3.1 Small mammals as biosensors

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

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

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 partic-

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

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

biosensors to detect HM in the environment.

ular pollutant but also allows for easy scoring.

adapt to changes in the environment [118].

2.3 Transgenic plants as biosensors

Biosensors for Environmental Monitoring

organisms [13].

102

Small mammals (SM) are frequently used to monitor environmental contamination 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 minor concern [126]. A total of 30 subspecies have been reported. It has a wide geographical distribution that includes the British islands, Europe, and Russia. To the north, it can be found beyond the Arctic Circle; to the south, they are found in northern Turkey and Kazakhstan. This species is not found in southern Iberia and the Mediterranean islands. It inhabits altitudes of 2400 m, including open forests, bushes, and hedges [126]. Bank voles are mainly herbivorous, consuming fleshy fruits, seeds, tender leaves, mushrooms, moss, flowers, and roots. They gnaw the bark of young trees and feed on the cambium, but they can also consume earthworms. It is predated by raptors such as the tawny owl (Strix aluco), the barn owl (Tyto alba), as well as small and medium carnivores [127]. Its home range is estimated to be up to 1000 m<sup>2</sup> , and its activity ranges up to 35.7 m [125]. During breeding seasons, the males cover large areas that include the territories of several females. The females have exclusive territories. The mating system is polygynous. The females have 3 or 4 l/reproductive period, with an average of 4 or 5 pups/l. Gestation lasts between 18 and 22 days; the lactation period lasts approximately 18 days. The average life span is between 12 and 13 months, but under extreme conditions they can live for 3 months [127].

mention that shrews can bioaccumulate more metals and metalloids than

The Use of Biosensors for Biomonitoring Environmental Metal Pollution

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

material deposit), the shrew Sorex araneus bioaccumulates more Cd than A. sylvaticus and C. glareolus. This could be explained by the dietary habits of the studied species, since the diet of Sorex araneus consists of invertebrates, including insects and molluscs, while A. sylvaticus and C. glareolus are mainly

communities and the dynamics of the ecosystem [86].

herbivorous [131].

species [135].

105

A. sylvaticus. Mertens and collaborators found that, in contaminated sites (a dredged

Studies by Wijnhoven and collaborators on floodplain species (A. sylvaticus, C. glareolus, C. russula, M. agrestis, M. arvalis, M. minutus, S. araneus) found that two species of shrew had higher concentrations of HM compared to the other species; the highest concentrations were found in the shrew S. araneus, which has insectivorous and carnivorous habits. Only Cu concentrations were higher in C. glareolus than in A. sylvaticus and M. agrestis. The differences in the concentrations of HM may be due to variations in exposure time (age of the individual), the heterogeneity of the concentrations of HM in soil, the movement of the animals to the other sites, and their feeding patterns. The accumulation of HM in the studied species could also be a risk factor for their predators, potentially altering the structure of their

Cooke and collaborators studied three mammalian species, A. sylvaticus, M. agrestis, and S. araneus, associated with a site contaminated with Pb, Cd, and F. The total accumulation levels of these three compounds in the studied species had the following order: S. araneus > M. agrestis > A. sylvaticus. The stomach contents of S. araneus showed that it had the highest intake of Pb, F, and Cd [132]. The differences in bioaccumulation are due to differences in daily intake, in the efficiency of digestion and assimilation, and to other physiological, biochemical, and behavioral factors. Similarly, Drouhot and collaborators found that Crocidura russula accumulated more As than A. sylvaticus, Mus spretus, and Microtus arvalis. They also mention that the differences in the accumulation of As between species and within the same species are due to variations in diet, foraging behavior, differences in metab-

