**4. Assessment of toxicity in fish by biomarkers**

The biological response of an organism to xenobiotics following absorption and distribution starts with toxicant induced changes at the cellular and biochemical levels, leading to changes in the structure and function of the cells, tissues, physiology and behavior of the organism. These changes can perhaps ultimately affect the integrity of the population and ecosystem. For the biomonitoring and management of the aquatic ecosystems, these biologi‐ cal responses (biomarkers) have been proposed to complement and enhance the reliability of the chemical analysis data (Parvéz; Raisuddin, 2005).

There are very few pollutants that have been confirmed to cause adverse effects. In most cases, casual relationships have not been established to a large group of persistent pollu‐ tants, due the complex chemical contamination of environmental compartments, which dif‐ ficulty to attribute harmful effects to any particular pollutant or category of pollutants.

Several definitions have been given for the term 'biomarker', which is generally used in a broad sense to include almost any measurement reflecting an interaction between a biologi‐ cal system and a potential hazard, which may be chemical, physical or biological (WHO, 1993). A biomarker is defined as a change in a biological response (ranging from molecular through cellular and physiological responses to behavioral changes) that can be related to exposure to or toxic effects of environmental chemicals (Van Der Oost, R et al., 2003).

Pesticides are the substances most extensively researched in the aquatic ecotoxicology due to the large amounts used in agriculture and livestock in the whole world. Melo (2004) exposed the silver catfish to the organophosphate methyl parathion at the suble‐ thal concentration of 6 mg/L for 96 hours with the aim of histological and ultrastructural analysis of liver tissue. Melo (2004) suggested that the histological changes are related to the intoxication by methyl parathion that is likely to bring the individuals metabolic, cel‐ lular and subcellular problems. It was observed in *Rhamdia quelen* loss of the contour of the hepatocytes and endothelial cells, as well as changes in the nuclear chromatin. Ac‐ cording to Melo (2004), changes in the organization of rough endoplasmic reticulum and mitochondrial disruption within four hours of intoxication and strongly eosin stained granulation in the cytosol within 24 hours of exposure could be noticed. Ultrastructural‐ ly, a high incidence of lipid droplets was observed in the cytosol of hepatocytes when compared with control within 48 hours of exposure to fipronil. Increased foci of necrosis in the liver of silver catfish after 48 and 72 hours of exposure in relation to the control group, which presents only occasional foci of necrosis, were noted, among other structur‐ al changes. After 96 hours of exposure, Melo (2004) describes cells indistinguishable con‐ tour, presence necrosis focus, in addition of damaged blood vessels, vacuolization of the cytosol and the presence of an unknown material strongly eosin stained in the cytoplasm of the hepatocytes. Blood leukocyte infiltration and congestion were observed in all ani‐ mals analyzed. Melanomacrophage were found in various locations between the hepato‐

Evaluation of Toxicity in Silver Catfish http://dx.doi.org/10.5772/53899 203

Ghisi (2010) studied the effects of the phenyl pyrazolefipronil in the gills of the silver catfish after 60 days of intoxication in the sublethal concentrations 0.05; 0.10 and 0.23 μg/L. The au‐ thor reports hyperplasia, lamellar fusion and aneurysms in all groups treated, including the control group that can impair the gill function. However, Ghisi (2010) considers the injuries of low severity and possible regression if the source of stress is eliminated, since the concen‐

Cattaneo (2009) studied the effects of 2,4 - dichlorophenoxiacetic acid (2,4-D) herbicide in *Rhamdia quelen* fingerlings in an acute toxicity assay. After acclimation, silver catfish were intoxicated with concentrations of 0, 400, 600 and 700 mg/mL of 2,4 – D for 96 hours. The author states that the histological analysis showed alterations in the liver of silver catfish af‐ ter exposure to 2,4-D herbicide, such as abnormal arrangement of hepatocytes cords, cell

The effects of the herbicide clomazone in teleost fish *Rhamdia quelen* were studied by Cresta‐ ni et al. (2007). The silver catfish were exposed to the concentrations 0.5 and 1.0 mg/L of clo‐ mazone for 12, 24, 48, 96 and 192 h. Histological analysis showed vacuolation in the liver after herbicide exposure, some of that were completely restored after a recovery period. Fer‐ reira (2010) studied the sublethal effects of glyphosate (1.21 mg/L), methyl parathion (0.8 mg/L) and tebuconazole (0.88 mg/L) herbicides to silver catfish. The fingerlings were intoxi‐ cated during 96 hours and then sampled after anesthesia. The mapathologicalcal changes in liver histology were: diffuse hepatocyte degeneration, bile stagnation, granules on the cyto‐ plasm, hyperemia and vacuoles in the cytoplasm and nucleus of fingerlings exposed to

cytes as well as pyknotic nuclei cells.

tration of fipronil used was very low.

membrane rupture and hepatocytes vacuolation.

