemical processes [6]. Cd does not appear to have any physiological function, with the exception of the marine diatom Thalassiosira weissflogii that possesses a carbonic anhydrase with Cd as its metal center [7]. Cu, Ni, Co, Zn, and Mn are all plant micronutrients. They participate in prosthetic groups and as cofactors of many proteins and are therefore essential for growth and development and, however, at high concentrations cause oxidative stress [8]. Al is another toxic element with significant implications for agriculture, because 30% of the world's land areas consist of acid soils [9].

Exposure to toxic metals can result in inhibition of seed germination, photosynthesis, and plant growth and consequently causes yield losses. These symptoms are normally related with overproduction or reactive oxygen species (ROS), changes in the permeability and structure of cell membranes, imbalance of mineral nutrients, incorporation of the metal into S-containing molecules, and cell cycle disruption [10]. Also, environmental metal exposure can affect all levels of biological organization. HM bioaccumulation in plants, lichens, small mammals, and bacteria might respond to this chemical stress on the molecular, cellular, or morphological scale and, at population, community and even ecosystem levels [11]. These different types of responses to toxic stress induced by HM (biomarkers) offer a powerful tool for documenting the extent of exposure and the effects of environmental metal contamination [12], revealing the potential use of plant, lichens, small mammals,

The use of plants as biosensors has a long history. For decades, they were used as a part of ecological risk assessment of agricultural and industrial chemicals, solid wastes, food additives, and chemically and radioactively polluted soil and water. However, the use of plants as environmental biomonitors has some drawbacks. Despite being higher eukaryotes, plants have completely different mechanisms of uptake, distribution, storage, compartmentalization, and metabolism of various

Vegetal species have the ability to absorb metals, particularly those essential for their development and growth through their root system from the soil, water, and overground vegetative organs from the atmosphere [14]. Also, these chemicals may be transported, transformed, stored, and accumulated in different cells and plant tissues. Genotoxicity or DNA damage is an early effect biomarker at the molecular level that has been used in several plant species exposed to HM. Metal binding to the cell nucleus causes damage including DNA base modifications, inter- and intramolecular cross-linkage of DNA and proteins, DNA strand breaks, rearrangements,

2. The use of plants as biosensors of heavy metal pollution

systems are Cd, chromium (Cr), Hg, Pb, As, and antimony (Sb).

Biosensors for Environmental Monitoring

and bacterial species as biosensors.

pollutants [13].

98

2.1 Plants as biosensors

reduces the frequency of mitotic cells and increases the frequency of aberrant cells in onion (Allium cepa) [56]. Also, in corn plants (Zea mays), Eun and colleagues found that HM intervene in cellular division affecting the microtubules, which produces weakness in the cellular structure and the formation of binucleate cells in metaphase [57]. The aforementioned effects have been related mainly with the Pb and their synergies with other metals such as Al and Cu.

bioaccumulated in the ingested plant biomass as a defense against subsequent predation [80]. Even, metals can also affect plant-pollinator interactions as reported in Impatiens capensis (Balsaminaceae) in sites contaminated with Al and Ni [81]. At the ecosystem level, bioaccumulation within successive trophic levels (biomagnification) has been well documented for some metals. Under this scenario, the plants (primary producers) represent an important step in metal transfer since

