**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 aeneus* **in 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 [37]. 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 [38]. 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 *A. aeneus*, analyzing responses between lower and higher levels of organization, and linking spatial and seasonal fish responses with water quality features. A Water Quality Index (WQI) was em‐ ployed as an indicator of environmental conditions, a set of sub-organismal biomarkers in *A. aeneus* lipid peroxidation (LPO), GST, EROD, and lactate dehydrogenase (LDH)) was moni‐ tored to determine the Integrated biomarker response (IBR), and organosomatic indices: GSI, HSI and CF were characterized. Three study sites were analyzed: San Juan Carpizo (SJC) in the upper portion of the river, San Antonio del Río (SAR) in the middle portion of the river (where there are a rustic swimming spot lacking of sanitary facilities), and downstream Ulumal (U); the study periods were: April, July, and November 2007 and February 2008.

been suggested that females with a lower body condition reduce reproductive investment yet

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 detoxifica‐ tion organ in fish [35]. 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 [35], 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

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

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

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 [37]. 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 [38]. 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 *A. aeneus*, analyzing responses between lower and higher levels of organization, and linking spatial and seasonal fish responses with water quality features. A Water Quality Index (WQI) was em‐ ployed as an indicator of environmental conditions, a set of sub-organismal biomarkers in *A. aeneus* lipid peroxidation (LPO), GST, EROD, and lactate dehydrogenase (LDH)) was moni‐ tored to determine the Integrated biomarker response (IBR), and organosomatic indices: GSI, HSI and CF were characterized. Three study sites were analyzed: San Juan Carpizo (SJC) in

**5. Specific case studies about fish as sentinel organisms**

evident the utility of freshwater fish as sentinels and are briefly exposed.

**5.1. Case of study of** *Astyanax aeneus* **in the Champotón river**

still have an increased risk of mortality.

156 New Advances and Contributions to Fish Biology

body weight as a percentage.

convenient organosomaitc index [36].

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 in‐ crease 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, partic‐ ularly during the dry season, when its rating ranged from contaminated to highly contami‐ nated; 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, hexachlor‐ ocyclohexanes, aldrin-related pesticides, heptachlor, and dichlorodiphenyltrichloroethane) have been detected in the Champotón river [41]. In reference [42] reported that sediments from several Champotón river sites contained two or more of the 16 PAHs considered by the Envi‐ ronmental Protection Agency United States as priority pollutants that represent a potential threat to exposed organisms. Seasonal variations in POCs were found by [41]; PCBs and hex‐ achlorocyclohexanes reached their highest values during the rainy season, while dichlorodi‐ phenyltrichloroethane, drines, and heptachlor peaked during the dry season. Additionally, high episodic loadings of contaminants have been detected in aquatic ecosystems following flooding events [43].

Regarding biomarkers in the sentinel fish *A. aeneus*, the highest LPO values were detected in November and February (post-hurricane and windy seasons), while the lowest values were detected in July at all study sites. GST activity was highest in November at site U and lowest in July and April at all study sites. EROD activity in general was highest in April, while the lowest means occurred in July at all sites. Spatial analysis revealed that SAR recorded the highest mean EROD activity, while the lowest occurred at sites SJC and U. LDH values peaked in November and February following the hurricane and windy seasons, while minimum LDH levels occurred in April during the dry season. Spatial variability occurred; sites in the middle reaches (U, SAR) had the highest LDH values, while the lowest were recorded at headwater site SJC [44].

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 [41], may be incorporated into the aquatic sys‐ tem. Similar results were found by [45] that detected the mobilization of agriculture-related xenobiotics, during the flooding of the river Elbe. Oxidative stress in fish after extensive flood‐ ing was also detected by [43] in the Pamlico Sound estuary. The observed increase in LPO levels in *A. aeneus* may be related to climate-induced stress and by exposure to the mixture of xenobiotics that may be mobilized during that period.

unable to regain homeostasis, the responses may become maladaptive and may pose a risk to

Freshwater Fish as Sentinel Organisms: From the Molecular to the Population Level, a Review