Some authors have used A. sylvaticus in distance gradient studies of contaminated areas. Scheirs and collaborators studied the concentration of metals (Cd, Co, Cr, Cu, Fe, Mn, Pb, and Zn) in soil and the genotoxicity found in A. sylvaticus along a distance gradient. The authors reported that the concentration of HM and the genetic damage found in A. sylvaticus was higher near the most contaminated areas [134]. Rogival and collaborators studied the accumulation of As, Cd, Cu, and Pb and Zn in A. sylvaticus mice inhabiting five sites along a distance gradient, in the soil of the sites, and in the mice's diet (acorns and two species of earthworms: Dendrodrilus rubidus and Lumbricus rubellus). They observed a gradient in the exposure to metals, beginning on the foundry (most contaminated site), in all the studied elements (soil, diet, and rodent), but not for the essential metals analyzed (Cu and Zn). The concentrations of As, Cd, and Pb in acorns were higher in the sites closest to the foundry. In earthworms, the concentrations of the five metals were higher near the foundry. The transfer of metals occurred mainly from the diet to the mice in the case of Pb and Cd [124]. Another study conducted by Tête and collaborators found that the concentrations of Pb in the liver and kidneys of A. sylvaticus followed a distance gradient from the contamination source (foundry). In contrast, the concentrations of Cd in the liver and kidneys of mice varied along the contamination gradient, forming a bell curve. Unlike the results of bioaccumulation, renal alterations (necrosis, lymphocyte infiltration) did not show an increase associated with a distance gradient. The results showed that A. sylvaticus is chronically exposed to Pb and Cd and that there is kidney damage present in the

olism, amount of ingested soil, and mobility of the organisms [133].

#### 3.2 Apodemus sylvaticus as a biosensor

There are studies that show that Apodemus sylvaticus populations inhabiting contaminated areas bioaccumulate metalloids and HM. For example, Erry and collaborators quantified the concentration of As in A. sylvaticus and C. glareolus in five sites contaminated with As. These authors found that the organisms of both species accumulate similar concentrations of As in contaminated sites. The concentration of As in the liver and kidneys of the animals inhabiting the contaminated sites was higher than those of animals inhabiting the control site. The concentration of As in those organs was associated with the concentration of As in the stomach contents. Thus, the authors suggest that the animals were exposed to As through the diet and that the two species of mice bioaccumulate As in various organs [128]. Sánchez-Chardi and collaborators compared a population A. sylvaticus population inhabiting a control (non-contaminated) site with a population inhabiting a site contaminated by leachates containing potentially toxic elements. They found that the mice inhabiting the leachate site bioaccumulated Cd, Fe, Zn, Cu, Mn, Mo, and Cr, compared with the animals inhabiting the control site. The mice in the leachate site also showed low weight index and a high relative weight of the kidney, as well as high plasma values of glutamic pyruvic transaminase (GPT), an indicator of liver damage. They also showed greater genotoxicity than the animals of the control site. The authors suggest that the morphological and physiological changes observed in the population of A. sylvaticus inhabiting the leachate site indicate that this species is more sensitive than Crocidura russula, the other studied species inhabiting the site, and that the leachates affected the health of A. sylvaticus [129].

The comparison between A. sylvaticus and species of the order Soricomorpha, particularly shrews, showed that A. sylvaticus is more sensitive to renal toxicity caused by exposure to HM than C. russula. Nevertheless, shrews can bioaccumulate more HM. Sánchez-Chardi and collaborators compared populations of A. sylvaticus and C. russula inhabiting a non-contaminated site (control) with populations of the same species inhabiting a site contaminated by leachates containing potentially toxic elements. In both species inhabiting the contaminated the site, the histological analysis of the liver showed signs of necrosis and apoptosis, inflammation of preneoplastic nodules, and vacuolization. The kidneys were altered mainly in A. sylvaticus (necrosis and tubular inflammation), which suggested that this species is more sensitive to renal toxicity than C. russula [130]. However, some authors

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

mention that shrews can bioaccumulate more metals and metalloids than A. sylvaticus. Mertens and collaborators found that, in contaminated sites (a dredged material deposit), the shrew Sorex araneus bioaccumulates more Cd than A. sylvaticus and C. glareolus. This could be explained by the dietary habits of the studied species, since the diet of Sorex araneus consists of invertebrates, including insects and molluscs, while A. sylvaticus and C. glareolus are mainly herbivorous [131].

