Fish around the world are found occupying almost any aquatic habitat. In particular, freshwater fish are severely threatened as the freshwater ecosystems are considered the most endangered of the world . The ultimate destination of most contaminants is water; rivers, lakes, aquifers, or sea, are receptors of wastewaters with a complex mixture of xenobiotics. The variety of contaminants and their mixtures that daily reach the water bodies coupled with a multitude of irresponsible water management practices and destructive land uses, are currently threatening freshwater ecosystems , such is the case of discharge of municipal and industrial wastewaters, deforestation, increase of land crops, and water extraction from water bodies to human consumption and other uses. The impact of contaminants in an aquatic ecosystem is complex, therefore has increased the need for determining the ambient status in order to provide an indication of changes induced by anthropogenic activities and their influence on aquatic organisms. As physicochemical analyses shed no light on the biological status of ecosystems, a biological approach is needed to evaluate environmental health; moreover, the biological effects of contaminant interactions cannot be expressed by physicochemical investigations .The aquatic ecosystem health is often reflected by the health of organisms that reside in that system. Fish in their natural environments are typically exposed to numerous stressors including unfavorable or fluctuating temperatures, high water velocities and sediment loads, low dissolved oxygen concentrations, limited food availability, and among other types of natural episodic variables. In addition, anthropogenic stressors such as contaminant loading can add to the insults that fish may already experience in many systems. All these factors, individually or together, can impose considerable stress on physiological systems of fish and impair their health [4, 5]. Environmental contaminants are known to induce measurable biochemical changes in exposed aquatic organisms . Likewise, stressors can load or limit physiological systems, reduce growth, impair reproduction, predispose fish to disease, and reduce the capacity of fish to tolerate additional stressors. Many species of fish, in particular those species near the top of the food chain, are generally regarded as integrators of environmental conditions and may reflect, therefore, the health of aquatic ecosystems  and therefore, they are excellent indicators of the relative health of aquatic ecosystems and their surrounding watersheds . Thus, effects of contaminants on aquatic organisms may be manifested at all levels of biological organization (in a hierarchical scale that can be at cellular level, organisms, populations, communities, and ecosystems). In this way, the measuring of a suite of indicators across such levels of organization is often necessary to assess ecological integrity; these indicators also should include molecular, biochemical, physiological, population, community, and ecosystem responses.
The indicators allow us to isolate key aspects of the environment from an overwhelming array of signals . Ecological indicators have been defined as measurable characteristics of the structure (e.g. genetic, population, habitat, and landscape pattern), composition (e.g., genes, species, populations, communities, and landscape types), or function (e.g., genetic, demographic/life history, ecosystem, and landscape disturbance processes) of ecological systems . On the other hand, other authors , established that bioindicators are organisms or communities of organisms, which reactions are observed representatively to evaluate a situation, giving clues for the condition of the whole ecosystem; Gerhardt also indicate that bioindicators are species reacting to anthropogenical effects on the environment, concluding that a biological indicator would be: a 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. In this sense, the primary role of ecological indicators is to measure the response of the ecosystem to anthropogenic disturbances . A sentinel species can be defined as any domestic or wild microorganism, plant or animal, that can be used as an indicator of exposure to and toxicity of a xenobiotic that can be used in assessing the impact on human and/or environmental health because of the organism's sensitivity, position in a community, likelihood of exposure, geographic and ecological distribution or abundance .
The specific objective of this review is to provide a short framework of effects of xenobiotics on the responses of freshwater fish across molecular to population level when have been exposed to environmental stressors. Likewise, the present review considers the use of fish as sentinel organisms to assess the anthropogenic impacts over the freshwater ecosystems. The review asks whether fish can be able to reflect the environmental damage from molecular to population levels. Also, the present review offers a selection of examples of studies employing fish as sentinel organisms in ecological, toxicological and environmental risk assessments.