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159

Regarding somatic indices, GSI displayed the reproductive period of *A. aeneus* occurred from April (end of the dry season) to July (early rainy season), with the reproductive peak in July. HSI was highest in *A. aeneus* during periods of reproductive inactivity, which is interpreted as an increase in liver-stored reserve materials for later use during gametogenesis. Furthermore, low HSI values prior to and during reproduction may result from the transfer of liver energy reserves toward gonadal maturation and the reproductive event, with consequent depletion of these reserves and a decrease in HSI values [56]. CF remained constant among sites and study periods, with maximum values during July. CF reflects the interactions between abiotic and biotic factors in the physiological conditions of the fish [57]. Observations demonstrate that *A. aeneus* maintains a stable, robust condition. CF and HSI trends may reflect physiological conditions in *A. aeneus* that enable oocyte maturation and release, suggesting that oocyte pro‐ duction relies on energy stored in the liver and not on energy stored in the musculature [58, 59]. GSI, HSI, and CF revealed that the reproductive success of the sentinel species had not been affected, since the GSI values concurred with those reported for other species in the genus *Astyanax*. HSI values showed the transfer of energy during the reproductive period, and CF remained stable throughout the study, evidencing no effects on the general condition of the fish during periods of higher stress and indicating that the stress was temporal and the fish

Despite WQI scores suggest that the Champotón river water is not highly polluted, the set of *A. aeneus* biomarkers constitutes a more sensitive and effective tool for identifying periods of environmental conditions adverse to fish health. Markers of oxidative damage (LPO), energy processes (LDH), detoxification (EROD), and antioxidant activity (GST) suggested that two stress periods affected the health condition of *A. aeneus* in different ways. These biomarkers can be used as early warning signals of environmental change prior to the onset of irreversible damage at the population level. Indeed, the IBR values highlighted the two periods of high biomarker response. Overall, this study provides evidence supporting the use of a biomarker set in assessing the health of aquatic systems, corroborating the suitability of *Astyanas aeneus*

*Chirostoma jordani* is an Atherinopsid fish endemic of Central México. Data presented are from a population living in Yuriria Lake, one of the most important lakes in México, located in the Central Plateau (hydrologic region Lerma-Chapala-Santiago 20 ° 20'24'' -19 ° 04'48'' N and 101

depth of 3.2 m) feed by a diversion of Lerma river (their main tributary), that carries wastes from mining activities, livestock, industrial, urban and rural areas [60]. In the western end the lake receives water from two small an intermittent tributaries. Yuriria Lake supplies water for the surrounding farming areas, harbors migratory bird populations, and supports fisheries

and maximum

° 55'48'' -100 ° 48 '36'' W). It is an artificial, small and shallow lake (area= 66 km2

the health and wellbeing of the fish [54].

were able to compensate for it.

as a sentinel species.

**5.2. Study case of** *Chirostoma jordani* **in Yurira Lake**

and tourism of several human settlements on the littoral zone.

Produced ROS are detoxified by antioxidant defense mechanisms, which are essential for pro‐ tection of cellular systems against xenobiotic-induced oxidative stress [46]. GST is particularly interesting, since it is involved in elimination of reactive compounds and is the transport sys‐ tem for glutathione [22]. GST activity in *A. aeneus* peaked during the same period that LPO increased, following the hurricane season, which may reflect a defense of the fish against ox‐ idative stress, as reported by [47] in *Prochilodus lineatus* in Argentina and by [48] in three subspecies of *Salmo truta* in Turkey. The liver usually contains high levels of antioxidant enzyme activity, which may be due to high rates of free radical generation in this tissue [49].

*A. aeneus* exhibited seasonal fluctuations in EROD activity, peaking in April at the SAR site. Several authors [43, 50] point out that POCs (including PAHs) typically cause elevated levels of EROD activity, and fossil fuel spills and infiltrations are a major source of PAH input [51]. In *A. aeneus* EROD activity was highest at the SAR site, a recreational spot reached only by unpaved road where motor vehicles park on the riverbank. EROD induction may thus be due to surface runoff carrying PAHs.

LDHs are cytoplasmic enzymes that catalyze the reversible reduction of pyruvate to lactate [52], 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 timedependent and may vary with the pollutant and organism involved [41]. LDH in *A. aeneus* increased at all sites in November and February, coinciding with the period of maximum LPO as well as the post-hurricane season and highest river flow.