Studies by Wijnhoven and collaborators on floodplain species (A. sylvaticus, C. glareolus, C. russula, M. agrestis, M. arvalis, M. minutus, S. araneus) found that two species of shrew had higher concentrations of HM compared to the other species; the highest concentrations were found in the shrew S. araneus, which has insectivorous and carnivorous habits. Only Cu concentrations were higher in C. glareolus than in A. sylvaticus and M. agrestis. The differences in the concentrations of HM may be due to variations in exposure time (age of the individual), the heterogeneity of the concentrations of HM in soil, the movement of the animals to the other sites, and their feeding patterns. The accumulation of HM in the studied species could also be a risk factor for their predators, potentially altering the structure of their communities and the dynamics of the ecosystem [86].

Cooke and collaborators studied three mammalian species, A. sylvaticus, M. agrestis, and S. araneus, associated with a site contaminated with Pb, Cd, and F. The total accumulation levels of these three compounds in the studied species had the following order: S. araneus > M. agrestis > A. sylvaticus. The stomach contents of S. araneus showed that it had the highest intake of Pb, F, and Cd [132]. The differences in bioaccumulation are due to differences in daily intake, in the efficiency of digestion and assimilation, and to other physiological, biochemical, and behavioral factors. Similarly, Drouhot and collaborators found that Crocidura russula accumulated more As than A. sylvaticus, Mus spretus, and Microtus arvalis. They also mention that the differences in the accumulation of As between species and within the same species are due to variations in diet, foraging behavior, differences in metabolism, amount of ingested soil, and mobility of the organisms [133].

Some authors have used A. sylvaticus in distance gradient studies of contaminated areas. Scheirs and collaborators studied the concentration of metals (Cd, Co, Cr, Cu, Fe, Mn, Pb, and Zn) in soil and the genotoxicity found in A. sylvaticus along a distance gradient. The authors reported that the concentration of HM and the genetic damage found in A. sylvaticus was higher near the most contaminated areas [134]. Rogival and collaborators studied the accumulation of As, Cd, Cu, and Pb and Zn in A. sylvaticus mice inhabiting five sites along a distance gradient, in the soil of the sites, and in the mice's diet (acorns and two species of earthworms: Dendrodrilus rubidus and Lumbricus rubellus). They observed a gradient in the exposure to metals, beginning on the foundry (most contaminated site), in all the studied elements (soil, diet, and rodent), but not for the essential metals analyzed (Cu and Zn). The concentrations of As, Cd, and Pb in acorns were higher in the sites closest to the foundry. In earthworms, the concentrations of the five metals were higher near the foundry. The transfer of metals occurred mainly from the diet to the mice in the case of Pb and Cd [124]. Another study conducted by Tête and collaborators found that the concentrations of Pb in the liver and kidneys of A. sylvaticus followed a distance gradient from the contamination source (foundry). In contrast, the concentrations of Cd in the liver and kidneys of mice varied along the contamination gradient, forming a bell curve. Unlike the results of bioaccumulation, renal alterations (necrosis, lymphocyte infiltration) did not show an increase associated with a distance gradient. The results showed that A. sylvaticus is chronically exposed to Pb and Cd and that there is kidney damage present in the species [135].

minor concern [126]. A total of 30 subspecies have been reported. It has a wide geographical distribution that includes the British islands, Europe, and Russia. To the north, it can be found beyond the Arctic Circle; to the south, they are found in northern Turkey and Kazakhstan. This species is not found in southern Iberia and the Mediterranean islands. It inhabits altitudes of 2400 m, including open forests, bushes, and hedges [126]. Bank voles are mainly herbivorous, consuming fleshy fruits, seeds, tender leaves, mushrooms, moss, flowers, and roots. They gnaw the bark of young trees and feed on the cambium, but they can also consume earthworms. It is predated by raptors such as the tawny owl (Strix aluco), the barn owl (Tyto alba), as well as small and medium carnivores [127]. Its home range is esti-

breeding seasons, the males cover large areas that include the territories of several females. The females have exclusive territories. The mating system is polygynous. The females have 3 or 4 l/reproductive period, with an average of 4 or 5 pups/l. Gestation lasts between 18 and 22 days; the lactation period lasts approximately 18 days. The average life span is between 12 and 13 months, but under extreme

