**6. Holistic approach**

the Mixed-Function Oxidase (MFO). Since MFO facilitates the excretion of aromatic con‐ taminants 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 agricul‐ tural and urban wastewater [83]. Also, there are other factors, such as UV radiation, that causes increase in MFO activity [84], moreover, damaged livers, like those of LV organ‐

Responses to environmental stress also were reflected in bioindicators in both species studied. The major HSI values in LV concurs with [86] 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

Species may differ in the nature of their physiological response and reproductive consequences to stressors [87]. Tolerant species to environmental stress, like *G. atripinnis*, are more abundant in more disturbed environments, like LV [74, 88]; on the other hand, *A. splendens* is more abundant in a more stable environment, like ER. In LV females/male ratio for *A. splendens* could be affected by environmental estrogens, like pesticides [89]; these could act by merging re‐ ceptor binding properties of estradiol, alteration of estradiol/testosterone ratios or estrogen

The higher SL, weight and CF values in LV are in concordance with [71], 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 [89]; in the present study, *A splendens* in LV presented these features; only in *G. atripinnis* organisms GSI was higher in the ER. [90] found a decrease in GSI as a result of the exposition of chubs to effluents carrying out organic pollutants and metals. [71] revealed a negative re‐ lation between CF and HSI with GSI, but this relation was observed only in *G. atripinnis*. The larger size, higher growth, longevity and reproductive success of organisms living at LV sug‐ gest a tactic to compensate for the stress to which the populations of both species studied are

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. Bioindica‐ tors showed evidences of physiological changes due to contaminants exposure and make evi‐ dent 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 bio‐

resulted from exposition to hydrocarbons which cause hypertrophia in liver [86].

subjected; however, offspring is smaller and has a lower weight.

isms, are less capable to MFO induction [85].

164 New Advances and Contributions to Fish Biology

receptor levels [12].

Environmental stressors can cause several and different damages over aquatic organisms. These damages could be from molecular to population levels, likewise community and eco‐ system levels. Through the biomarkers such as defined in this document (any biological meas‐ urable 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 or‐ ganisms. 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 [17]. In this index, the biomarker data must first be normal‐ ized 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 [17].

An other case of index based on a battery of biomarkers was proposed by [91]. 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 [4] 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 end‐ points measured in the liver of fish [21]. 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, orga‐ nochlorines, 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.

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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 (bio‐ markers and bioindicators) to identify potential sources of stress and damage to which organ‐ isms 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.