The Use of Biosensors for Biomonitoring Environmental Metal Pollution

DOI: http://dx.doi.org/10.5772/intechopen.84309

they constitute the base of the food chain. Therefore, certain metals can be transported from plants to higher levels of the food chain, representing a threat to biodiversity and to ecosystem integrity [82]. For example, HM transfer along the trophic chain has been reported for the Ni hyperaccumulator plant Alyssum pintodalsilvae (Brassicaceae) that transfers Ni to grasshoppers (herbivore) and spiders (carnivorous insect), the spiders having higher Ni concentrations [71]. Boyd and Wall found similar results suggesting that Ni could be passed from herbivorous to carnivorous insects [83]. Even, it has been documented that the transfer of HM reaches animals such as small mammals, reporting higher HM levels in carnivorous or omnivorous mammals in comparison to those that feed only by plants [84–86]. This process has also been reported in plants not considered hyperaccumulators. For example, Notten and collaborators report that Urtica dioica (Urticaceae) that is distributed in areas with elevated metal concentrations contained only very low metal concentrations [87]. However, the snail Cepaea nemoralis, which is the main herbivore feeding on these plants, did contain metal concentrations that were much higher than background values [87, 88]. These studies demonstrate that HM can be transferred among invertebrate species, mobilizing metals from one trophic level to another [84–86]. Finally, these studies evidenced the importance of vegetal species for the evaluation of HM impact on the trophic chain levels, as well as their incor-

Lichens may be considered as one of the most commonly applied organisms as biosensors [89]. They are symbiotic organisms of fungi and algae and have been widely used in biomonitoring of air pollution [90]. Some of the most commonly used lichen species for toxic metals biomonitoring are Parmelia sulcata, P. caperata, Hypogymnia physodes, and Xanthoria parietina [89, 91]. Lacking a protective cuticle and roots, lichens absorb and retain nutrients and trace elements, including HM from dry and wet atmospheric deposition that exceed their physiological requirements. They tolerate these high concentrations by sequestering elements extracel-

A plethora of metal toxicity symptoms, including loss of cell membrane integrity, potassium leakage, disruption of ultrastructure, chlorophyll degradation, and oxidative stress, have been reported in lichens [94–96]. However, their expression depends on the metal and lichen species involved [97, 98]. For example, it is widely known that Cu causes cell membrane damage and adversely influences the photosynthetic apparatus of lichens [99] and also affects fungal and algal ultrastructure [100]. Also GSH (reduced glutathione), the precursor of PCs, is the principal lowmolecular thiol and nonenzymatic antioxidant in lichens [101]. It plays a critical role in cellular defense against oxidative damage caused also by HM. For example, in a laboratory study, Pawlik-Skowronska and collaborators found that Cd, Pb, and Zn induced biosynthesis of PCs in the widespread epiphytic lichens Xanthoria

Toxic metal effects reported on lichens include discolored or pinkish necrotic patches and an absence of growth [103]. However, there are tolerant species, some normally associated with HM. For example, Stereocaulon species colonized polluted

poration and biomagnification patterns.

lularly as oxalate crystals or lichen acid complexes [92, 93].

parietina, Physconia grisea, and Physcia adscendens [102].

2.2 Lichens as biosensors

101

Likewise, HM interfere with ionic homeostasis and enzyme activity, resulting in physiological alterations which involve single organs (such as nutrient uptake by the roots) followed by more general processes such as germination, growth, photosynthesis, plant water balance, primary metabolism, and reproduction [30, 58]. Indeed, visible symptoms of heavy metal toxicity include chlorosis, leaf rolling and necrosis, senescence, wilting and stunted growth, low biomass production, limited numbers of seeds, and eventually death [58]. For example, reduction in morphological attributes of height, coverage, or biomass derived from exposure to HM has been reported in Arundo donax (Poaceae), exposed to As, Cd, and Pb [59]; Zea mays (Graminaceae) exposed to Cd, Fe, Ni, and Zn [60]; Prosopis laevigata (Fabaceae [61]), Pithecellobium dulce (Fabaceae [22]), Vachellia farnesiana (Fabaceae [21]), and Wigandia urens [23] exposed to Cu, Pb, and Zn.

Although the immediate effects of exposure to metals occur at the molecular and cellular levels, they can be extended to higher levels of biological organization: populations, communities, and ecosystems.