The IBR enables evaluation of the global variations of biomarkers, taking into account the contributions and variations in the biomarkers assessed [17]. IBR data revealed seasonal fluc‐ tuations, the maximum total IBR values occurred in April and November (April, 15.73; No‐ vember, 14.73) and the minimum values in February (2.53). Response in April and November suggested that *A. aeneus* was exposed to greater stress during this period, one at the end of the dry season and the other coinciding with the post-hurricane season that also led to high LPO values (oxidative stress), high GST levels (antioxidant responses), and high levels of LDH (high energetic need). Multiple stressors are involved during flooding, such as altered habitat, changes in hydrological regime, and mobilization of pollutants. Responses of biota to envi‐ ronmental stressors are the integrated result of natural and anthropogenic stressors that can be ultimately manifested in biotic changes at several levels of organization [53]. IBR differences among the study periods may reflect a compensatory mechanism by which the fish regains homeostasis following the period of highest stress. Fish display a large variety of physiological stress responses that manifest as an increase in certain biomarkers after a stressful event [54]. These responses are considered adaptations of the fish to adjust itself to the disturbance and regain homeostasis [55]. Fish have also been found to exhibit recovery responses dependent on stress duration and magnitude. If the stressors are too severe or persistent and the fish is unable to regain homeostasis, the responses may become maladaptive and may pose a risk to the health and wellbeing of the fish [54].

xenobiotics, during the flooding of the river Elbe. Oxidative stress in fish after extensive flood‐ ing was also detected by [43] in the Pamlico Sound estuary. The observed increase in LPO levels in *A. aeneus* may be related to climate-induced stress and by exposure to the mixture of

Produced ROS are detoxified by antioxidant defense mechanisms, which are essential for pro‐ tection of cellular systems against xenobiotic-induced oxidative stress [46]. GST is particularly interesting, since it is involved in elimination of reactive compounds and is the transport sys‐ tem for glutathione [22]. GST activity in *A. aeneus* peaked during the same period that LPO increased, following the hurricane season, which may reflect a defense of the fish against ox‐ idative stress, as reported by [47] in *Prochilodus lineatus* in Argentina and by [48] in three subspecies of *Salmo truta* in Turkey. The liver usually contains high levels of antioxidant enzyme

*A. aeneus* exhibited seasonal fluctuations in EROD activity, peaking in April at the SAR site. Several authors [43, 50] point out that POCs (including PAHs) typically cause elevated levels of EROD activity, and fossil fuel spills and infiltrations are a major source of PAH input [51]. In *A. aeneus* EROD activity was highest at the SAR site, a recreational spot reached only by unpaved road where motor vehicles park on the riverbank. EROD induction may thus be due

LDHs are cytoplasmic enzymes that catalyze the reversible reduction of pyruvate to lactate [52], 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 timedependent and may vary with the pollutant and organism involved [41]. LDH in *A. aeneus* increased at all sites in November and February, coinciding with the period of maximum LPO

The IBR enables evaluation of the global variations of biomarkers, taking into account the contributions and variations in the biomarkers assessed [17]. IBR data revealed seasonal fluc‐ tuations, the maximum total IBR values occurred in April and November (April, 15.73; No‐ vember, 14.73) and the minimum values in February (2.53). Response in April and November suggested that *A. aeneus* was exposed to greater stress during this period, one at the end of the dry season and the other coinciding with the post-hurricane season that also led to high LPO values (oxidative stress), high GST levels (antioxidant responses), and high levels of LDH (high energetic need). Multiple stressors are involved during flooding, such as altered habitat, changes in hydrological regime, and mobilization of pollutants. Responses of biota to envi‐ ronmental stressors are the integrated result of natural and anthropogenic stressors that can be ultimately manifested in biotic changes at several levels of organization [53]. IBR differences among the study periods may reflect a compensatory mechanism by which the fish regains homeostasis following the period of highest stress. Fish display a large variety of physiological stress responses that manifest as an increase in certain biomarkers after a stressful event [54]. These responses are considered adaptations of the fish to adjust itself to the disturbance and regain homeostasis [55]. Fish have also been found to exhibit recovery responses dependent on stress duration and magnitude. If the stressors are too severe or persistent and the fish is

activity, which may be due to high rates of free radical generation in this tissue [49].

xenobiotics that may be mobilized during that period.

as well as the post-hurricane season and highest river flow.

to surface runoff carrying PAHs.