2. Suborganismal responses
When an organism is exposed to stressors like contaminants or a mixture of them, energy is demanded to deal with that stress ; stressors tend to impact ecosystems at lower levels of organization first . One of the methods to quantify the exposure to xenobiotics and its potential impact on living organisms is the monitoring by the use of the so-called biomarkers . Biomarkers have been defined by several authors, all of them, in reference to biological responses to contaminants exposure, as a) measurements in body fluids, cells or tissues indicating biochemical or cellular modifications due to the presence and magnitude of toxicants, or of host response ; b) a change in a biological response (ranging from molecular through cellular and physiological responses to behavioral changes) which can be related to exposure to or toxic effects of environmental chemicals ; c) any biological response to an environmental chemical at the subindividual level, measured inside an organism or in its products (urine, faeces, hair, feathers, etc.), indicating a deviation from the normal status that cannot be detected in the intact organism ; d) a xenobiotically induced variation in cellular or biochemical components or processes, structures, or functions that is measurable in a biological system or samples ; e) contaminant-induced physiological, biochemical, or histological response of an organism, and f) as functional measures of exposure to stressors expressed at the sub-organismal, physiological or behavioural level. Considering these definitions of biomarkers, we could adopt our own definition: “
In concordance with  and other authors [19, 20], biomarkers have been classified in three different categories: a) biomarkers of exposure, which represent responses such as induction or inhibition of specific enzymes involved in biotransformation and detoxification as a consequence of chemical exposure , b) biomarkers of effect, are any changes in a biological system that reflects qualitative or quantitative impairment resulting from exposure , including responses measurable at level biochemical, physiological or some other alterations within tissues or body fluids of an organism that can be recognized as associated with an established or possible health impairment or disease , and c) biomarkers of susceptibility, which serve as indicators of a particular sensitivity of individuals to respond to the challenge of exposure to a effect of a xenobiotic or to the effects of a group of such compounds, in this case, individual changes included genetic factors and changes in receptors which alter the susceptibility of an organism to that exposure . However, other authors have been subdivided the biomarkers in exposure biomarkers, effects biomarkers and predictive biomarkers.
Responses of fish at suborganismal level to xenobiotic exposure are complex and varied and depending of type of contaminant and time of exposure. The most general effect of xenobiotics on fish is oxidative stress, which is experienced when antioxidant defenses are overcome by prooxidant compounds. Oxidative stress include a variety of oxidative reactions, usually started by free radicals and propagated by molecular oxygen, which results in the oxidation of lipids, proteins, and nucleic acids . Free radicals are atoms, molecules, or ions with unpaired electrons on an otherwise open shell configuration. These unpaired electrons are usually highly reactive due to which radicals are likely to take part in chemical reactions. Very often free radicals are confused with reactive oxygen species (ROS) such as molecular and singlet oxygen, superoxide anion, hydroxyl radical and some their derivatives; however, hydrogen peroxide is not a radical, but it is a reactive species because has higher activity than molecular oxygen . Hydroxyl radical is the most important free radical of biological importance, because of its potent oxidative potential and indiscriminate reactivity with cellular components of enzymes and DNA [24, 25]; likewise, being oxidant, all ROS are agents which at high concentrations are toxic to cells. Oxidative stress is a risky condition in which increases in free radical production, and/or decreases in antioxidant levels can lead to potential damage. The antioxidant system in aerobic organisms includes several biochemical safety mechanisms such as antioxidant enzymes and other compounds like vitamins, glutathione, matallothioneins, and others.