There are studies that show that Apodemus sylvaticus populations inhabiting contaminated areas bioaccumulate metalloids and HM. For example, Erry and collaborators quantified the concentration of As in A. sylvaticus and C. glareolus in five sites contaminated with As. These authors found that the organisms of both species accumulate similar concentrations of As in contaminated sites. The concentration of As in the liver and kidneys of the animals inhabiting the contaminated sites was higher than those of animals inhabiting the control site. The concentration of As in those organs was associated with the concentration of As in the stomach contents. Thus, the authors suggest that the animals were exposed to As through the diet and that the two species of mice bioaccumulate As in various organs [128]. Sánchez-Chardi and collaborators compared a population A. sylvaticus population inhabiting a control (non-contaminated) site with a population inhabiting a site contaminated by leachates containing potentially toxic elements. They found that the mice inhabiting the leachate site bioaccumulated Cd, Fe, Zn, Cu, Mn, Mo, and Cr, compared with the animals inhabiting the control site. The mice in the leachate site also showed low weight index and a high relative weight of the kidney, as well as high plasma values of glutamic pyruvic transaminase (GPT), an indicator of liver damage. They also showed greater genotoxicity than the animals of the control site. The authors suggest that the morphological and physiological changes observed in the population of A. sylvaticus inhabiting the leachate site indicate that this species is more sensitive than Crocidura russula, the other studied species inhabiting the site,

and that the leachates affected the health of A. sylvaticus [129].

The comparison between A. sylvaticus and species of the order Soricomorpha, particularly shrews, showed that A. sylvaticus is more sensitive to renal toxicity caused by exposure to HM than C. russula. Nevertheless, shrews can bioaccumulate more HM. Sánchez-Chardi and collaborators compared populations of A. sylvaticus and C. russula inhabiting a non-contaminated site (control) with populations of the same species inhabiting a site contaminated by leachates containing potentially toxic elements. In both species inhabiting the contaminated the site, the histological analysis of the liver showed signs of necrosis and apoptosis, inflammation of preneoplastic nodules, and vacuolization. The kidneys were altered mainly in A. sylvaticus (necrosis and tubular inflammation), which suggested that this species is more sensitive to renal toxicity than C. russula [130]. However, some authors

, and its activity ranges up to 35.7 m [125]. During

mated to be up to 1000 m<sup>2</sup>

Biosensors for Environmental Monitoring

104

conditions they can live for 3 months [127].

3.2 Apodemus sylvaticus as a biosensor

### 3.3 Myodes glareolus as biosensor

Myodes glareolus, or Clethrionomys glareolus, has been used mainly in studies of bioaccumulation of metals and metalloids. Wijnhoven and collaborators analyzed several species of small mammals living in a contaminated floodplain. They found that, in almost 40% of the population of C. glareolus, the concentration of Cd exceeded the lowest level at which adverse effects are produced. The other two species, Microtus agrestis and M. arvalis, showed less ecotoxicological effects [136]. Topashka-Ancheva and collaborators evaluated other small mammals: A. flavicollis, M. macedonicus, C. glareolus, P. subterraneus, M. arvalis, M. rossiaemeridionales, and C. nivalis. They found that C. glareolus had a higher concentration of Cu and Cd in the body compared to the other species. The concentrations of Cu, Zn, Pb, and Cd in C. glareolus were significantly higher than in A. flavicollis in both the whole body and in the liver (except for Pb in the liver, which was higher in A. flavicollis). The authors suggest that the differences between species are due to the position of each species in the trophic chain, their diet, and lifestyle [137]. Damek-Poprawa and Sawicka-Kapusta compared the populations of a control site and two contaminated sites close to a steel and zinc foundry. No damage was found in C. glareolus inhabiting the control site, but there were histopathological changes in the kidneys and liver of the rodents inhabiting the contaminated sites. The concentration of Pb and Cd in liver, kidney, and femur tissues was higher in the rodents living in contaminated areas [138]. Erry and collaborators studied populations of A. sylvaticus and C. glareolus in a site contaminated with As and in a control site. Many species of rodents living in the contaminated site accumulated more As in the spleen, lung, muscle, and femur than those living in the control site. The concentrations of As in the liver, femur, and hair were higher in A. sylvaticus than in C. glareolus in both the contaminated and the control sites. The authors mention that these results could be due to the high water exchange and urinary excretion of C. glareolus compared to A. sylvaticus, which could make C. glareolus susceptible to renal toxicity [139].

