Usefulness of Bioindicators and Biomarkers in Pollution Biomonitoring

We have different possibilities and tools to assess the impact of pollution on marine ecosystems. The ecotoxicological approaches are based on the use of biomonitors and biomarkers. They aim to study the effect of toxic chemicals on the biological organisms especially at the population, community and ecosystem levels. The ultimate goal of ecotoxicology is to be able to predict the effects of pollution so that the most efficient and effective action to prevent or remediate any detrimental effect. In order to assess the impact of anthropogenic activities on the aquatic ecosystem and to insure compliance with regulation or guidelines, we use biomonitoring. This kind of approach is based on the use of biological responses in order to assess anthropogenic changes in the environment. Biomonitoring involves the use of indicator species such as filter feeding mollusk bivalves. These organisms tend to accumulate pollutants in their tissues without showing any apparent detrimental effect. Moreover, they could reflect the real bio available fraction of the pollutant. In order to have an early warning system predicting the pollution effects even at low levels, biomarkers were extensively studied. Some of them were validated in both field and in vivo conditions. In the present paper, the usefulness of bioindicators and biomarkers in pollution monitoring are discussed. An overview of results from case studies dealing with in situ, in vivo and transplantation experiments is presented.

The marine environment is exposed to different anthropogenic pollutants generated by industrial, domestic and agricultural activities.In marine organisms, many pollutants are susceptible to interact with the physiological processes such as growth and reproduction.In fact pollutants can alter their life and lead to serious disruptions such as reduction of the animal populations, changes of the reproductive functions.
In order to provide an optimal use of marine resources, one of the major preoccupations of governments and researchers consists to distinguish between "clean" and polluted ecosystems.For this reason, monitoring programs are used to evaluate pollution state of coastal zones and implement short term and long term strategies for marine resources protection.Different compartments could be involved: sea water, sediment and marine organisms.
Measuring pollutant concentrations in sea water presents some disadvantages such as the low concentrations and the random spatial and temporal variations.The sediment is a long-term integrator of pollution where concentrations are higher than in *Address correspondence to this author at the ES11ES90 Marine Ecotoxicology, IPEIS, Sfax University-Tunisia, BP1172-3018-SFAX, Tunisia; Tel: +21620333622; E-mail: amel.chaffai@tunet.tnseawater, but contaminants are not always bioavailable for organisms owing to their physico-chemical forms.Moreover, heterogeneity of sediment (particle size and organic matter) could make comparison between sites difficult.That is why the use of living organisms called biomonitors is preferable for pollutant quantification.In fact, these organisms accumulate contaminants usually from water and food, a fact reflecting only the bio-available fraction that is of potential ecotoxicological significance and could therefore interest environmental managers.The use of biomonitors to evaluate pollution impact is called biomonitoring.
Biomonitoring programs based on measuring contaminants in marine organisms are interesting from a human health point of view.However, they could not give information about the toxicological significance of pollutants accumulated and do not indicate the health status of the organisms [1].Consequently, recent biomonitoring programs are now involving biomarkers, which are are measurable parameters at different levels of biological organisation (molecular, cellular or physiological).Biomarkers traduce changes in the metabolic regulatory processes resulting from the effect of anthropogenic stressors [2].
In this paper we will focus on biomonitors and biomarkers and discuss their usefulness in biomonitoring pollution.