A bioindicator is defined as organism giving information on the environmental conditions of its habitat by its presence or absence or by its behavior, and an ecological indicator is an ecosystem parameter, describing the structure and functioning of ecosystems.

According to the WHO (1993), biomarkers can be subdivided into three classes:


#### **4.1. Histopathology**

Histopathological characteristics of specific organs express condition and represent timeintegrated endogenous and exogenous impacts on the organism stemming from altera‐ tions at lower levels of biological organization. Therefore, histological changes occur earlier than reproductive changes and are more sensitive than growth or reproductive parameters and, as an integrative parameter, provide a better evaluation of organism health than a single biochemical parameter. Histopathological biomarkers signal the ef‐ fects of exposures both acute and chronic to toxic agents in high or low levels. The pathological changes may be adaptive or degenerative and will determine the survival or death of the organism. The organs most commonly damaged by toxic agents are gills, liver and kidneys, which may be also affected by bacteria, viruses and parasites. That is why a health assessment of the animal is important to differentiate damage induced by toxic agents and diseases. Pharmaceuticals of many categories may be detected on the environment contaminating water flows. In a study for the evaluation of the effect of the analgesic dipyron in surface waters, Pamplona (2009) observed important histological disorders in the kidneys of silver catfish. The fish were exposed to three concentrations of dipyrone (0.5, 5 and 50 μg/L) in the water for 15 days and then evaluated. According to the author, the histological parameters of the intoxicated animals showed important tissue damage in the silver catfish kidneys, such as necrosis, fibrous deposition, vacuoli‐ zation and constriction of blood vessels of the renal parenchyma, hyperplasia, and even necrosis of great renal vessels and glomeruli in the higher concentration of dipyron. Such alterations were not observed in the control group.

Pesticides are the substances most extensively researched in the aquatic ecotoxicology due to the large amounts used in agriculture and livestock in the whole world. Melo (2004) exposed the silver catfish to the organophosphate methyl parathion at the suble‐ thal concentration of 6 mg/L for 96 hours with the aim of histological and ultrastructural analysis of liver tissue. Melo (2004) suggested that the histological changes are related to the intoxication by methyl parathion that is likely to bring the individuals metabolic, cel‐ lular and subcellular problems. It was observed in *Rhamdia quelen* loss of the contour of the hepatocytes and endothelial cells, as well as changes in the nuclear chromatin. Ac‐ cording to Melo (2004), changes in the organization of rough endoplasmic reticulum and mitochondrial disruption within four hours of intoxication and strongly eosin stained granulation in the cytosol within 24 hours of exposure could be noticed. Ultrastructural‐ ly, a high incidence of lipid droplets was observed in the cytosol of hepatocytes when compared with control within 48 hours of exposure to fipronil. Increased foci of necrosis in the liver of silver catfish after 48 and 72 hours of exposure in relation to the control group, which presents only occasional foci of necrosis, were noted, among other structur‐ al changes. After 96 hours of exposure, Melo (2004) describes cells indistinguishable con‐ tour, presence necrosis focus, in addition of damaged blood vessels, vacuolization of the cytosol and the presence of an unknown material strongly eosin stained in the cytoplasm of the hepatocytes. Blood leukocyte infiltration and congestion were observed in all ani‐ mals analyzed. Melanomacrophage were found in various locations between the hepato‐ cytes as well as pyknotic nuclei cells.

cal system and a potential hazard, which may be chemical, physical or biological (WHO, 1993). A biomarker is defined as a change in a biological response (ranging from molecular through cellular and physiological responses to behavioral changes) that can be related to

A bioindicator is defined as organism giving information on the environmental conditions of its habitat by its presence or absence or by its behavior, and an ecological indicator is an

**• Biomarkers of exposure:** covering the detection and measurement of an exogenous sub‐ stance or its metabolite or the product of an interaction between a xenobiotic agent and some target molecule or cell that is measured in a compartment within an organism; **• Biomarkers of effect:** including measurable biochemical, physiological or other altera‐ tions within tissues or body fluids of an organism that can be recognized as associated