158 New Advances and Contributions to Fish Biology

Regarding somatic indices, GSI displayed the reproductive period of *A. aeneus* occurred from April (end of the dry season) to July (early rainy season), with the reproductive peak in July. HSI was highest in *A. aeneus* during periods of reproductive inactivity, which is interpreted as an increase in liver-stored reserve materials for later use during gametogenesis. Furthermore, low HSI values prior to and during reproduction may result from the transfer of liver energy reserves toward gonadal maturation and the reproductive event, with consequent depletion of these reserves and a decrease in HSI values [56]. CF remained constant among sites and study periods, with maximum values during July. CF reflects the interactions between abiotic and biotic factors in the physiological conditions of the fish [57]. Observations demonstrate that *A. aeneus* maintains a stable, robust condition. CF and HSI trends may reflect physiological conditions in *A. aeneus* that enable oocyte maturation and release, suggesting that oocyte pro‐ duction relies on energy stored in the liver and not on energy stored in the musculature [58, 59]. GSI, HSI, and CF revealed that the reproductive success of the sentinel species had not been affected, since the GSI values concurred with those reported for other species in the genus *Astyanax*. HSI values showed the transfer of energy during the reproductive period, and CF remained stable throughout the study, evidencing no effects on the general condition of the fish during periods of higher stress and indicating that the stress was temporal and the fish were able to compensate for it.

Despite WQI scores suggest that the Champotón river water is not highly polluted, the set of *A. aeneus* biomarkers constitutes a more sensitive and effective tool for identifying periods of environmental conditions adverse to fish health. Markers of oxidative damage (LPO), energy processes (LDH), detoxification (EROD), and antioxidant activity (GST) suggested that two stress periods affected the health condition of *A. aeneus* in different ways. These biomarkers can be used as early warning signals of environmental change prior to the onset of irreversible damage at the population level. Indeed, the IBR values highlighted the two periods of high biomarker response. Overall, this study provides evidence supporting the use of a biomarker set in assessing the health of aquatic systems, corroborating the suitability of *Astyanas aeneus* as a sentinel species.

#### **5.2. Study case of** *Chirostoma jordani* **in Yurira Lake**

*Chirostoma jordani* is an Atherinopsid fish endemic of Central México. Data presented are from a population living in Yuriria Lake, one of the most important lakes in México, located in the Central Plateau (hydrologic region Lerma-Chapala-Santiago 20 ° 20'24'' -19 ° 04'48'' N and 101 ° 55'48'' -100 ° 48 '36'' W). It is an artificial, small and shallow lake (area= 66 km2 and maximum depth of 3.2 m) feed by a diversion of Lerma river (their main tributary), that carries wastes from mining activities, livestock, industrial, urban and rural areas [60]. In the western end the lake receives water from two small an intermittent tributaries. Yuriria Lake supplies water for the surrounding farming areas, harbors migratory bird populations, and supports fisheries and tourism of several human settlements on the littoral zone.

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 *C. jorda‐ ni* in Yuriria Lake. The study was carried on in a period with scarce pluvial precipitation and prolonged drought (May, August, November 2009 and February and May 2010).

thermore, in May 2009, there were higher levels of LPO in gills, which may indicate that water

Freshwater Fish as Sentinel Organisms: From the Molecular to the Population Level, a Review

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161

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 in‐ creased or depleted CAT, SOD, and GPx activities [12]; in both cases, the result is damage to the antioxidant system. In *C. jordani* inhabiting Yuriria Lake, both responses were detected: 1) stimulation of the activity, when LPO levels in liver and muscle were higher (November and February) and antioxidant activity showed the highest values; and 2) depletion in antioxidant activity during May 2009 with high levels of LPO in the gills, and May 2010 with increased LPO in liver and muscle. These highly variable responses of the antioxidant system depend on the type and concentration of contaminant to which fish are exposed, as well as on the intensity and duration of exposure [65]. The clear decrease in the activities of CAT, SOD, and GPx in *C. jordani* is extremely important because it suggests severe damage to the antioxidant system of fish. These damages have been previously documented by [66], who recorded a collapse of the antioxidant defense system of *Liza aurata* in the Ria de Aveiro, Portugal, with exposure to mercury. In addition, [67] reported damage to the antioxidant system of *Oreo‐*

in the lake at the beginning of the rainy season provoke oxidative stress in gills.

