**5. Impact of toxic agents in the reproductive system**

The use of fish embryos is gaining popularity for research in the area of toxicology and tera‐ tology. Particularly embryos of the zebrafish, catfish and rainbow trout offer an array of dif‐ ferent applications ranging from regulatory testing to mechanistic research.

Recently, much attention has been given to possible adverse effects resulting from exposure of aquatic animals to chemicals during the prenatal and perinatal (Tramujas et al., 2006; Montanha; Pimpão, 2012).

The impairment of reproductive function in humans and animal species has been of particu‐ lar concern in recent years. Many factors can interact with the components and the repro‐ ductive function and cause infertility and other functional and structural changes. Illness, stress, hormonal changes and exposure to chemicals, are some factors that contribute to the emergence of reproductive system disorders (Sanchez, 2006).

Fish are one of the most thoroughly studied organisms in terms of effect of substance with estrogenic activity in the development of abnormalities in the reproductive system. Accord‐ ing to Sumpter (1998), research on how estrogenic substances affect the sexual system of fish began in the 1980s (Bila; Dezotti, 2003).

The fecundity, the spawning period and type-specific characteristics are essential for the maintenance of any species of fish (Gomiero et al., 2007; Montanha; Pimpão, 2012). Repro‐ duction in fish, as in other vertebrates, is affected by environmental factors, social and nutri‐ tional (Parra et al., 2008; Montanha, 2010). Reproductive parameters are more complex indicators of exposure and accumulation of chemicals, hampered for several reasons, the two main effects of pollutants on reproduction caused directly and indirectly and physiolog‐ ical process (Bernardi et al., 2008).

Few data exist regarding the potential sublethal effects of pesticides on reproduction and long-term viability of fish populations (Moore; Waring, 2001).

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

The use of fish embryos is gaining popularity for research in the area of toxicology and tera‐ tology. Particularly embryos of the zebrafish, catfish and rainbow trout offer an array of dif‐

Recently, much attention has been given to possible adverse effects resulting from exposure of aquatic animals to chemicals during the prenatal and perinatal (Tramujas et al., 2006;

The impairment of reproductive function in humans and animal species has been of particu‐ lar concern in recent years. Many factors can interact with the components and the repro‐ ductive function and cause infertility and other functional and structural changes. Illness, stress, hormonal changes and exposure to chemicals, are some factors that contribute to the

Fish are one of the most thoroughly studied organisms in terms of effect of substance with estrogenic activity in the development of abnormalities in the reproductive system. Accord‐ ing to Sumpter (1998), research on how estrogenic substances affect the sexual system of fish

The fecundity, the spawning period and type-specific characteristics are essential for the maintenance of any species of fish (Gomiero et al., 2007; Montanha; Pimpão, 2012). Repro‐ duction in fish, as in other vertebrates, is affected by environmental factors, social and nutri‐ tional (Parra et al., 2008; Montanha, 2010). Reproductive parameters are more complex indicators of exposure and accumulation of chemicals, hampered for several reasons, the two main effects of pollutants on reproduction caused directly and indirectly and physiolog‐

naphthalene tested for the species.

208 New Advances and Contributions to Fish Biology

Montanha; Pimpão, 2012).

began in the 1980s (Bila; Dezotti, 2003).

ical process (Bernardi et al., 2008).

**5. Impact of toxic agents in the reproductive system**

emergence of reproductive system disorders (Sanchez, 2006).

ferent applications ranging from regulatory testing to mechanistic research.

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

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 even humans (Sanchez, 2006).

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

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

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

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

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

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

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‐ ters, being accumulated in fish. Many are potentially harmful to health since they are diffi‐ cult excretion, may accumulate in the body and cause changes in all systems, especially in the endocrine system (Lara et al., 2011).

Cypermethrin proved toxic to the catfish during embryonic development in concentra‐ tions of 0.001, 0.01 and 0.1 mg/L relative to the fertilization rate and hatching. As re‐ gards the survival rate at 12 hours of exposure to cypermethrin after hatching, the concentration 10 mg/L performed toxic to larvae of catfish and 24 hours, concentrations

Evaluation of Toxicity in Silver Catfish http://dx.doi.org/10.5772/53899 211

Some studies have reported the influence of heavy metals in fish breeding, referring to the influences of contaminants on the quality of the gametes on the fertilization of the oocytes

The water contamination by lead, the moment of fertilization to the first 8 hours of incuba‐ tion eggs artificially catfish gray (quelen) at a concentration greater than 0.25 mg/L cause a

Water contamination fertilization with cadmus at levels up to 28.65 mg/L does not reduce rates of fertilization and hatching. However, concentrations above 20 mg/L cause reduction

In the study by Routledge et al. (1998), two fish species, *Oncorhynchus mykiss* and *Rutilus ru‐ tilus*, were exposed for 21 days at concentrations of 17 β-estradiol and estrone environmen‐ tally relevant. According to these researchers, the results confirmed that estrogens identified in effluents are present in sufficient amounts to cause the synthesis of vitellogenin, a protein that plays an important role in the reproductive system of female oviparous vertebrates, in fish species. Kang et al. (2002) clearly show that exposure to estrogen concentration of 17 βestradiol environmentally relevant (in the range of 30-500 ng/L), for three weeks, induces high levels of vitellogenin hermaphrodite and the incidence of male fish species *Oryzias lat‐*

According to studies by Panter et al. (1998), low concentrations of 17 β-estradiol and estrone caused effects in male fish species *Pimephales promelas*, as vitellogenin synthesis and testicu‐