**• Biomarkers of susceptibility:** indicating the inherent or acquired ability of an organism to respond to the challenge of exposure to a specific xenobiotic substance, including ge‐ netic factors and changes in receptors which alter the susceptibility of an organism to that

Histopathological characteristics of specific organs express condition and represent timeintegrated endogenous and exogenous impacts on the organism stemming from altera‐ tions at lower levels of biological organization. Therefore, histological changes occur earlier than reproductive changes and are more sensitive than growth or reproductive parameters and, as an integrative parameter, provide a better evaluation of organism health than a single biochemical parameter. Histopathological biomarkers signal the ef‐ fects of exposures both acute and chronic to toxic agents in high or low levels. The pathological changes may be adaptive or degenerative and will determine the survival or death of the organism. The organs most commonly damaged by toxic agents are gills, liver and kidneys, which may be also affected by bacteria, viruses and parasites. That is why a health assessment of the animal is important to differentiate damage induced by toxic agents and diseases. Pharmaceuticals of many categories may be detected on the environment contaminating water flows. In a study for the evaluation of the effect of the analgesic dipyron in surface waters, Pamplona (2009) observed important histological disorders in the kidneys of silver catfish. The fish were exposed to three concentrations of dipyrone (0.5, 5 and 50 μg/L) in the water for 15 days and then evaluated. According to the author, the histological parameters of the intoxicated animals showed important tissue damage in the silver catfish kidneys, such as necrosis, fibrous deposition, vacuoli‐ zation and constriction of blood vessels of the renal parenchyma, hyperplasia, and even necrosis of great renal vessels and glomeruli in the higher concentration of dipyron.

exposure to or toxic effects of environmental chemicals (Van Der Oost, R et al., 2003).

ecosystem parameter, describing the structure and functioning of ecosystems. According to the WHO (1993), biomarkers can be subdivided into three classes:

with an established or possible health impairment or disease;

Such alterations were not observed in the control group.

exposure.

**4.1. Histopathology**

202 New Advances and Contributions to Fish Biology

Ghisi (2010) studied the effects of the phenyl pyrazolefipronil in the gills of the silver catfish after 60 days of intoxication in the sublethal concentrations 0.05; 0.10 and 0.23 μg/L. The au‐ thor reports hyperplasia, lamellar fusion and aneurysms in all groups treated, including the control group that can impair the gill function. However, Ghisi (2010) considers the injuries of low severity and possible regression if the source of stress is eliminated, since the concen‐ tration of fipronil used was very low.

Cattaneo (2009) studied the effects of 2,4 - dichlorophenoxiacetic acid (2,4-D) herbicide in *Rhamdia quelen* fingerlings in an acute toxicity assay. After acclimation, silver catfish were intoxicated with concentrations of 0, 400, 600 and 700 mg/mL of 2,4 – D for 96 hours. The author states that the histological analysis showed alterations in the liver of silver catfish af‐ ter exposure to 2,4-D herbicide, such as abnormal arrangement of hepatocytes cords, cell membrane rupture and hepatocytes vacuolation.

The effects of the herbicide clomazone in teleost fish *Rhamdia quelen* were studied by Cresta‐ ni et al. (2007). The silver catfish were exposed to the concentrations 0.5 and 1.0 mg/L of clo‐ mazone for 12, 24, 48, 96 and 192 h. Histological analysis showed vacuolation in the liver after herbicide exposure, some of that were completely restored after a recovery period. Fer‐ reira (2010) studied the sublethal effects of glyphosate (1.21 mg/L), methyl parathion (0.8 mg/L) and tebuconazole (0.88 mg/L) herbicides to silver catfish. The fingerlings were intoxi‐ cated during 96 hours and then sampled after anesthesia. The mapathologicalcal changes in liver histology were: diffuse hepatocyte degeneration, bile stagnation, granules on the cyto‐ plasm, hyperemia and vacuoles in the cytoplasm and nucleus of fingerlings exposed to methyl parathion and tebuconazole. The liver of fish exposed to glyphosate did not show any visible histological changes.