*chromis niloticus* from acute and chronic exposure to Cd, Cr, Cu, Zn, and Fe.

quently reduced adult stocks and serious effects on fisheries.

productive peak in May 2010.

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 deg‐ radation. The standard length of *C. jordani* ranges from 21.16 to 77.61 mm. Three size classes were determined: Class I with a mean size of 26 mm, represented only in the month of May 2010; Class II with a mean size of 56 mm; and class III with a mean size of 62 mm. There was a gap in the size frequencies in the class of 32–50 mm from May to August 2009, whose abun‐ dances were not sufficient to form this cohort. The lack of smaller sizes from May to November 2009 can be interpreted as a possible reproductive failure during that year, resulting in low recruitment and consequently precluding estimation of the cohort [68]. These gaps or missing cohorts have been previously documented for other fish as a result of overfishing and/or en‐ vironmental degradation [69]. Several causes could explain this event. According to [70], the hydrological cycle plays an important role in the development of different biological attributes such as gonadal maturation, migration, spawning, larval development, growth, and feeding. Prolonged periods of drought are also associated with failures in the recruitment and subse‐

The somatic indices revealed that K displayed small variation between sites and between pe‐ riods. 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 re‐

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

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 [61]. 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 pol‐ lutants in Yuriria Lake [61]; 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 *C. jorda‐ ni.* The biomarker response showed pronounced seasonal variations. The gills presented high‐ er values of LPO. May 2009 displayed the highest levels and November the lowest. In the liver the higher levels of LPO were detected during November and February and May 2010 (the end of the rainy season and the dry season). In muscle, the highest level of LPO was observed during February and May 2010. Gills being the first organ of contact with water are exposed directly to any xenobiotic in the aquatic environment and their biomarker responses are the result of the exposure to stressors. In addition, toxics can also enter via the intake of water and food and be absorbed and transported by the portal system to the liver before entering the general circulatory system; in consequence, the liver is one of the most sensitive organs to environmental stressors [63].

The activity of the antioxidant enzymes in liver and gills also showed a marked seasonal var‐ iation. 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; how‐ ever, 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 bod‐ ies, can promote the formation of ROS and induce oxidative stress [12]. 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. Fur‐ thermore, 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.

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 *C. jorda‐ ni* in Yuriria Lake. The study was carried on in a period with scarce pluvial precipitation and

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 [61]. 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 pol‐ lutants in Yuriria Lake [61]; however, the effect of the mixtures of these pollutants on the

The biomarker assessment suggests that the lake conditions exert stress on the fish *C. jorda‐ ni.* The biomarker response showed pronounced seasonal variations. The gills presented high‐ er values of LPO. May 2009 displayed the highest levels and November the lowest. In the liver the higher levels of LPO were detected during November and February and May 2010 (the end of the rainy season and the dry season). In muscle, the highest level of LPO was observed during February and May 2010. Gills being the first organ of contact with water are exposed directly to any xenobiotic in the aquatic environment and their biomarker responses are the result of the exposure to stressors. In addition, toxics can also enter via the intake of water and food and be absorbed and transported by the portal system to the liver before entering the general circulatory system; in consequence, the liver is one of the most sensitive organs to

The activity of the antioxidant enzymes in liver and gills also showed a marked seasonal var‐ iation. 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; how‐ ever, its highest value was found in gills during February. In general, the activity of antioxidant

Exposure to various xenobiotics, such as metals and organic compounds that enter water bod‐ ies, can promote the formation of ROS and induce oxidative stress [12]. 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. Fur‐

prolonged drought (May, August, November 2009 and February and May 2010).

aquatic biota inhabiting the lake had not been analyzed.

environmental stressors [63].