sensitive to different pollutants such as antibiotics, pesticides, toxins, and organic compounds, which makes it a non-specific method for the detection of heavy metals [145]. Another bacterium proposed as a bioindicator is Vogesella indigofera; under normal conditions this bacterium develops a blue color due to the indigoidine production; when it grows in the presence of Cr6, the bacteria decreases the pigment production, and this decrease is dependent on the concentration of Cr<sup>6</sup>

The Use of Biosensors for Biomonitoring Environmental Metal Pollution

150 μg/ml the bacteria are entirely white and rough [146]. Serratia marcescens is a Gram-negative bacterium that produces a red pigment known as prodigiosin; when the bacterium grows in sub-inhibitory concentrations of Cd, Cr, and Pb, the pigment production decreases drastically, so the authors propose it as a bioindicator of

The presence of heavy metals in the environment exerts an intense selection pressure on the organisms that live there; an increase in its concentration and the high rate of horizontal gene transfer can select heavy metal-resistant microorganisms. Therefore, the resistance and detoxification genes have been used as biomarkers for the study of contaminated environments using molecular techniques such as quantitative PCR and real-time quantitative reverse transcription PCR. Within these genes are those involved in the resistance to As, ACR3(1) (arsenite efflux pump), aioA (arsenite oxidase), arsB (arsenical efflux pump), arsC (arsenate reductase), and arsM (arsenic methyltransferase) [148–151]; those that confer resistance to Cu, copA (Copper-exporting P-type ATPase), and cusA (copper export system) [152, 153]; for Cd, Zn, and Co resistance, czcA (Cd/Zn/Co efflux pump) [154]; for Hg, hgcA (mercury methylating protein) and merA (mercuric reductase) [155]; the mr [140] that encodes to metallothionein a cysteine-rich and heavy metalbinding protein [156]; and sodA [140] which codes for a superoxide dismutase, involved in the protection of toxicity against heavy metals [157]. Another technique to measure the presence and abundance of genes involved in resistance to heavy metals is through the use of genetic microarrays such as the GeoChip, commercially available [158]. With the use of this microarray, it was possible to correlate the presence of arsC, copA, cueO (multicopper oxidase), merB (alkylmercury lyase), metC (cystathionine beta-lyase), tehB (tellurite methyltransferase), and terC (tellurium resistance protein) genes in sediments and waters contaminated with Cd, Cr,

High concentrations of heavy metals affect microbial populations and therefore their processes. Thus, the evaluation of microbial processes represents good biomarkers of exposure in different environments. Within the parameters most used are the monitoring of enzymatic activities of the carbon and nitrogen cycle, soil respiration, microbial mass, and the ecosystem biodiversity [160, 161]. Microbial biodiversity is drastically affected by contamination with heavy metals. In general, it is observed that a higher concentration of heavy metals decreases bacterial species. However, with the massive sequencing of DNA, some bacterial groups that could serve as biosensors of contamination were identified, for example, the study carried out by Schneider and collaborators finds that the bacterial groups γ-Proteobacteria, Verrucomicrobia, and Chlamydiae showed a consistent response to

Pb content across contrasting ecosystems. The phyla Chlamydiae and γ-

Proteobacteria were more abundant, while Verrucomicrobia were less abundant at high contamination level. So, they conclude that such groups and ratios thereof can be considered as relevant bioindicators of Pb contamination [162]. In soils contaminated with Cu, it was observed that at increased concentrations, bacterial richness was negatively impacted and enhanced relative abundance of Nitrospira and Acidobacteria members and a lower representation of Verrucomicrobia,

Proteobacteria, and Actinobacteria, suggesting a promising role as bioindicators of

heavy metal contamination [147].

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

Cu, Hg, and S [158, 159].

copper contamination in soils [163].