**Diets containing aflatoxins \* Treatment with diflubenzuron in 24-hour immer‐**

**204ppb/ aflatoxin/ kg**

11,16 ± 0,88

**Chemical substance**

Erythrocytes (x 106/µL)

Hemoglobin (g/dL)

Hematocrit (%)

Leucocytes (x 103/µL)

Lymphocytes (%)

Neutrophils (%)

Granulocytic especial cell (%)

Monocytes (%)

Eosinophils (%)

Glucose (mg/dL)

\* Vieira, et al. (2006). \*\*Mabilia; Souza (2006) \*\*\*Tavares-Dias et al. (2002)

45,00 ± 6,28

36,40 ± 2,42

24,40 ± 6,69

23,20 ± 5,93

**Table 1.** Some hematological and blood chemistry results in catfish (*Rhamdia quelen*) exposed to toxic chemicals.

24,51 ± 1,51

**Control 41ppb/**

**aflatoxin/ kg**

13,16 ± 1,47

**90ppb/ aflatoxin/ kg**

11,75 ± 0,41

**sion baths \*\***

8,2 ± 0,5 5,5 ± 0,3 4,0 ± 0,3 3,5 ± 0,2 6,73 ± 1,15

32,7 ± 3,62

**Control 0,01 mg/ l -1difluben‐ zuron**

**0,1 mg/ l -1difluben‐ zuron**

2,71 ± 0,3 2,67 ± 0,4 2,56 ± 0,3 2,6 ± 0,4 1,95 ± 0,40

32,9 ± 2,61 31,1 ± 2,8 30,1 ± 1,7 26,5 ± 5,3

13,4 ± 2,7 13,2 ± 2,9 12,6 ± 2,1 12,2 ± 2,4

62,1 ± 5,7 61,9 ± 6,8 62,4 ± 6,3 62,6 ± 6,4 11,61 ±

25,3 ± 5,5 26,9 ± 5,8 25,9 ± 6,7 25,9 ± 6,7 6,02 ± 3,33

2,7 ± 1,2 2,0 ± 0 2,2 ± 0 4,0 ± 2,0 1,24 ± 1,96

11,6 ± 2,9 10,9 ± 4,5 11,1 ± 3,5 10,6 ± 4,3 1,13 ± 1,09

2,0 ± 0 2,0 ± 0 2,0 ± 0 2,0 ± 0 0,02 ± 0,14

**1,0 mg/ l -1difluben‐ zuron**

Evaluation of Toxicity in Silver Catfish http://dx.doi.org/10.5772/53899

**\*\*\***

205

**None**

6,57

#### **4.2. Hematological and biochemical analyses**

Hematological analysis in fish is not routinely used for fish diseases diagnosis. Hema‐ tology of fish lags behind that of other classes of vertebrates, but analysis of blood still can be informative about disease processes in teleosts and elasmobranchs (Clauss et al., 2008). The most important factor limiting accurate hematological analysis in different fish species is the variation in types, numbers, and appearance of leukocytes. In addi‐ tion, the considerable variation in reported leukocyte values from healthy fish, even within a species, is also partly caused by differences in the methodology used as well as by subjective interpretation of cell types by the investigator. Nevertheless, some ef‐ fort should be made to try to surpass these difficulties since these parameters can be a useful tool for evaluation of the effects of pesticides in the cellular components of blood and even in the immune system. Therefore, the analysis of hematological and biochemi‐ cal parameters in fish can contribute to the assessment of the animal's health and also the habitat conditions (Pimpão et al., 2007).

Table 1 provides an overview of some hematological and blood chemistry results in catfish (*Rhamdia quelen*) exposed to toxic chemicals.

#### **4.3. Enzymatic analyses**

One of the more sensitive effect biomarkers is alteration in levels and activities of biotrans‐ formation enzymes. The activity of these enzymes may be induced or inhibited upon in fish exposure to xenobiotic. Enzyme induction is an increase in the amount or activity of these enzymes, or both and inhibition is the opposite of induction. In this case, enzymatic activity is blocked, possibly due to a strong binding or complex formation between the enzyme and the inhibitors.