In the last decade, one of the emergent effects at the population level that has been evaluated in environmentally exposed populations to HM is shifts in their genetic pool, which were defined by Mussali-Galante and collaborators as permanent biomarkers [12]. Particularly, populations can undergo changes in their diversity and genetic structure [62] in two ways: (1) increased genetic variation as a result of mutations induced by genotoxic agents or (2) decreased genetic variability as a result of bottlenecks or selection [12]. In fact, these changes in the genetic reservoir of populations exposed to HM have been proposed as an indicator of ecosystem health [12]. In general, plant species in which alterations on its genetic diversity have been reported as a consequence of HM exposure include Taraxacum officinale (Asteraceae [63]), Silene paradoxa (Caryophyllaceae [64]),Thlaspi caerulescens (Brassicaceae [65]), Pinus sylvestris (Pinaceae [66]),Thlaspi caerulescens (Brassicaceae [67]), Cistus ladanifer (Cistaceae [68]), Arabidopsis halleri (Brassicaceae [69]), and Prosopis laevigata (Fabaceae [70]).

HM exposure may have consequences in higher levels of biological organization such as plant communities; however, few studies have examined these effects [71–73]. Shifts in diversity and species richness, changes in dominant species, changes in species composition, and biodiversity loss may be some of the emergent effects. For example, in a study carried out by Martínez-Becerril, it was documented that the vegetal community (trees, shrubs, and herbaceous) associated to mine tailings differed in its species composition compared to reference sites. Likewise, a significant reduction in species richness and diversity was documented [74]. Similarly, a reduction in plant species diversity in grassland contaminated by Cu and Cd [75] and a reduction in vegetal abundance with the increase of HM soil concentration in a metallophyte plant community [76] has been reported using plant communities as the point of interest.

Additionally, HM bioaccumulation in plants may also affect its interactions with other species. It has been suggested that metal accumulation by plants may be a defense strategy to discourage consumption by herbivores [77]. This defensive role of HM has been reported, for example, in the hyperaccumulator Stanleya pinnata (Brassicaceae) against black-tailed prairie dog herbivory in seleniferous habitats [78, 79]. Similarly, some species of herbivores have evolved to use metals

#### The Use of Biosensors for Biomonitoring Environmental Metal Pollution DOI: http://dx.doi.org/10.5772/intechopen.84309

bioaccumulated in the ingested plant biomass as a defense against subsequent predation [80]. Even, metals can also affect plant-pollinator interactions as reported in Impatiens capensis (Balsaminaceae) in sites contaminated with Al and Ni [81].

At the ecosystem level, bioaccumulation within successive trophic levels (biomagnification) has been well documented for some metals. Under this scenario, the plants (primary producers) represent an important step in metal transfer since they constitute the base of the food chain. Therefore, certain metals can be transported from plants to higher levels of the food chain, representing a threat to biodiversity and to ecosystem integrity [82]. For example, HM transfer along the trophic chain has been reported for the Ni hyperaccumulator plant Alyssum pintodalsilvae (Brassicaceae) that transfers Ni to grasshoppers (herbivore) and spiders (carnivorous insect), the spiders having higher Ni concentrations [71]. Boyd and Wall found similar results suggesting that Ni could be passed from herbivorous to carnivorous insects [83]. Even, it has been documented that the transfer of HM reaches animals such as small mammals, reporting higher HM levels in carnivorous or omnivorous mammals in comparison to those that feed only by plants [84–86]. This process has also been reported in plants not considered hyperaccumulators. For example, Notten and collaborators report that Urtica dioica (Urticaceae) that is distributed in areas with elevated metal concentrations contained only very low metal concentrations [87]. However, the snail Cepaea nemoralis, which is the main herbivore feeding on these plants, did contain metal concentrations that were much higher than background values [87, 88]. These studies demonstrate that HM can be transferred among invertebrate species, mobilizing metals from one trophic level to another [84–86]. Finally, these studies evidenced the importance of vegetal species for the evaluation of HM impact on the trophic chain levels, as well as their incorporation and biomagnification patterns.