Antioxidant defense enzymes are induced by various environmental pollutants under pro-oxydant conditions among these enzymes we can found superoxide dismutase (SOD), Catalase (Cat) and Glutation Peroxidase (GPx), Glutation reductase and Glutation S Transferase (GST, catalyze the nucleophilic conjugation of different biologically and potentially carcinogenic compounds ). The role of SOD is to catalyze the reaction of superoxide radical (O2-) to peroxide (H2O2). CAT detoxifies H2O2 to H2O and O2. GPx detoxifies mainly organic peroxides. CAT is an enzyme with high biological relevance because reduce the concentration of peroxide, a precursor of OH-, which is a highly reactive toxic form of ROS . Cytochrome P450 monooxygenases (CYPs) are a multi-gene family of enzymes that play a key role in the biotransformation of pollutants, such as dioxins, pesticides, polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs). One of the most common and highly conserved is the CYP1A subfamily. The CYP1A biomarker is widely used as a biomarker of effect both in vertebrates and invertebrates for environmental biomonitoring, especially in marine bivalves and fish. The induction of CYP1A is triggered via the cytosolic aryl hydrocarbon (Ah) receptor due to exposure to pollutants, such as PCBs, dioxins, and numerous PAHs. CYP1A activity is typically measured using the substrate ethoxyresorufin, which is o-deethylated by ethoxyresorufin-O-deethylase (EROD) to a fluorescent product, resorufin, which can be easily measured. Because EROD activities are generally measured using liver homogenates that also tend to accumulate numerous CYP1A substrates, activity may be inhibited by residual substrates or metals .
3. Individual responses
Morphological alterations is one of the most individual level parameters that are measure to identified damage in sentinel organisms [27, 28]. In reference  identified severe histological damage in gills and liver of
Also, haematological parameters, such as erythrocyte and leucocyte count, erythrocyte indices and thrombocyte number vis-a-vis coagulation of blood has been considered bioindicators of toxicosis in fish following exposure to xenobiotics . Any alteration in normal cellular components (morphology and number), and level of fluids of blood is named dyscrasia. Some authors  carry out an extensive review over dyscrasia in fish. They show several examples over alterations in morphological changes in blood cells, total count, haemoglobin content, thrombocytes and clotting time, concluding that haematological parameters are not specific to faced acute and chronic exposure of fish to xenobiotics, mainly organochlorinated, organophosphonate, pyrethroid and carbamate pesticides.
4. Population responses
The status of a fish population is a reflection of the overall condition of the aquatic environment in which that population resides. As such, fish population characteristics can be used as indicators of environmental health. Although changes in population structure may act as a sensitive indicator of changing environmental conditions, the timing, degree and nature of the feedback response to altered conditions will vary with the intensity, identity and the number of stressors, as well as the availability of energy .
Bioindicators are responses to environmental effects that occur at higher levels of biological organization than sub-organism (biomarkers); This kind of responses can be measured at different high levels of biological organization, from individual, through population (reproductive success, mortality, size distribution, reduction in abundance and biomass), community (primary production, disruption of the nutrient cycle) to ecosystem levels [31, 32], whose main characteristic is that the measure change with exposure to negative environmental factors. Organosomatic indices are common approaches for assessing fish health . In this review we consider three organosomatic indices: Condition factor (CF), hepatosomatic index (HSI) and gonadosomatic index (GSI).
Since the nutritional status of fish can change according to different factors, such as season, food availability, and among they the exposure to xenobiotics, is important to have a measure of corporal condition of fish. The chronic exposure to contaminants may cause changes in feeding behavior, leading to a deterioration of the health. The CF is a frequently used index for fish biology study, as it furnishes important information related to fish physiological state . It is a measure of corporal condition since measuring the body mass associated with the body length. A fish that is heavier for a given length (higher CF) is considered to be a healthier fish, because extra weight means extra energy reserves. While a lighter fish lack energy reserves and therefore, tend to be more susceptible to environmental stressors. A low body condition may also suggest muscle wasting (proteolysis) indicating a starvation response . It has also been suggested that females with a lower body condition reduce reproductive investment yet still have an increased risk of mortality.
The liver plays a major role in the metabolism of xenobiotic compounds with biochemical alterations occurring under some toxic conditions; likewise, the liver is a primary detoxification organ in fish . Therefore, this strong activity can lead to an increase in liver size, from hypertrophy (an increase in size) to hyperplasia (an increase in number) of hepatocytes , or both. Studies evaluating the relative liver size of fishes from contaminated and reference sites often utilize the Hepatosomatic Index (HIS), which expresses the ratio of liver weight to body weight as a percentage.