BIOINDICATORS OR BIOMONITORS
The first definition was proposed by Phillips and Rainbow [3] who considered biomonitors as animal (s) or plant(s) which accumulate contaminants in their tissues and organs from their surroundings.The quantification of such contaminants (in fish, crustaceans, bivalves, etc…) can therefore reflect the concentration of contaminants in the surrounding environment.
In 1999, Gerhardt [4] proposed another definition; bioindicators are defined as species or group of species that readily reflects the abiotic or biotic state of an environment represents the impact of environmental change on a habitat, community or ecosystem or is indicative of the diversity of a subset of taxa or the whole diversity within an area.The author has considered that bioindicators are useful in three situations: (i) where the indicated environmental factor cannot be measured, (ii) where the indicated factor is difficult to measure, e.g.pesticides and their residues or complex toxic effluent (iii) where the environmental factor is easy to measure but difficult to interpret, e.g.whether the observed changes have ecological significance.
According to the aim of bioincation, three types of biomonitors were proposed, (i) compliance indicators, (ii) diagnostic indicators (iii) early warning indicator.
According to various authors, bioindicators are organisms that indicate the long-term interaction of several environmental conditions, but also react to a sudden change of important combinations of factors.Other authors consider only those organisms that react to changes in the environment by alterations in their metabolism, activity, or other aspects of their biology or that accumulate toxic substances [5].
In order to monitor pollution, bio-monitors or bioindicators are organisms that can be used to provide information on the variation of pollutants over time and space.
Taking the case of metal pollution, each bioindicator shows the special merits for the biomonitoring aquatic ecosystem when compared to the others [6].
In the practice, the organisms living in the aquatic systems are sampled for the analysis of various biological responses to chemical exposures.A "perfect" bioindicator is expected to have several characters.The most important are : (a) it can accumulate high levels of pollutants without death ; (b) it has enough abundance and wide distribution for the repetitious sampling and comparison ; (c) its life is long enough for the comparison between various ages ; (d) it can afford suitable target tissue or cell for the further research at microcosmic level ; (e) easy sampling and easy raising in the lab ; (f) well dose-effect relationship can be observed in it [6].
For aquatic pollution, the common used bioindicators mainly contained organisms including mollusks bivalves, mollusks gastropods and fishes.Zooplankton species can accumulate and metabolize pollutants, offering the possibility to be used as bioindicators of water quality and show their special advantages in biomonitoring [6].Aquatic algae, considered as the important elementary producers in marine water plays key role to the whole ecosystem, and can directly reflect the water quality.As an example of bioindicators algae we can note Chlorella ellipsoidea, C. ellipsoidea [7], in addition to the algae Cystoseira stricta and the famous posidonia oceanica which are very sensible and disappear in polluted surroundings.Moreover they are used to outline the history of pollution [7].
Bivalve mollusks, such as mussels and oysters are characterized by their aptitude to concentrate both metals and organic contaminants, their immobility, their limited ability to metabolize accumulated contaminants, their abundance, their persistence, and their ease of collection They have become recognized bio-monitors of pollutants in coastal waters and have been used in different international monitoring programs such as the Mussel Watch (USA) and the RNO (France) [8][9][10].
In the case of Tunisia, Mussels and oysters are available only in the northern coast.As a substitute, we have proposed and validate the use of Ruditapes decussatus or Cerastodema glaucum.In fact these two species are sedentary filter feeding marine bivalves that are widely available along the Tunisian coast and satisfying criteria required for good bio-monitors of pollution.The first species Ruditapes decussatus represents an important economic endpoint since it is a natural resource in the gulf of Gabès, the stock being mostly exported to Europe.The second bivalve is the cockle Cerastoderma glaucum is also a filter feeding organism living in the superficial sediment and available in many sites along the Tunisian coast.
Nevertheless, mussel and oysters are not available everywhere.In such circumstances, each country should adapt its monitoring program according to available species.
Before considering these two bivalve species as Biomonitors, we need to check if the conditions above cited are satisfied.
Since many years we have undertaken both in vivo [11][12][13], in situ studies [14][15][16] as well as transplantation experiments [17].Our choice was guided by their availability along the Tunisian coasts, their sedentary filter feeding habits.While Ruditapes decussates presents a long siphon and lives inside the sediments, Cerastoderma glaucum has a short siphon and lives in the superficial sediment.Together these two bivalve species could reflect the pollution of their surrounding water.We have demonstrated both in vivo and in situ, the relationship between trace metal concentration in the studied sites and in clam and cockle tissues.We also identify target organs for metal accumulation.Biotic variations linked to the reproductive cycle, size, sex were also studied [18,19].