### 2.2 Lichens as biosensors

reduces the frequency of mitotic cells and increases the frequency of aberrant cells in onion (Allium cepa) [56]. Also, in corn plants (Zea mays), Eun and colleagues found that HM intervene in cellular division affecting the microtubules, which produces weakness in the cellular structure and the formation of binucleate cells in metaphase [57]. The aforementioned effects have been related mainly with the Pb

Likewise, HM interfere with ionic homeostasis and enzyme activity, resulting in physiological alterations which involve single organs (such as nutrient uptake by the roots) followed by more general processes such as germination, growth, photosynthesis, plant water balance, primary metabolism, and reproduction [30, 58]. Indeed, visible symptoms of heavy metal toxicity include chlorosis, leaf rolling and necrosis, senescence, wilting and stunted growth, low biomass production, limited numbers of seeds, and eventually death [58]. For example, reduction in morphological attributes of height, coverage, or biomass derived from exposure to HM has been reported in Arundo donax (Poaceae), exposed to As, Cd, and Pb [59]; Zea mays (Graminaceae) exposed to Cd, Fe, Ni, and Zn [60]; Prosopis laevigata (Fabaceae [61]), Pithecellobium dulce (Fabaceae [22]), Vachellia farnesiana (Fabaceae [21]),

Although the immediate effects of exposure to metals occur at the molecular and

In the last decade, one of the emergent effects at the population level that has been evaluated in environmentally exposed populations to HM is shifts in their genetic pool, which were defined by Mussali-Galante and collaborators as permanent biomarkers [12]. Particularly, populations can undergo changes in their diversity and genetic structure [62] in two ways: (1) increased genetic variation as a result of mutations induced by genotoxic agents or (2) decreased genetic variability as a result of bottlenecks or selection [12]. In fact, these changes in the genetic reservoir of populations exposed to HM have been proposed as an indicator of ecosystem health [12]. In general, plant species in which alterations on its genetic diversity have been reported as a consequence of HM exposure include Taraxacum officinale (Asteraceae [63]), Silene paradoxa (Caryophyllaceae [64]),Thlaspi caerulescens (Brassicaceae [65]), Pinus sylvestris (Pinaceae [66]),Thlaspi caerulescens

cellular levels, they can be extended to higher levels of biological organization:

(Brassicaceae [67]), Cistus ladanifer (Cistaceae [68]), Arabidopsis halleri

HM exposure may have consequences in higher levels of biological organization such as plant communities; however, few studies have examined these effects [71–73]. Shifts in diversity and species richness, changes in dominant species, changes in species composition, and biodiversity loss may be some of the emergent effects. For example, in a study carried out by Martínez-Becerril, it was documented that the vegetal community (trees, shrubs, and herbaceous) associated to mine tailings differed in its species composition compared to reference sites. Likewise, a significant reduction in species richness and diversity was documented [74]. Similarly, a reduction in plant species diversity in grassland contaminated by Cu and Cd [75] and a reduction in vegetal abundance with the increase of HM soil concentration in a metallophyte plant community [76] has been reported using plant communities as the

Additionally, HM bioaccumulation in plants may also affect its interactions with other species. It has been suggested that metal accumulation by plants may be a defense strategy to discourage consumption by herbivores [77]. This defensive role of HM has been reported, for example, in the hyperaccumulator Stanleya pinnata (Brassicaceae) against black-tailed prairie dog herbivory in seleniferous habitats [78, 79]. Similarly, some species of herbivores have evolved to use metals

(Brassicaceae [69]), and Prosopis laevigata (Fabaceae [70]).

and their synergies with other metals such as Al and Cu.

Biosensors for Environmental Monitoring

and Wigandia urens [23] exposed to Cu, Pb, and Zn.

populations, communities, and ecosystems.

point of interest.

100

Lichens may be considered as one of the most commonly applied organisms as biosensors [89]. They are symbiotic organisms of fungi and algae and have been widely used in biomonitoring of air pollution [90]. Some of the most commonly used lichen species for toxic metals biomonitoring are Parmelia sulcata, P. caperata, Hypogymnia physodes, and Xanthoria parietina [89, 91]. Lacking a protective cuticle and roots, lichens absorb and retain nutrients and trace elements, including HM from dry and wet atmospheric deposition that exceed their physiological requirements. They tolerate these high concentrations by sequestering elements extracellularly as oxalate crystals or lichen acid complexes [92, 93].