160 New Advances and Contributions to Fish Biology

enzymes decreased from November to May 2010.

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 in‐ creased or depleted CAT, SOD, and GPx activities [12]; in both cases, the result is damage to the antioxidant system. In *C. jordani* inhabiting Yuriria Lake, both responses were detected: 1) stimulation of the activity, when LPO levels in liver and muscle were higher (November and February) and antioxidant activity showed the highest values; and 2) depletion in antioxidant activity during May 2009 with high levels of LPO in the gills, and May 2010 with increased LPO in liver and muscle. These highly variable responses of the antioxidant system depend on the type and concentration of contaminant to which fish are exposed, as well as on the intensity and duration of exposure [65]. The clear decrease in the activities of CAT, SOD, and GPx in *C. jordani* is extremely important because it suggests severe damage to the antioxidant system of fish. These damages have been previously documented by [66], who recorded a collapse of the antioxidant defense system of *Liza aurata* in the Ria de Aveiro, Portugal, with exposure to mercury. In addition, [67] reported damage to the antioxidant system of *Oreo‐ chromis niloticus* from acute and chronic exposure to Cd, Cr, Cu, Zn, and Fe.

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 deg‐ radation. The standard length of *C. jordani* ranges from 21.16 to 77.61 mm. Three size classes were determined: Class I with a mean size of 26 mm, represented only in the month of May 2010; Class II with a mean size of 56 mm; and class III with a mean size of 62 mm. There was a gap in the size frequencies in the class of 32–50 mm from May to August 2009, whose abun‐ dances were not sufficient to form this cohort. The lack of smaller sizes from May to November 2009 can be interpreted as a possible reproductive failure during that year, resulting in low recruitment and consequently precluding estimation of the cohort [68]. These gaps or missing cohorts have been previously documented for other fish as a result of overfishing and/or en‐ vironmental degradation [69]. Several causes could explain this event. According to [70], the hydrological cycle plays an important role in the development of different biological attributes such as gonadal maturation, migration, spawning, larval development, growth, and feeding. Prolonged periods of drought are also associated with failures in the recruitment and subse‐ quently reduced adult stocks and serious effects on fisheries.

The somatic indices revealed that K displayed small variation between sites and between pe‐ riods. 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 re‐ productive 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 nu‐ tritional status [71]. *C. jordani* in Yuriria Lake show that K was maintained at stable levels. Furthermore, there was no alternation of energy storage between the liver and gonads; GSI and HSI correlated positively, indicating that the reproductive period did not compromise energy reserves or the liver or the soma.

two fish species were assessed, according to the main objectives: a) to assess water quality of a spring (ER reference site) and De LaVega reservoir (LV impacted site) where *G. atripinnis* and *A. splendens* coexist; b) to examine the health of both fish species in the reference and impacted sites by means of a battery of biomarkers; and c) to analyze physiological condition indices and population level assessment by mean of bioindicators (population measurements). A WQI was assessed. The set of biomarkers was composed by: enzyme activities of gammaglutamyl-transpeptidase (γ-GTP), acetylcholinesterase (AChE), EROD and the LPO were de‐

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163

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

Regarding biomarkers highest values of LPO were found in September (rainy season); the highest values were detected for female livers and gills of *A. splendens* in the impacted site LV and for female livers and male gills of *G. atripinnis* in LV. The LPO activity in the upper Ameca River displayed the stress to which organisms are subjected, since in the reservoir LPO showed the highest values, being *A. splendens* the most affected species. Many environmental pollu‐ tants and their metabolites have shown to exert toxic effects associated to oxidative stress, producing free radicals that initiate the LPO and cause damage to membrane proteins [76].

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 [77]. On the other hand, an increase of γ-GTP activity towards March in LV could be to prevent increases in LPO [76]; 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

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 [79]. In nervous tissue AchE is responsible for the breakdown of acetylcholine (Ach) during transmission of an impulse; if the enzyme is inhib‐ ited, Ach is accumulated and thus a prolonged transmission of impulses could result in tetani and often in respiratory failure and death. In September in *A. splendens* living at LV, AchE exhibited lower values related to reference site that could have resulted from fish exposure to diverse pesticides used in the adjacent agricultural lands that run off in the rainy season. In March during the dry season, in *G. atripinnis*, living at LV, AchE also exhibited lower values related to ER, may be the effect of evaporation and consequently the concentration of total

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 detox‐ ification mechanism in fishes living there [80]. This mechanism is considered as the main measure of the CYP1A activity, which in turn constitutes a part of the enzyme complex of

solid dissolved at LV, including pesticides and other xenobiotics.

termined. Additionally, somatic index were analyzed: CF, HSI and GSI.

both sites.

products [78].