Gonadosomatic index (GSI) is also a percentage relationship between the gonad weight and fish weight. Depending on the severity of exposure to xenobiotics, the sublethal effects can be to limit physiological capacity, reduce growth, and impair reproduction, therefore GSI is a convenient organosomaitc index .
5. Specific case studies about fish as sentinel organisms
In México some studies have analyzed the use of freshwater fish as sentinels. These studies include the use of biomarkers and sentinel fish and the use of the whole fish population as indicators of environmental change. Furthermore, these studies are in areas of contrasting environmental conditions, in the case of biomarkers studies are in: the course of a river in the Atlantic slope (Río Champotón), a Lake in the Central Plateau (Yuriria Lake), a spring and a reservoir in the upper portion of a river of the Pacific slope (Ameca river). These studies make evident the utility of freshwater fish as sentinels and are briefly exposed.
5.1. Case of study of
Astyanax aeneusin the Champotón river
The Champotón river, located in the humid subtropics of southeastern Mexico in terrain with a high content of karstic material, is the main surface stream in the Yucatán Peninsula; is a coastal river with 48 km in length to its outlet with a drainage basin surface area of 650 km2. The fish studied inhabit the fresh water zone of the river with salinity up to 1.2 practical salinity units . The climatic regime is hot subhumid with summer rain (June to September) and occasional winter precipitation as a result of the windy (northerly) and hurricane seasons. The main anthropogenic activity in the basin is agriculture and livestock raising . During the study period (2007-2008) the region was affected by several hurricanes (mostly from August to October), that caused the river overflow. This study assessed the effects of the environmental conditions along the freshwater portion of the Champotón river on the native fish
The WQI scores exhibited spatial and temporal variations (from 53.21 to 78.49) on a scale of 0 to 100. The lowest value was recorded in July at site SAR, where the lack of sanitary facilities provoke fecal materials are swept away by runoff during the rainy season (July), increasing coliform numbers and lowering the WQI. In addition, this river flows through a region in which calcareous substrates predominate; with a high content of calcium carbonate and increase in conductivity both provoked decreases WQI scores, particularly during the drought. However, WQI scores were higher in November and February, following the hurricane season that brought large amounts of precipitation and increased river flow favoring dilution. As a result, the values of several WQI parameters (including hardness and conductivity) decreased while the river was in flood. Several studies reported similar WQI fluctuation patterns. WQI scores in the Lerma-Chapala Basin, Mexico, indicated severe degradation of the basin, particularly during the dry season, when its rating ranged from contaminated to highly contaminated; however, WQI improved during the wet season, [39, 40].
Although the WQI scores indicated that the Champotón river had acceptable water quality, some pollutants (residues of persistent organic compounds (POCs), such as PCBs, hexachlorocyclohexanes, aldrin-related pesticides, heptachlor, and dichlorodiphenyltrichloroethane) have been detected in the Champotón river . In reference  reported that sediments from several Champotón river sites contained two or more of the 16 PAHs considered by the Environmental Protection Agency United States as priority pollutants that represent a potential threat to exposed organisms. Seasonal variations in POCs were found by ; PCBs and hexachlorocyclohexanes reached their highest values during the rainy season, while dichlorodiphenyltrichloroethane, drines, and heptachlor peaked during the dry season. Additionally, high episodic loadings of contaminants have been detected in aquatic ecosystems following flooding events .