The according by Van Der Oost, R et al. (2003) enzymatic analyzes to fish are used more:


Table 2 provides an overview of some enzymatic results in catfish exposed to toxic chemicals.


methyl parathion and tebuconazole. The liver of fish exposed to glyphosate did not show

Hematological analysis in fish is not routinely used for fish diseases diagnosis. Hema‐ tology of fish lags behind that of other classes of vertebrates, but analysis of blood still can be informative about disease processes in teleosts and elasmobranchs (Clauss et al., 2008). The most important factor limiting accurate hematological analysis in different fish species is the variation in types, numbers, and appearance of leukocytes. In addi‐ tion, the considerable variation in reported leukocyte values from healthy fish, even within a species, is also partly caused by differences in the methodology used as well as by subjective interpretation of cell types by the investigator. Nevertheless, some ef‐ fort should be made to try to surpass these difficulties since these parameters can be a useful tool for evaluation of the effects of pesticides in the cellular components of blood and even in the immune system. Therefore, the analysis of hematological and biochemi‐ cal parameters in fish can contribute to the assessment of the animal's health and also

Table 1 provides an overview of some hematological and blood chemistry results in catfish

One of the more sensitive effect biomarkers is alteration in levels and activities of biotrans‐ formation enzymes. The activity of these enzymes may be induced or inhibited upon in fish exposure to xenobiotic. Enzyme induction is an increase in the amount or activity of these enzymes, or both and inhibition is the opposite of induction. In this case, enzymatic activity is blocked, possibly due to a strong binding or complex formation between the enzyme and

The according by Van Der Oost, R et al. (2003) enzymatic analyzes to fish are used more:

(AHH); NADPH cytochrome P450 reductase (P450 RED);

**•** Phase I enzymes: Total cytochrome P450 (cyt P450); Cytochrome P450 1A ; Cytochrome b5 (cyt b5); Ethoxyresorufin O-deethylase (EROD) and arylhydrocarbon hydroxylase

**•** Phase II enzymes and cofactors: Reduced and oxidized glutathione (GSH and GSSG);

**•** Oxidative stress parameters: Superoxide dismutase (SOD); Catalase (CAT); Glutathione peroxidase (GPOX); Glutathione reductase (GRED); Non-enzymatic antioxidants

Table 2 provides an overview of some enzymatic results in catfish exposed to toxic

Glutathione S-transferases (GSTs); UDP-glucuronyltransferases (UDPGTs);

**•** Biochemical indices of oxidative damage: Lipid peroxidation; DNA oxidation;

any visible histological changes.

204 New Advances and Contributions to Fish Biology

**4.2. Hematological and biochemical analyses**

the habitat conditions (Pimpão et al., 2007).

(*Rhamdia quelen*) exposed to toxic chemicals.

**4.3. Enzymatic analyses**

the inhibitors.

chemicals.

**Table 1.** Some hematological and blood chemistry results in catfish (*Rhamdia quelen*) exposed to toxic chemicals.


genetic damages occurred in the lowest concentration of dipyron (0.5 μg/L). DNA fragmen‐ tation may be triggered by the release of free radicals in response to a stressor such as a xen‐ obionte that may damage the nucleus chromatin, or by lipoperoxidation that causes the loss of cellular membrane integrity, subjecting the attack of the nucleic acid by oxygen radicals. In a study of genotoxicity by copper sulphate in four different doses (5, 30, 50 and 500 mg/Kg) using the catfish, Costa (2011) induced DNA breakage triggered by comet assay. The fish were intoxicated by trophic copper sulphate during 60 days and suffered genetic damage in brain tissue, kidney, liver and blood tissues. Blood was less sensitive tissue to levels of copper used in this study. The piscine micronucleus test developed by Costa (2011), however, showed no significant changes in relation to the negative control, as well as the polychromatic erythrocytes frequency in relation to the total number of erythrocytes. There was an increase in the frequency of viable cells in blood tissue, according to the author.

Evaluation of Toxicity in Silver Catfish http://dx.doi.org/10.5772/53899 207

Piancini (2011) performed the piscine micronucleus test and the comet assay in erythrocytes and gill cells of the silver catfish to evaluate the genotoxic effects of atrazine in concentra‐ tions 2, 10 and 100 μg/L for 96 hours. The author observed an increased frequency of nuclear morphological changes at all concentrations, a dose-dependent effect of atrazine on DNA trough the comet assay in erythrocytes: damage was significantly higher on 10 and 100 μg/L concentrations compared to control. In gills, there was no difference between treatments. Piancini (2011) observed a dose-dependent effect of atrazine in relation to genotoxicity and its capacity of damaging the DNA of the silver catfish in concentration considered safe by the Brazilian legislation. The same author evaluated the genotoxic effects of copper on *Rhamdia quelen* through the piscine micronucleus test and the comet assay in erythrocytes and gills. The silver catfish were exposed to concentrations of 2.0; 9.0 and 18 μg/L of cupr‐ ous chloride for 96 hours. Dose-dependent breaks to the DNA were detected by the comet