According to [72], 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, en‐ abling them to compensate for the environmental impairments. This scenario is likely occur‐ ring in Lake Yuriria, where *C. jordani*, having enough food, can complete reproductive cycles and reach larger sizes despite the presence of stressors in the lake.

According to the results critical periods in the health of *C. jordani* occur from November, Feb‐ ruary to May 2010 (end of the rainy season and the dry season) because in this period, the higher LPO, the lowest antioxidant response, and the lowest K were observed in contrast to the higher WQI scores detected in the same period. This result indicates that fish health as‐ sessed by biological indicators as oxidative stress biomarkers and the lack of a cohort, are highly sensitive to environmental conditions imposed by the dry season. The drought has been recognized as one of the critical periods in fish health because during this period, the dilution capacity of aquatic ecosystems is low, which increases the risk of exposure to high concentra‐ tions of pollutants [73].

The findings suggest that *C. jordani* faces oxidative stress resulting from the presence of prooxidant agents in Yuriria Lake. At the population level, *C. jordani* has adapted to the conditions in Yuriria Lake, with mean values of HSI and GSI greater than those in other sites and K values stable throughout the year. Changes were observed in recruitment and reproductive success associated with low water levels in the lake in 2009 which shows that the fish population is highly dependent on water levels in the lake and climate changes. Previous studies indicate that Yuriria Lake receives various xenobiotics with levels that vary both spatially and tempo‐ rally. Biomarkers of oxidative stress, somatic indices, and monitoring of the size classes in the fish *C. jordani* are appropriate indicators of Yuriria Lake conditions.

#### **5.3. Study case of** *Ameca splendens* **and** *Goodea atripinnis* **in 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 in‐ puts 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 *Ameca splendens*, have suffered a reduction in their range, and have become more prone to extinction (NOM-059, 2002) than those with a broad distribution (like *Goodea atripinnis*).

The authors of this paper [32] analyze biomarkers and bioindicators of two viviparous fish species, *A. splendens* and *G. atripinnis* living in a reservoir of the upper portion of the Ameca River, which receives wastewater, and in a spring of the same river that is free from such polluting water. In this study a comparison of the biomarker responses and bioindicators in two fish species were assessed, according to the main objectives: a) to assess water quality of a spring (ER reference site) and De LaVega reservoir (LV impacted site) where *G. atripinnis* and *A. splendens* coexist; b) to examine the health of both fish species in the reference and impacted sites by means of a battery of biomarkers; and c) to analyze physiological condition indices and population level assessment by mean of bioindicators (population measurements). A WQI was assessed. The set of biomarkers was composed by: enzyme activities of gammaglutamyl-transpeptidase (γ-GTP), acetylcholinesterase (AChE), EROD and the LPO were de‐ termined. Additionally, somatic index were analyzed: CF, HSI and GSI.

estimating the possible balances or energy transfer between the reproductive period and nu‐ tritional status [71]. *C. jordani* in Yuriria Lake show that K was maintained at stable levels. Furthermore, there was no alternation of energy storage between the liver and gonads; GSI and HSI correlated positively, indicating that the reproductive period did not compromise

According to [72], 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, en‐ abling them to compensate for the environmental impairments. This scenario is likely occur‐ ring in Lake Yuriria, where *C. jordani*, having enough food, can complete reproductive cycles

According to the results critical periods in the health of *C. jordani* occur from November, Feb‐ ruary to May 2010 (end of the rainy season and the dry season) because in this period, the higher LPO, the lowest antioxidant response, and the lowest K were observed in contrast to the higher WQI scores detected in the same period. This result indicates that fish health as‐ sessed by biological indicators as oxidative stress biomarkers and the lack of a cohort, are highly sensitive to environmental conditions imposed by the dry season. The drought has been recognized as one of the critical periods in fish health because during this period, the dilution capacity of aquatic ecosystems is low, which increases the risk of exposure to high concentra‐