Regarding biomarkers in the sentinel fish
The increased LPO values in the post-hurricane season (November) and in the windy season (February) was associated with the hurricane season in the Champotón river which provokes flooding of adjacent areas where field crops are treated with agrochemicals that, along with the POCs and PAHs detected in sediments by , may be incorporated into the aquatic system. Similar results were found by  that detected the mobilization of agriculture-related xenobiotics, during the flooding of the river Elbe. Oxidative stress in fish after extensive flooding was also detected by  in the Pamlico Sound estuary. The observed increase in LPO levels in
Produced ROS are detoxified by antioxidant defense mechanisms, which are essential for protection of cellular systems against xenobiotic-induced oxidative stress . GST is particularly interesting, since it is involved in elimination of reactive compounds and is the transport system for glutathione . GST activity in
LDHs are cytoplasmic enzymes that catalyze the reversible reduction of pyruvate to lactate , an important step in the energy processes of many animal groups. Its use as a biomarker is based on the assumption that organisms subjected to chemical stress must obtain additional energy rapidly, thus increasing anaerobic glycolysis. The LDH response is apparently time-dependent and may vary with the pollutant and organism involved . LDH in
The IBR enables evaluation of the global variations of biomarkers, taking into account the contributions and variations in the biomarkers assessed . IBR data revealed seasonal fluctuations, the maximum total IBR values occurred in April and November (April, 15.73; November, 14.73) and the minimum values in February (2.53). Response in April and November suggested that
Regarding somatic indices, GSI displayed the reproductive period of
Despite WQI scores suggest that the Champotón river water is not highly polluted, the set of
5.2. Study case of
Chirostoma jordaniin Yurira Lake
This study shows an assessment of water quality by means of a WQI and a battery of oxidative stress: LPO, and the activity of antioxidant enzymes, SOD, CAT, and GPx along with somatic indices, such as the GSI, HSI, and CF were analyzed to assess the health condition of
Yuriria Lake is characterized by a high deterioration in their water quality, WQI values ranged from 55 to 70, with a global mean of 65.85. The lake has spatial differences in water quality, the limnetic zone has higher scores (63 to 70), and the tributaries have the lower quality (55 to 58). Yuriria Lake being located in the Central Plateau, one of the most highly populated areas in México, displays the general problem of water quality of the basin (the Lerma-Chapala basin), where urban and industrial wastewater discharges, and leachates of agrochemicals are the main pollutants that diminish the water quality . Particularly the middle Lerma (where Yuriria Lake is located) is recognized as the most affected area, with WQI scores between 41.1 and 54.2 in 1999 [40, 61, 62]. Furthermore, previous studies have recognized the entry of pollutants in Yuriria Lake ; however, the effect of the mixtures of these pollutants on the aquatic biota inhabiting the lake had not been analyzed.
The biomarker assessment suggests that the lake conditions exert stress on the fish
The activity of the antioxidant enzymes in liver and gills also showed a marked seasonal variation. SOD and GPx significantly increased during November, mainly in the gills, compared to the rest of the seasons. CAT also showed higher values in activity during November; however, its highest value was found in gills during February. In general, the activity of antioxidant enzymes decreased from November to May 2010.
Exposure to various xenobiotics, such as metals and organic compounds that enter water bodies, can promote the formation of ROS and induce oxidative stress . The increase in the level of LPO in liver observed in November 2009 and February and May 2010, suggests the existence of pro-oxidant agents in Yuriria Lake and indicates increased oxidative stress in these seasons. Seasonal variation in LPO values could be related to the rainy and the dry seasons; the rainy season can promote dilution of xenobiotics that induce less stress during this season. Rainfall also increases leaching and runoff that enhance the entry of xenobiotics (chemicals), in this study results show that the damage generated in the fish liver became evident from November (the end of the rainy season) until February and May 2010 (dry season), when the processes of evaporation and consequent concentration of xenobiotics could be higher. Furthermore, in May 2009, there were higher levels of LPO in gills, which may indicate that water in the lake at the beginning of the rainy season provoke oxidative stress in gills.