Ghisi (2010) did not observe micronuclei in the catfish cells, but did confirm nuclear mor‐ phologic changes, after 60 days of intoxication in the sublethal concentrations 0.05; 0.10 and 0.23 μg/L of fipronil. The control group was not significantly different from the group in‐ toxicated by the lower concentration of the insecticide. The higher concentrations were showed significantly higher among of micronuclei compared to the control group, not in‐ toxicated by fipronil, suggesting that the pesticide in higher concentrations (0.10 e 0.23 μg/L) may induce damages to the DNA of *Rhamdia quelen*. The comet assay, also performed by Ghisi (2010) in the same study, showed no significant difference between the intoxicated

The aim of the study performed by Salvagni et al. (2011) was to determine, by micronucleus testing on silver catfish, the risk of genotoxic impact by the use of pesticides such as glypho‐ sate, cyhalothrin, atrazine, simazine and azoxystrobinon in farms located in the Lambedor River watershed in Guatambu, State of Santa Catarina. Blood samples were collected from several species of fish, including *Rhamdia quelen*, captured in 10 different dams existing in agricultural rural properties. Micronucleate erythrocytes were found in blood samples of sil‐ ver catfish close to the overall average of the other species. Salvagniet at. (2011) believe through *in vivo* piscine micronucleus testing, that water from the Lambedor watershed can

groups and the control, corroborating the results of histopathological analysis.

assays in all treatments, as well as in gills.

Van Der Oost, R et al. (2003)

Symbols: --, strong inhibition (<20% of control); -, inhibition; =, no (significant) response; +, induction; ++, strong in‐ duction (>500% of control);

**Table 2.** Laboratory studies on responses of organic trace pollutants on fish enzymes

#### **4.4. Genetic analyses**

Genotoxic substances produce chemical or physical changes inDNA, commonly measured as breaks or DNA adducts, respectively (Nacci et al., 1996). The exposure of an organism to genotoxic substances can induce a cascade of events such as changes in the structure of DNA and the expression of damaged DNA using modified products. Thus, biological conse‐ quences may be triggered in cells, organs, body and finally in community and population (Lee; Steinert, 2003; Van Der Oost et al., 2003).

Many biomarkers have been used as tools for the detection of exposure and to evaluate the genotoxic effects of pollution. Among the major genetic biomarkers, we can mention the evaluation of the frequency of chromosomal aberrations, analysis of the frequency of sister chromatid exchanges, formation of DNA adducts, DNA breakage assessed by measuring the comet assay and micronucleus frequency and nuclear morphological abnormalities (Bombail et al., 2001).

According to the studies of Pamplona (2009) by the Comet Assay, widely used to measure DNA damage in aquatic organisms, after the intoxication of silver catfish by dipyrone, the genetic damages occurred in the lowest concentration of dipyron (0.5 μg/L). DNA fragmen‐ tation may be triggered by the release of free radicals in response to a stressor such as a xen‐ obionte that may damage the nucleus chromatin, or by lipoperoxidation that causes the loss of cellular membrane integrity, subjecting the attack of the nucleic acid by oxygen radicals.

**Species Pollutants cyt**

206 New Advances and Contributions to Fish Biology

2 Aminocanthracene

(AA)

Ictaluruspuncta‐

CatfishIctalurus‐ *nebulosus*

Channel catfish Ictaluruspuncta‐

Van Der Oost, R et al. (2003)

duction (>500% of control);

**4.4. Genetic analyses**

(Bombail et al., 2001).

*tus*

*tus*

**P450**

Channel catfish PCB Aroclor1254 = ++ ++ = = Ankley et al., 1986

**Species Pollutants SOD GPOX GSSG CAT LPOX GSH References**

**Table 2.** Laboratory studies on responses of organic trace pollutants on fish enzymes

(Lee; Steinert, 2003; Van Der Oost et al., 2003).