BIOMAKERS
The term biomarker has been defined as a xenobiotically induced variation in cellular or biochemical components or processes, structures, or functions that is measurable in a biological system or sample [20].Biomarkers were originally developed in the medical and veterinarian sciences and there has been an increasing emphasis on the use of invertebrate and particularly bivalve biomarkers to assess marine pollution [21].

Biomarkers
occurs at different levels of organization, from subcellular to whole organisms and ecosystem.Effect at molecular level trend occurs first, followed by responses at the cellular (biochemical), tissue/organ and whole-body levels.Responses that occur at individual, population and ecosystem level are generally accepted to have ecological relevance and tend to be less reversible and more detrimental than effects at lower levels.In fact, much attention is given towards identifying and understanding toxic effects initiated at the sub-organism level (molecular, biochemical or physiological changes) and towards developing biomarkers at this level to be incorporated into routine biomonitoring programs [22,23].
Biomarkers can be generally, broadly categorized as markers of exposure and effect.
1. Biomarkers of exposure are the product of integration between a xenobiotic and some target molecule or cell that is measured within a compartment of an organism.In general, biomarkers of exposure are used to predict the dose received by an individual, which can be related to change resulting in a disease state.
2. Biomarkers of effect are defined as measurable biochemical, physiological, behavioral, or other alterations within an organism that, according to their magnitude, can be recognized as established or potential health impairment or disease.
Some biomarkers are highly specific for individual chemicals; such biomarkers include inhibition of cholinesterase by organophosphate or carbamate [24] metallothioneines by toxic trace metals [14,[25][26][27] and ethoxyresorufin-o-deethylase (EROD) respond to organic chemicals, particularly PAHs and PCBs.Others biomarkers are also well validated, but they have wider application and tend to respond to broader classes of chemicals.Example of these biomarkers are the induction of the multiplexenobiotic Resistance (MXR) protein [28,29], Stress on stress [30], the formation of DNA adduct and other DNA alteration [31][32][33], and also lysosomal alteration in molluscan digestive gland cell [34,35], etc… These assays required either additional biomarker studies or chemical residue analysis in order to link causative agent to adverse effect.
An important application of biomarkers is their ability to integrate multiple chemical exposure across an area with variety of chemical contaminants; the CYP A1 responses to sediment contaminated with dioxin, polychlorited biphenyl (PCBs), or polynuclear aromatic hydrocarbons (PAHs) can provide insight to the status of the contaminants on site, their bioavailability, and overall risk that they pose [36][37][38].Similarly, metallothionein content and immune function can provide insight to the combined effect of metals found on metal contaminated sites [39].

Some Examples
We below present some biomarkers which were studied in our laboratory: metallothioneins (MTs), malonedialdehyde (MDA), acetylcholinesterase (AchE) glycogen and stress on stress test.

Metallothioneins (MTs):
Most scientists agree that biochemical and physiological mechanisms allowing mollusk bivalve species to accumulate and tolerate high amounts of heavy metals are based on their metal handling by metallothioneins (MTs).These are low molecular weight, cysteine-rich, cytosolic proteins of ubiquitous occurrence which are suggested to inactivate toxic metal ions by binding them to sulfur atoms of the peptide cysteine residues [40].In fact, it has repeatedly been shown that concentrations levels of MT can be correlated to accumulated fractions of toxic metal ions such as copper or cadmium in animals' tissues [41].In addition, metallothionein concentrations in molluscs may also vary due to influence by nonmetallic pollutants.Hence, the idea was raised that MTs might be used as biomarkers for environmental pollution by measuring their concentrations in bivalves from contaminated habitats.
Earlier work in our laboratory has shown the presence of metallothioneins in the gills and digestive gland of Ruditapes decussatus and Cerastoderma glaucumin field conditions and after an experimental contamination.
Malonedialdehyde (MDA), is a product of lipid peroxidation due to over production of oxyradicals in cells, following contaminant exposure or stress due to natural conditions [42].Lipid peroxidation is considered an important feature in cellular injury.It results from free radical reactions in biological membranes, which are rich in polyunsaturated fatty acids.MDA has been used extensively to assess detrimental effects of various pollutants.
Glycogen, is the fuel for different metabolic and physiological processes.Glycogen has been shown to respond quite well to complex and diffuse contamination situations [43].Decreased levels of glycogen related to an altered growth were observed in mussels Mytilus edulis and clams (Mya arenaria) after an exposure to pulp and paper mills effluents as well as in a mesocosms where bivalves were exposed to oil and silicon based polymer [44].
Acetylcholinesterase activity (AchE) is an enzyme essential to the correct transmission of nerve impulse.A reduction or inhibition of this enzymatic activity has been used to detect and measure the biological effect of organophosphorus and carbamates in the marine environment [45].
The stress on stress test is considered as a nonspecific biomarker allowing the evaluation of the general health status of bivalves.It consists in exposing animals to anoxia by air and to evaluate survival time.Measuring the lethal time for 50% or the organisms (LT50) provides information about the health status [46].
According to our research work, the assessment of both environmental and biological variables that may affect the biomarker responses should be investigated