An increase in LPO levels in fish can trigger an antioxidant response as a defense mechanism to prevent cell damage caused by pro-oxidant agents [19, 64] and could be expressed as increased or depleted CAT, SOD, and GPx activities ; in both cases, the result is damage to the antioxidant system. In
At the population level the assessment can reveal changes in the fish biology and ecology resulting from natural fluctuations and/or ecosystem changes caused by environmental degradation. The standard length of
The somatic indices revealed that K displayed small variation between sites and between periods. HSI showed significant differences between sites in August, and between periods in May, values were significantly lower than those of other periods. The GSI showed the greatest variation, with a clear reproductive peak during May 2010. There was a positive correlation between the GSI and HSI. By size class, only the GSI showed variations between seasons. Class I was significantly lower than the rest of the classes. Classes II and III the GSI showed a reproductive peak in May 2010.
In fish, the cost of reproduction may be considerable; thus, fish can express different patterns of energy storage and depletion in relation to reproductive cycles, with an alternation in energy storage (56). The comparison of K and the HSI with the GSI could therefore be useful for estimating the possible balances or energy transfer between the reproductive period and nutritional status .
According to , fish living in waters contaminated with domestic sewage exhibit higher K and GSI, these authors suggest it is because these sites have more available food for fish, enabling them to compensate for the environmental impairments. This scenario is likely occurring in Lake Yuriria, where
According to the results critical periods in the health of
The findings suggest that
5.3. Study case of
Ameca splendensand Goodea atripinnisin Ameca river
The Ameca River is located in the western slope of Mexico which drains to the Pacific Ocean. It is a river characterized by their great fish biodiversity, however, the upper portion of Ameca River, is affected by several environmental disturbers: the construction of a reservoir, the inputs of wastewater from a sugar-processing facility and water extraction, which have resulted in a drastic reduction in fish biodiversity [74, 75]. In addition, some endemic fish species such as
The authors of this paper  analyze biomarkers and bioindicators of two viviparous fish species,
Data of WQI scores showed spatial and temporal variations, the spring ER achieved the highest scores in all months over the course of the study and the highest values occurred in March in both sites.
Regarding biomarkers highest values of LPO were found in September (rainy season); the highest values were detected for female livers and gills of
Results of γ-GTP showed less marked seasonal differences than LPO. In this study, in most comparisons between sites, γ-GTP activity was slightly higher at the spring; the inhibitory effect of this activity at LV could indicate a diminution in the amount of membrane proteins caused by LPO . On the other hand, an increase of γ-GTP activity towards March in LV could be to prevent increases in LPO ; this increase coincides with the rainy season. Several authors have found seasonal variations in the response of this enzyme as a result of exposition to alkylphenols, the final degradation products of pesticides, detergents and other formulated products .
There was a seasonal variation in AchE activity. Organophosphates and carbamates, as well as PAHs, have been widely recognized for causing AchE inhibition, through their reaction with the serine at active site of the enzyme . In nervous tissue AchE is responsible for the breakdown of acetylcholine (Ach) during transmission of an impulse; if the enzyme is inhibited, Ach is accumulated and thus a prolonged transmission of impulses could result in tetani and often in respiratory failure and death. In September in
Regarding EROD, the hydrocarbons discharged by the sugar-processing facility and other effluents into LV from December to June, could be responsible for activating EROD detoxification mechanism in fishes living there . This mechanism is considered as the main measure of the CYP1A activity, which in turn constitutes a part of the enzyme complex of the Mixed-Function Oxidase (MFO). Since MFO facilitates the excretion of aromatic contaminants from the body induction of this complex is an effective biomarker of exposure [81, 82]; there are several studies reporting elevated levels of MFO activity in liver fishes as a result of exposure to organic contaminants, such as PAHs, dioxins, PCBs and agricultural and urban wastewater . Also, there are other factors, such as UV radiation, that causes increase in MFO activity , moreover, damaged livers, like those of LV organisms, are less capable to MFO induction .
Responses to environmental stress also were reflected in bioindicators in both species studied. The major HSI values in LV concurs with  who reported major HSI values, related with higher EROD induction, at contaminated sites in comparison with a reference site. Moreover, in our study, higher LSI in LV concurs with higher IBR values. High values of HSI could have resulted from exposition to hydrocarbons which cause hypertrophia in liver .