107

; at

As shown in the studies on A. sylvaticus and C. glareolus, the differences between both species are a function of diet, metabolism, mobility, and lifestyle; thus, the monitoring of environmental contamination with metals and metalloids should use small mammals belonging to different taxa in order to determine the real impact of HM on organisms and on trophic chains.

### 4. The use of bacteria as biosensors of heavy metal pollution

#### 4.1 Bacteria as biosensors

Microorganisms are primary producers in many environmental ecosystems and play an essential role in the nutrient cycle, and they are very abundant and ubiquitous. The microbes proliferate rapidly, are easily detectable and easy to sample, and respond quickly to environmental changes, like temperature, pH, or the presence of contaminants including heavy metals. These characteristics make microorganisms good candidates as pollution biosensors [140]. In this sense, bioluminescent bacteria such as Aliivibrio fischeri and Photobacterium phosphoreum have been used to monitor water and soil contaminated with HM [141, 142]. This bioassay is carried out using the natural bioluminescence emitted by these bacteria and is based on the decrease of this fluorescence when the bacteria grow in samples of water or soil contaminated with different heavy metals such as Zn, Cu, Cd, Hg, and Cr, among others [141–144]. In the case of A. fischeri, the test has been developed commercially and is distributed under the name of Microtox®. However, this method is

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

sensitive to different pollutants such as antibiotics, pesticides, toxins, and organic compounds, which makes it a non-specific method for the detection of heavy metals [145]. Another bacterium proposed as a bioindicator is Vogesella indigofera; under normal conditions this bacterium develops a blue color due to the indigoidine production; when it grows in the presence of Cr6, the bacteria decreases the pigment production, and this decrease is dependent on the concentration of Cr<sup>6</sup> ; at 150 μg/ml the bacteria are entirely white and rough [146]. Serratia marcescens is a Gram-negative bacterium that produces a red pigment known as prodigiosin; when the bacterium grows in sub-inhibitory concentrations of Cd, Cr, and Pb, the pigment production decreases drastically, so the authors propose it as a bioindicator of heavy metal contamination [147].

The presence of heavy metals in the environment exerts an intense selection pressure on the organisms that live there; an increase in its concentration and the high rate of horizontal gene transfer can select heavy metal-resistant microorganisms. Therefore, the resistance and detoxification genes have been used as biomarkers for the study of contaminated environments using molecular techniques such as quantitative PCR and real-time quantitative reverse transcription PCR. Within these genes are those involved in the resistance to As, ACR3(1) (arsenite efflux pump), aioA (arsenite oxidase), arsB (arsenical efflux pump), arsC (arsenate reductase), and arsM (arsenic methyltransferase) [148–151]; those that confer resistance to Cu, copA (Copper-exporting P-type ATPase), and cusA (copper export system) [152, 153]; for Cd, Zn, and Co resistance, czcA (Cd/Zn/Co efflux pump) [154]; for Hg, hgcA (mercury methylating protein) and merA (mercuric reductase) [155]; the mr [140] that encodes to metallothionein a cysteine-rich and heavy metalbinding protein [156]; and sodA [140] which codes for a superoxide dismutase, involved in the protection of toxicity against heavy metals [157]. Another technique to measure the presence and abundance of genes involved in resistance to heavy metals is through the use of genetic microarrays such as the GeoChip, commercially available [158]. With the use of this microarray, it was possible to correlate the presence of arsC, copA, cueO (multicopper oxidase), merB (alkylmercury lyase), metC (cystathionine beta-lyase), tehB (tellurite methyltransferase), and terC (tellurium resistance protein) genes in sediments and waters contaminated with Cd, Cr, Cu, Hg, and S [158, 159].