The findings suggest that *C. jordani* faces oxidative stress resulting from the presence of prooxidant agents in Yuriria Lake. At the population level, *C. jordani* has adapted to the conditions in Yuriria Lake, with mean values of HSI and GSI greater than those in other sites and K values stable throughout the year. Changes were observed in recruitment and reproductive success associated with low water levels in the lake in 2009 which shows that the fish population is highly dependent on water levels in the lake and climate changes. Previous studies indicate that Yuriria Lake receives various xenobiotics with levels that vary both spatially and tempo‐ rally. Biomarkers of oxidative stress, somatic indices, and monitoring of the size classes in the

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 in‐ puts 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 *Ameca splendens*, have suffered a reduction in their range, and have become more prone to extinction (NOM-059, 2002) than those with a broad distribution (like *Goodea atripinnis*).

The authors of this paper [32] analyze biomarkers and bioindicators of two viviparous fish species, *A. splendens* and *G. atripinnis* living in a reservoir of the upper portion of the Ameca River, which receives wastewater, and in a spring of the same river that is free from such polluting water. In this study a comparison of the biomarker responses and bioindicators in

and reach larger sizes despite the presence of stressors in the lake.

fish *C. jordani* are appropriate indicators of Yuriria Lake conditions.

**5.3. Study case of** *Ameca splendens* **and** *Goodea atripinnis* **in Ameca river**

energy reserves or the liver or the soma.

162 New Advances and Contributions to Fish Biology

tions of pollutants [73].

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 *A. splendens* in the impacted site LV and for female livers and male gills of *G. atripinnis* in LV. The LPO activity in the upper Ameca River displayed the stress to which organisms are subjected, since in the reservoir LPO showed the highest values, being *A. splendens* the most affected species. Many environmental pollu‐ tants and their metabolites have shown to exert toxic effects associated to oxidative stress, producing free radicals that initiate the LPO and cause damage to membrane proteins [76].

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 [77]. On the other hand, an increase of γ-GTP activity towards March in LV could be to prevent increases in LPO [76]; 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 [78].

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 [79]. In nervous tissue AchE is responsible for the breakdown of acetylcholine (Ach) during transmission of an impulse; if the enzyme is inhib‐ ited, Ach is accumulated and thus a prolonged transmission of impulses could result in tetani and often in respiratory failure and death. In September in *A. splendens* living at LV, AchE exhibited lower values related to reference site that could have resulted from fish exposure to diverse pesticides used in the adjacent agricultural lands that run off in the rainy season. In March during the dry season, in *G. atripinnis*, living at LV, AchE also exhibited lower values related to ER, may be the effect of evaporation and consequently the concentration of total solid dissolved at LV, including pesticides and other xenobiotics.

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 detox‐ ification mechanism in fishes living there [80]. 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 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‐ isms, are less capable to MFO induction [85].

indicators revealed that *A. splendens* is a less tolerant species than *G. atripinnis* to environmental stress. Differences in biological response could be attributed to different physiological status of each fish species during the wet and dry season as well as to differences in the type and quantity of the xenobiotics that input at LV due to the period of maximum and minimal activity of the sugar industry; the lixiviation of the agrochemicals from the adjacent lands to the water bodies and also to the complexity of the mixtures of pollutants that are conform at LV that

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165

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].

synergistic or antagonistic assumptions were incorporated into this index.

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

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

provoke several biological responses.

**6. Holistic approach**

in aquatic ecosystems.

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

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 receptor levels [12].

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

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‐ indicators revealed that *A. splendens* is a less tolerant species than *G. atripinnis* to environmental stress. Differences in biological response could be attributed to different physiological status of each fish species during the wet and dry season as well as to differences in the type and quantity of the xenobiotics that input at LV due to the period of maximum and minimal activity of the sugar industry; the lixiviation of the agrochemicals from the adjacent lands to the water bodies and also to the complexity of the mixtures of pollutants that are conform at LV that provoke several biological responses.