Species may differ in the nature of their physiological response and reproductive consequences to stressors . Tolerant species to environmental stress, like
The higher SL, weight and CF values in LV are in concordance with , that found higher CF and GSI in fishes living in waters polluted with untreated domestic sewage; they suggested that fishes in these sites could find abundant food availability, and they are able to compensate for environmental changes caused by untreated domestic sewage discharges. Fishes could have major GSI values, higher fecundity and lower maturity age even under conditions of high pH ; in the present study,
Throughout this study, water quality was higher in ER than in LV due to human activities; but in both sites there were different spatial and temporal factors that produced stress on fishes living there. Therefore, fishes had responses at biomarker and population levels of biological organization. Every biomarker and IBR in this study showed seasonal variation and they were useful environmental tools to demonstrate that, as consequence of pollution, LV is a more stressing place to organisms living them in comparison with ER. In general, organisms in LV presented oxidative stress by the LPO levels, and then neurotoxic impacts by the AchE and some detoxification mechanisms were evident by the γ-GTP and EROD activities. Bioindicators showed evidences of physiological changes due to contaminants exposure and make evident the plasticity of the organisms to survive in this site, in turn the responses should be considered as tactics to survive under stress condition. Moreover, both biomarkers and bioindicators revealed that
6. Holistic approach
Environmental stressors can cause several and different damages over aquatic organisms. These damages could be from molecular to population levels, likewise community and ecosystem levels. Through the biomarkers such as defined in this document (any biological measurable response from an organism, induced by the exposure to a xenobiotic or complex mixture of them) we can determine only some of possible causal relationships. Therefore, it is necessary always, measure a set of biomarkers to identify different stressors or damage on sentinel organisms. Several indices have been proposed to try to integrate the multi responses of different biomarkers in a single number that is indicative of the severity of the damage or stress. Such is the case of the IBR proposed by . In this index, the biomarker data must first be normalized and standardized; then the score is represented by the area of a star plot. IBR considers the responses of activation or inhibition of the biomarkers assessed. IBR is an exploratory tool and should be appropriate only if an a priori justification exists for each biomarker used and if the physiological significance of the changes to each biomarker is well known .
An other case of index based on a battery of biomarkers was proposed by . This biomarker index was obtained by summing the biomarker values expressed in term of classes. Classes were determined by a distribution-free approach derived from the theory of rough sets. No synergistic or antagonistic assumptions were incorporated into this index.
In  the authors proposed a quantitative health assessment index for rapid evaluation of fish condition in the field named Health Assessment Index (HAI). This index is not based on a battery of biomarkers; however, it is a quantitative index that allows statistical comparisons of fish health among data sets. Index variables are assigned numerical values based on the degree of severity or damage incurred by an organ or tissue from environmental stressors.
The Bioeffect Assessment Index (BAI), is based on the integration of several pathological endpoints measured in the liver of fish . The BAI represents a modification of the HAI since it includes solely validated biomarkers reflecting toxically induced alterations at different levels of biological organisation in order to quantify the effects of environmental pollution. BAI is able to reflect deleterious effects of several classes of xenobiotics such as heavy metals, organochlorines, pesticides, PAHs, and therefore is also considered as an integrative index of health in aquatic ecosystems.
When we use sentinel organisms, a key point is the study of baseline or natural variation of responses of the sentinel organism selected, or characterizing the response of the same sentinel organism in reference sites.
The use of fish as sentinel organism is feasible for pollution monitoring in aquatic systems; however, the survey should consider the application of a suite of measurable responses (biomarkers and bioindicators) to identify potential sources of stress and damage to which organisms are exposed, as shown in the case studies presented above. The set of biomarkers or bioindicators should also, consider several levels of biological organization in order to identify effects of environmental stressors, spatio temporal trends in environmental conditions and to identify early warning signals to prevent that damage continue from low biological organizing levels to higher levels of organization.