A plethora of metal toxicity symptoms, including loss of cell membrane integrity, potassium leakage, disruption of ultrastructure, chlorophyll degradation, and oxidative stress, have been reported in lichens [94–96]. However, their expression depends on the metal and lichen species involved [97, 98]. For example, it is widely known that Cu causes cell membrane damage and adversely influences the photosynthetic apparatus of lichens [99] and also affects fungal and algal ultrastructure [100]. Also GSH (reduced glutathione), the precursor of PCs, is the principal lowmolecular thiol and nonenzymatic antioxidant in lichens [101]. It plays a critical role in cellular defense against oxidative damage caused also by HM. For example, in a laboratory study, Pawlik-Skowronska and collaborators found that Cd, Pb, and Zn induced biosynthesis of PCs in the widespread epiphytic lichens Xanthoria parietina, Physconia grisea, and Physcia adscendens [102].

Toxic metal effects reported on lichens include discolored or pinkish necrotic patches and an absence of growth [103]. However, there are tolerant species, some normally associated with HM. For example, Stereocaulon species colonized polluted road-site during the period of high Pb emissions [104], and Vezdaea leprosa occurs alongside motorway crash barriers in Germany and the UK [105]. So, characteristic lichen assemblages occur on metalliferous soils polluted by industrial emissions and on abandoned mine wastes [105–107] or lichen communities growing on trees [108]. Hence, bark and soil HM contents play a major influence in determining the composition of epiphytic lichen floras [108–110]. Several studies have evidenced the metal influences on epiphytic lichen abundance, cover, richness, and species diversity. For example, in some studies in coniferous forests, correlations between epiphytic lichen abundance and Mn supply were detected [98, 111]. Specifically, a decreasing cover value of the foliose lichen Hypogymnia physodes with increasing Mn concentrations in bark or stemflow were repeatedly found in stands of Picea abies. In another example, the lichen community associated to Vachellia farnesiana, Prosopis laevigata, and Pithecellobium dulce in exposed sites to HM (Pb, Cu, and Zn) showed higher richness and species diversity values as compared with a reference site [108]. In conclusion, lichens have proved to be very effective organisms as biosensors to detect HM in the environment.

additives [118]. With the increasing availability of portable meters for measuring fluoresce [121], it is now feasible to transit this technology from the lab to the

The Use of Biosensors for Biomonitoring Environmental Metal Pollution

3. The use of small mammals as biosensors of heavy metal pollution

nation with HM such as Pb, Cd, Cr, Zn, Al, silver (Ag), As, Co, Cu, Fe, Mn, magnesium (Mg), nickel (Ni), Hg, selenium (Se), strontium (Sr), and Mo. These animals have been used mainly because they are found in the intermediate positions of trophic chains and they are small, have diverse diets, are relatively easy to capture, and have wide geographic distribution (which allows to compare between populations of contaminated and non-contaminated sites). The liver, kidneys, bone,