**CYP1A AHH EROD cyt b5 P450**

BKME = ++ Mather-Mihaich;DiGiulio, 1991

PAH (BNF) ++ Hasspieler et al., 1994 2,4D+ Picloram Gallagher and Di Giulio, 1991

PAH (BaP) + + Ploch et al., 1998 PCB126 ++ ++ Rice and Roszell, 1998

OPP (dichlorvos) = = = + = Hai et al., 1995

PAH + + + Di Giulio et al., 1989

Symbols: --, strong inhibition (<20% of control); -, inhibition; =, no (significant) response; +, induction; ++, strong in‐

Genotoxic substances produce chemical or physical changes inDNA, commonly measured as breaks or DNA adducts, respectively (Nacci et al., 1996). The exposure of an organism to genotoxic substances can induce a cascade of events such as changes in the structure of DNA and the expression of damaged DNA using modified products. Thus, biological conse‐ quences may be triggered in cells, organs, body and finally in community and population

Many biomarkers have been used as tools for the detection of exposure and to evaluate the genotoxic effects of pollution. Among the major genetic biomarkers, we can mention the evaluation of the frequency of chromosomal aberrations, analysis of the frequency of sister chromatid exchanges, formation of DNA adducts, DNA breakage assessed by measuring the comet assay and micronucleus frequency and nuclear morphological abnormalities

According to the studies of Pamplona (2009) by the Comet Assay, widely used to measure DNA damage in aquatic organisms, after the intoxication of silver catfish by dipyrone, the

BKME = = + = - Mather-Mihaich and Di Giulio,

**RED**


1991

**References**

In a study of genotoxicity by copper sulphate in four different doses (5, 30, 50 and 500 mg/Kg) using the catfish, Costa (2011) induced DNA breakage triggered by comet assay. The fish were intoxicated by trophic copper sulphate during 60 days and suffered genetic damage in brain tissue, kidney, liver and blood tissues. Blood was less sensitive tissue to levels of copper used in this study. The piscine micronucleus test developed by Costa (2011), however, showed no significant changes in relation to the negative control, as well as the polychromatic erythrocytes frequency in relation to the total number of erythrocytes. There was an increase in the frequency of viable cells in blood tissue, according to the author.

Piancini (2011) performed the piscine micronucleus test and the comet assay in erythrocytes and gill cells of the silver catfish to evaluate the genotoxic effects of atrazine in concentra‐ tions 2, 10 and 100 μg/L for 96 hours. The author observed an increased frequency of nuclear morphological changes at all concentrations, a dose-dependent effect of atrazine on DNA trough the comet assay in erythrocytes: damage was significantly higher on 10 and 100 μg/L concentrations compared to control. In gills, there was no difference between treatments. Piancini (2011) observed a dose-dependent effect of atrazine in relation to genotoxicity and its capacity of damaging the DNA of the silver catfish in concentration considered safe by the Brazilian legislation. The same author evaluated the genotoxic effects of copper on *Rhamdia quelen* through the piscine micronucleus test and the comet assay in erythrocytes and gills. The silver catfish were exposed to concentrations of 2.0; 9.0 and 18 μg/L of cupr‐ ous chloride for 96 hours. Dose-dependent breaks to the DNA were detected by the comet assays in all treatments, as well as in gills.

Ghisi (2010) did not observe micronuclei in the catfish cells, but did confirm nuclear mor‐ phologic changes, after 60 days of intoxication in the sublethal concentrations 0.05; 0.10 and 0.23 μg/L of fipronil. The control group was not significantly different from the group in‐ toxicated by the lower concentration of the insecticide. The higher concentrations were showed significantly higher among of micronuclei compared to the control group, not in‐ toxicated by fipronil, suggesting that the pesticide in higher concentrations (0.10 e 0.23 μg/L) may induce damages to the DNA of *Rhamdia quelen*. The comet assay, also performed by Ghisi (2010) in the same study, showed no significant difference between the intoxicated groups and the control, corroborating the results of histopathological analysis.