Biomarker Use
The use of biomarkers to evaluate pollution has noticeably increased in the past few years.Indeed, the biomarker approach has now attracted the attention of international regulatory agencies as a new and potentially powerful tool for detecting exposure to and the effect of environmental contamination [47].This approach should be multiparametric, using different and complementary biomarkers to reflect the effects of different contaminants.However, we also need to consider variations linked to biotic and abiotic factors.In fact, the physiological state of an organism within an ecosystem is the result of equilibrium between the influences of anthropogenic, abiotic and biotic factors [14,48,49].
The use of biomarkers measured at molecular or cellular level have been proposed as sensitive 'early warning' tools for biologic effect measurement in environmental quality assessment [47].The selected biomarkers should indicate that organism has been exposed to pollutants (exposure biomarkers) and / or the magnitude of the organism's response to the pollutant (effect biomarkers).These early warning biomarkers can be used in a predictive way, allowing to initiation of bioremediation strategies before irreversible environmental damage of ecological consequence occurs.These biomarkers are then defined as shortterm indicators of long-term biological effects [34,50].
A practical and successful biomarker should satisfy a number of criteria: 1.The Biomarker response should be sensitive enough to detect early stage of the process of toxicity and should precede the effect at high levels of biological organization.
2. The Biomarker should be specific to a particular contaminant or for a class of contaminants.
3. The Biomarker should respond in a concentrationdependant manner to change in ambient levels of the contaminant.
4. Identification of the non toxicological variability identified in particular variations linked to biotic factors.
In practice, all of these characteristics are not satisfied; most biomarkers have limited specificity, because of the variety of pollutants present in an environment.That is why, a pool of biomarkers at different levels of biological organization is required to be effectively applicable in a biomonitoring program [47,51,52].Non-specific biomarkers provide information that may indicate environmental perturbations but not the causal agent.To identify causal relationship, analytical chemistry and specific biomarkers are needed.However, it is not cost-effective to use a suite of specific biomarkers and measure a wide range of contaminants in all monitoring programs.It is, therefore recommended that the effects of pollutants might be, initially, detected by relatively non-specific biomarkers, usually high in the hierarchy (e.g., behavioral and physiological biomarkers).Detection of abnormalities with these non-specific biomarkers at a site at risk from pollution might then justify the measurements of more costly, lower hierarchy, specific biochemical and cellular biomarkers (e.g., MFO activity, metallothioneins, genotoxic marker, and tissue lesions) to identify the class of pollutant responsible for the exposure [50].Another compelling reason to use general biomarkers in the initial stages of monitoring is that organism will undoubtedly be exposed to range of contaminants in the environment which have the potential to act antagonistically or synergistically.Specific biomarkers may miss the effect of certain chemical whereas non specific biomarkers will respond to the complex mixture of chemical present in the environment.
By the application of biomarker measurement the use of expensive and complex analytical chemical equipment can be reduced, as these analyses are relatively quick to perform.Furthermore, since biomarkers are a part of the detoxification mechanism.This provides not only early warning system about degradation in environmental quality, but also specific measures of the toxic, carcinogenic and mutagenic compound in the biological material.Many studies about biomarker response in organisms are being carried out in most of the developed biomonitoring program in Europe and USA.Moreover, different methods for biological effect measurement have been evaluated in a series of practical workshops organized by the International council for the Exploration of the Sea (ICES) and the Intergovernmental Oceanographic Commission (IOC), such as those in the North Sea.
In the Mediterranean Sea, the United Environment Program has funded a biomonitoring program including a variety of biomarkers [53].Biomarkers have also been included in the joint monitoring program of the OSPAR convention where Portugal, Spain and others European countries are the members [34].Several of biomarkers have been applied in a pollution monitoring program of the Mediterranean Sea [54].The program involved use of both biomarkers of effects (micronuclei frequency and lysosomal membrane stability) and biomarkers of exposure (cytochrome P450, EROD and metallothioneins).
The BEEP (Biological Effect of Environmental Pollution in Marine Coastal Ecosystems) project, involving 30 institutions from 12 countries in Europe and Scandinavia including the North Atlantic region, has focused on development of new biomarkers [55].
Finally, there is a long list of biomarkers that are under development or have been used with varying degrees or success but required further validation before can be used in hazard evaluation.
In natural ecosystem, this is very likely as changes in physicochemical and biological characteristics often vary over very small distances.Such heterogeneity will then be reflected in small-scale differences of the bioavailability of pollutants.However, an equally important source of variability may be inherent differences in the morphology and biochemical/ physiological status of exposure organisms [51].Variability in most of the biochemical and physiological biomarker responses may be, thus, attributed to abiotic factors (Temperature, salinity, dissolved oxygen, ect) or biotic factors (Size, age, genotype, ect.)All these factors can vary in both time and space [52].These sources of variability render the biomarker responses insensitive compared to traditional chemical monitoring techniques.
In order to integrate biomarkers in modern pollution problem, taking into account temporal and spatial variability in biomarker responses, and with various pollutant mixtures, Handy et al. [52] proposed several priorities in future researches.These include the following: 1. Funding of long term biomarker studies (minimum 5 year) as part of validation of chronic biomarkers 2. Primary research to mathematically quantify and model temporal and spatial variables that influence biomarker responses.These should include age, sex and nutritional status corrections for biomarkers in specific organism exposed to known contaminants in the laboratory, following by field validation.
3.More fundamental research in to chronic doseresponse relationship.