muscle, brain, testicles, teeth, and blood are the main target organs for HM. Conducting studies with SM is important because they allow to make inferences about the bioavailability and bioaccumulation of HM, the biotransformation mechanisms of HM among different species, and the sources of exposure associated with the diet; they also allow to determine which species are susceptible to HM, which is an important step for the evaluation of the biomagnification of HM. Most of the monitoring studies of HM use SM belonging to two orders: Soricomorpha (shrews and moles) and Rodentia (squirrels, rats, mice, voles). The present chapter will focus on two species of the order Rodentia that belong to the families Muridae and Cricetidae. The life history characteristics of both species are described below. Family Muridae: Apodemus sylvaticus; common names include long-tailed field mouse, small wood mouse, and wood mouse. Its conservation status is a minor concern [122]. It has 32 subspecies. Its geographical distribution includes Europe (with the exception of Finland and northern Russia) and some regions of North Africa. It is found at altitudes up to 3300 m.a.s.l. and has been recorded in a variety of seminatural habitats that include forests, moors, steppes, arid Mediterranean scrub, and sand dunes. It is also found in artificial habitats such as suburban and urban parks, gardens, vacant lots, pastures, crops, fields, and forest plantations. It is an omnivorous species that feeds at ground level; its diet includes plants/seeds (70–80%) and invertebrates (20%). It eats tree seeds, fleshy fruits, mushrooms, flowers, and aerial parts of plants. It also consumes fern leaves (Culcita macrocarpa) and oak acorns (Quercus) [123]. It has also been reported to eat worms, which could be an important source of HM for this species [124]. It is predated by snakes (Hemorrhois hippocrepis), eagle-owls (Bubo bubo), barn owls (Tyto alba), and foxes (Vulpes vulpes), among others. It is nocturnal and lives in galleries dug at shallow depths, in crevices, or tree holes. The home range of males is larger than that of females, and it becomes larger for males during the reproductive period. The home range of males can be up to 1.44 ha and 0.49 ha for females [123]. However, some studies estimate that the home range of the males of this species can be up to 2500 m2 and that its activity range is 56.4 m [125]. The males are polygamous, and, during the breeding season, they travel long distances in search of reproductive partners. There are reports of attacks against intruders and subordinate males, which are thus expelled from the territory and which are displaced from the territories. Unlike males, females have exclusive territories. Their fertility rate is 1–7 l/year, each with an average of 5 pups. The maximum recorded life span in the wild is 12 months [123]. Family Cricetidae: taxonomic name, Myodes glareolus; its synonym is

Clethrionomys glareolus. The common name is bank vole. Its conservation status is a

Small mammals (SM) are frequently used to monitor environmental contami-

greenhouses and the fields.

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3.1 Small mammals as biosensors

DOI: http://dx.doi.org/10.5772/intechopen.84309

#### 2.3 Transgenic plants as biosensors

The alternative use of transgenic plants in horticulture, forestry, and construction seems to be more appealing for the public. In this respect, design and production of transgenic plants for environmental biomonitoring and cleaning up polluted areas can be action for more favorable public perception of genetically modified organisms [13].

Recently, substantial progress in generation and exploitation of transgenic plants as biomonitors has been made [112, 113]. One of the important advantages of transgenic biosensors is the ability to customize the assay in accordance with monitoring needs. This not only makes transgenic biosensors more sensitive to a particular pollutant but also allows for easy scoring.

Classically, a major approach to addressing these issues has been based on selective breeding or genetic engineering of plants in order to increase their baseline hardiness and/or ability to efficiently utilize resources [114]. Concurrent with these approaches have been efforts to develop and apply technologies toward monitoring and understanding the physiological responses of plants to stress [115–117]. This second, more recent approach is based on leveraging the finely tuned and highly sensitive mechanism plants which have developed to sense, to respond, and to adapt to changes in the environment [118].

Appreciation of this internal decision-making process in plants has led to the development of methods to monitor relevant natural physical phenomena, such as changes in the chlorophyll fluorescence spectra [115, 116]. Another route has been the direct engineering of plants to act as "vital reporters" both of their own health and of internal decision-making processes (so-called biosensors). By coupling knowledge of the genetic cascade stress responses with reporter proteins [e.g., betaglucuronidase (GUS), luciferase (LUC), or fluorescent proteins (FP)], it is possible to visualize genetic events linked to/associated with stress responses [115, 119]. Indeed, prior research has demonstrated that both endogenous and synthetic promoters can be used as "biomarkers" for a variety of stress conditions, with the appropriate choice of promoter depending on a number of factors, including ease of interpretation, signal-to-noise ratio, and the timeliness of data acquisition [115, 120]. Thus, biomarkers have the potential of informing the researcher, in real time, of the magnitude of a wide variety of physiological events. In particular, the use of FPs has distinct advantages; namely, FP outputs are observable using widely available equipment (e.g., a fluorescent microscope) and require no exogenous

additives [118]. With the increasing availability of portable meters for measuring fluoresce [121], it is now feasible to transit this technology from the lab to the greenhouses and the fields.