The aim of the study performed by Salvagni et al. (2011) was to determine, by micronucleus testing on silver catfish, the risk of genotoxic impact by the use of pesticides such as glypho‐ sate, cyhalothrin, atrazine, simazine and azoxystrobinon in farms located in the Lambedor River watershed in Guatambu, State of Santa Catarina. Blood samples were collected from several species of fish, including *Rhamdia quelen*, captured in 10 different dams existing in agricultural rural properties. Micronucleate erythrocytes were found in blood samples of sil‐ ver catfish close to the overall average of the other species. Salvagniet at. (2011) believe through *in vivo* piscine micronucleus testing, that water from the Lambedor watershed can be considered genotoxic, with emphasis on the degree of genotoxicity from pollution in the area, since spontaneous formation of micronuclei in fish is normally very rare. Ferraro (2009) also exposed the silver catfish to different molecules of pesticides and the combinations of them in the aim to determine the bioindicator potential of this teleost species. After the ex‐ posure of the fish to the pesticides glyphosate (1.58 e 3.16 mg/L), tebuconazole (0.4 e 0.8 mg/L) and a mixture of them (3.16 e 0.8 mg/L) for 5, 10 and 15 days, were proceeded the micronucleus test and the comet assay in blood samples collected from the fish. DNA dam‐ age was confirmed by both techniques in all treatments, pointing the suitability of the spe‐ cies for biomonioring. In a similar way, Ramsdorf (2011) studied the genotoxic effects of fipronil (0.05; 0.10 and 0.23 μg/L), lead nitrate (0.01; 0.03 and 0.10 mg/L) and naphthalene (0.005; 0.06 and 3 mg/L) in the water for *Rhamdia quelen* during 60, 30 and 28 days, respec‐ tively. In silver catfish the concentrations 0.10 and 0.23 μg/L of fipronil increased the fre‐ quency of micronucleus, nuclear morphological changes, and damages to DNA observed after comet assay. After the intoxication by lead nitrate, it was observed that the concentra‐ tions 0.03 e 0.1 mg/L increased the levels of DNA breaks, as well as in all concentration of naphthalene tested for the species.

Few data exist regarding the potential sublethal effects of pesticides on reproduction and

Evaluation of Toxicity in Silver Catfish http://dx.doi.org/10.5772/53899 209

In 60 years, Rachel Carson published her famous book considered a landmark in the history of environmental pollution, Silent Spring, calling attention to the reproductive failure in birds and fish caused by bioaccumulation of persistent organochlorinepesticides such as di‐ clorofodifeniltricloroetano (DDT). This work brought out the fears of modern society in rela‐ tion to the introduction of synthetic substances in the environment and renewed public

In the mid-70s, researchers found that other chemicals, such as the Kepone and PCBs (poly‐ chlorinated biphenyls), also had hormonal effects. Thereafter the toxicological effects of the mixture of individual congeners have been studied mainly in fish, mammalian cells and

The structural integrity of the gonads can be altered by xenobiotics (Mayon et al., 2006). Chemical agents that can affect the endocrine system are called endocrine-active chemicals

The Environmental Protection Agency of the United States (USEPA) alternatively defines endocrine disrupters as chemicals that lead to toxic results as various types of cancer and a

The pollution in the aquatic environment can affect the reproductive potential, thus reduc‐ ing the spread of fish species. This can occur by the possible interaction between the game‐ tes and water pollutants, such as the blockage of the micropyle, which prevents the entry of sperm in the fertilization process (Hilbig et al., 2008). In addition, solutions containing cer‐ tain levels of pollutants can directly interfere with sperm motility and morphology, and sub‐

In teleost fish, such as quelen (catfish), with external fertilization, which occurs when spawning, the gametes are released into the environment for fertilization to occur. At that moment, the gametes are exposed to various contaminants in the water, including heavy metals mercury, zinc, lead, copper and cadmus. These trace elements, at certain levels, affect

The gonadotropins stimulate gonadal maturation and release of steroid hormones from the gonads. The steroid hormones and the pituitary determine the development of sexual char‐

In teleosts, there are two gonadotropin: R gonadotropin (GTH I) and II gonadotropin (GtH II). In females, the GTH I stimulates gonadal growth, gametogenesis and the entry of vitello‐ genin in the oocyte. GTH II is important for the final maturation of oocytes and spawning.

Endocrine disrupters are chemicals, natural or synthetic compounds also contained in fungi‐ cides, pesticides and insecticides, which have estrogenic and antiandrogenic action. Some of these compounds can maintain their chemical nature for many years contaminating the wa‐

(ECAs), expression adapted to Portuguese as "endocrine disruptors" (Sanchez, 2006).

wide range of adverse effects on the reproductive system (Sanchez, 2006).

the motility of spermatozoa and fertilization of oocytes (Witeck et al., 2011).

acteristics and various influencing courtship and parental care (SANCHEZ, 2006).

In males, GTH II acts in the testis, acting on testosterone production (Sanchez, 2006).

sequently at fertilization (Hilbig et al., 2008; Witeck et al., 2011).

long-term viability of fish populations (Moore; Waring, 2001).

interest in science and government toxicology (SANCHEZ, 2006).

even humans (Sanchez, 2006).