CONCLUSION
Aquatic ecosystems are under constant pressure of anthropogenic pollutants originating from various sources.In the recent decades, there have increasing concerns about pollutants entering the aquatic environment.A need to discover simple and reliable ways to monitor the level of particular chemicals such as heavy metals or other pollutants in the aquatic environment, and to elucidate the mechanisms of pollutants uptake and storage in organisms, has resulted in a proliferation of studies into the use of biomonitor organisms.The success of some Mollusc Bivalves, is due to some factors including their wide geographical distribution, abundance, sedentary, tolerance to environmental changes, high bioconcentration factors, population stability, and size, adaptability for field, cage and laboratory experiments.This was completed by the biomarker approach, based on early warning systems for pollution monitoring.A detailed knowledge of natural variation in biomarkers responses must, therefore, be ascertained before field application in order to prevent confounded interpretation when they are applied in environmental monitoring programs.

Table 1 : Overview of Research Work about Bioindicators and Biomarker Validation
using in pollution monitoring.It was necessary to combine; in vivo experiments based on pure contaminants and industrial effluents, in vivo and filed transplantations, and field studies approaches.The combination of these approaches helped us to identify the most reliable biomarkers in relation to pollution exposure and taking into account some biotic factors (size, age, reproductive state).Table1summarizes the main used approaches, bioindicators, biomarkers as well as the biotic factors studied.