High concentrations of heavy metals affect microbial populations and therefore their processes. Thus, the evaluation of microbial processes represents good biomarkers of exposure in different environments. Within the parameters most used are the monitoring of enzymatic activities of the carbon and nitrogen cycle, soil respiration, microbial mass, and the ecosystem biodiversity [160, 161]. Microbial biodiversity is drastically affected by contamination with heavy metals. In general, it is observed that a higher concentration of heavy metals decreases bacterial species. However, with the massive sequencing of DNA, some bacterial groups that could serve as biosensors of contamination were identified, for example, the study carried out by Schneider and collaborators finds that the bacterial groups γ-Proteobacteria, Verrucomicrobia, and Chlamydiae showed a consistent response to Pb content across contrasting ecosystems. The phyla Chlamydiae and γ-Proteobacteria were more abundant, while Verrucomicrobia were less abundant at high contamination level. So, they conclude that such groups and ratios thereof can be considered as relevant bioindicators of Pb contamination [162]. In soils contaminated with Cu, it was observed that at increased concentrations, bacterial richness was negatively impacted and enhanced relative abundance of Nitrospira and Acidobacteria members and a lower representation of Verrucomicrobia, Proteobacteria, and Actinobacteria, suggesting a promising role as bioindicators of copper contamination in soils [163].

3.3 Myodes glareolus as biosensor

Biosensors for Environmental Monitoring

Myodes glareolus, or Clethrionomys glareolus, has been used mainly in studies of bioaccumulation of metals and metalloids. Wijnhoven and collaborators analyzed several species of small mammals living in a contaminated floodplain. They found that, in almost 40% of the population of C. glareolus, the concentration of Cd exceeded the lowest level at which adverse effects are produced. The other two species, Microtus agrestis and M. arvalis, showed less ecotoxicological effects [136]. Topashka-Ancheva and collaborators evaluated other small mammals: A. flavicollis, M. macedonicus, C. glareolus, P. subterraneus, M. arvalis, M. rossiaemeridionales, and C. nivalis. They found that C. glareolus had a higher concentration of Cu and Cd in the body compared to the other species. The concentrations of Cu, Zn, Pb, and Cd in C. glareolus were significantly higher than in A. flavicollis in both the whole body and in the liver (except for Pb in the liver, which was higher in A. flavicollis). The authors suggest that the differences between species are due to the position of each species in the trophic chain, their diet, and lifestyle [137]. Damek-Poprawa and Sawicka-Kapusta compared the populations of a control site and two contaminated

sites close to a steel and zinc foundry. No damage was found in C. glareolus

sylvaticus, which could make C. glareolus susceptible to renal toxicity [139].

4. The use of bacteria as biosensors of heavy metal pollution

HM on organisms and on trophic chains.

4.1 Bacteria as biosensors

106

As shown in the studies on A. sylvaticus and C. glareolus, the differences between both species are a function of diet, metabolism, mobility, and lifestyle; thus, the monitoring of environmental contamination with metals and metalloids should use small mammals belonging to different taxa in order to determine the real impact of

Microorganisms are primary producers in many environmental ecosystems and play an essential role in the nutrient cycle, and they are very abundant and ubiquitous. The microbes proliferate rapidly, are easily detectable and easy to sample, and respond quickly to environmental changes, like temperature, pH, or the presence of contaminants including heavy metals. These characteristics make microorganisms good candidates as pollution biosensors [140]. In this sense, bioluminescent bacteria such as Aliivibrio fischeri and Photobacterium phosphoreum have been used to monitor water and soil contaminated with HM [141, 142]. This bioassay is carried out using the natural bioluminescence emitted by these bacteria and is based on the decrease of this fluorescence when the bacteria grow in samples of water or soil contaminated with different heavy metals such as Zn, Cu, Cd, Hg, and Cr, among others [141–144]. In the case of A. fischeri, the test has been developed commercially and is distributed under the name of Microtox®. However, this method is

inhabiting the control site, but there were histopathological changes in the kidneys and liver of the rodents inhabiting the contaminated sites. The concentration of Pb and Cd in liver, kidney, and femur tissues was higher in the rodents living in contaminated areas [138]. Erry and collaborators studied populations of A. sylvaticus and C. glareolus in a site contaminated with As and in a control site. Many species of rodents living in the contaminated site accumulated more As in the spleen, lung, muscle, and femur than those living in the control site. The concentrations of As in the liver, femur, and hair were higher in A. sylvaticus than in C. glareolus in both the contaminated and the control sites. The authors mention that these results could be due to the high water exchange and urinary excretion of C. glareolus compared to A.