Exposure to pesticides and other toxic substances during the prenatal and perinatal, can al‐ ter the reproductive system of fish without compromising growth and viability of offspring, but cause functional changes that become apparent later in adulthood (Tramujas et al., 2006; Montanha; Pimpão2012). Many xenobiotics are known to affect reproduction in many or‐

**6. The fish and the fish embryo toxicity test as an animal alternative**

Biological and ecological responses to contaminant stressors may range from changes at the molecular level, where genetic integrity and subcellular processes are evaluated, to popula‐ tion and community levels where dynamics and structure of entire foodwebs can be affect‐

**method in hazard and risk assessment and scientific research**

deleterious effect on the percentage of fertilized eggs (Hilbig et al., 2008).

of 0.001 to 10 mg/L (Montanha, 2010).

*ipes* (Bila; Dezotti, 2003).

lar inhibition (Bila; Dezotti, 2003).

ganisms (Mayonet al., 2006; Montanha, 2010).

and the quality of the larvae (Hilbig et al., 2008).

in sperm motility catfish (quelen) (Witeck et al., 2011).

Recently, the monitoring of drug residues in the environment has been gaining great inter‐ est due to the fact that many of these substances are frequently found in effluents from sew‐ age treatment plants (STPs) and natural waters. Some groups of residual drugs deserve special attention, among them are estrogen, because of its potential to adversely affect the reproductive system of aquatic organisms such as the feminization of male fish found in riv‐ ers contaminated with effluent disposal TEE. This was observed in fish species such as *Cyp‐ rinus carpio* and *Rutilus rutilus*. Similar effects (induction of hermaphroditism or complete feminization) were also observed when fish species *Oryzias latipes* were exposed to estrogen 17 β-estradiol (Bila; Dezotti, 2003).

Currently, there is growing concern about the presence of pharmaceuticals in aquatic envi‐ ronments and their possible environmental impacts. Ternes et al. (1999) identified the pres‐ ence of various estrogens in sewage effluent and wastwater treatment plant (WWTP) in Germany, Brazil and Canada (Bila; Dezotti, 2003).

These compounds are characterized by mimicking the structure and function of natural sex steroids leading to endocrine disorders, which can result in abnormal sexual and reproduc‐ tive function in animals and humans (LARA et al., 2011).

Deltamethrin, a pyrethroid pesticide widely used, is listed by the USEPA as endocrine dis‐ ruptor, interfering with the reproductive system (Tramujas et al., 2006). Concentrations of 0.01 and 0.1 mg/L of deltamethrin presented toxic to fertilization of eggs of silver catfish (*Rhamdia quelen*). Since the concentrations 0.1, 0.5 and 1.0 mg/L proved to be toxic in the hatching and survival rates of 12 and 24 hours for eggs and larvae of silver catfish (Monta‐ nha, 2010). Tramujas et al. (2006) reported histological changes in levels in the gonads of aquatic organisms subjected to different concentrations of deltamethrin.

In recent years, monitoring the impacts of pesticides and other pollutants on sperm produc‐ tion of various kinds of research has been aimed (Mabilia et al., 2008). According to Moore and Waring (2001), even at low levels, cypermethrin (synthetic pyrethroid) in water caused a negative effect on reproductive functions in Atlantic salmon (*Salmo salar*) (Begum, 2005; Mabilia et al., 2008; Montanha, 2010; Montanha; Pimpão, 2012), causing a reduction in fertili‐ zation rate (Moore; Waring, 2001). Tramujas et al. (2006) reported a reduction in egg hatch‐ ing rate of zebrafish (*Danio rerio*) exposed to deltamethrin. Rodrigues (2007) reported hatching in early zebrafish (*Danio rerio*) exposed to dichlorodiphenyltrichloroethane (DDT), with consequent reduction in survival rate. Aydin et al. (2005) and Rodrigues (2007) report‐ ed mortality of larvae of carp (*Cyprinus carpio*) exposed to cypermethrin and zebrafish (*Dan‐ io rerio*) exposed to DDT, respectively after 12 hours of exposure.

DDT at concentrations of 50 μg/L, and cause premature death hatching of larvae of *Danio rerio*, as well as parathion, from which 20 μg/L cause a decrease in the growth rates of fish of the same species (Rodrigues, 2007).

Cypermethrin proved toxic to the catfish during embryonic development in concentra‐ tions of 0.001, 0.01 and 0.1 mg/L relative to the fertilization rate and hatching. As re‐ gards the survival rate at 12 hours of exposure to cypermethrin after hatching, the concentration 10 mg/L performed toxic to larvae of catfish and 24 hours, concentrations of 0.001 to 10 mg/L (Montanha, 2010).

ters, being accumulated in fish. Many are potentially harmful to health since they are diffi‐ cult excretion, may accumulate in the body and cause changes in all systems, especially in

Recently, the monitoring of drug residues in the environment has been gaining great inter‐ est due to the fact that many of these substances are frequently found in effluents from sew‐ age treatment plants (STPs) and natural waters. Some groups of residual drugs deserve special attention, among them are estrogen, because of its potential to adversely affect the reproductive system of aquatic organisms such as the feminization of male fish found in riv‐ ers contaminated with effluent disposal TEE. This was observed in fish species such as *Cyp‐ rinus carpio* and *Rutilus rutilus*. Similar effects (induction of hermaphroditism or complete feminization) were also observed when fish species *Oryzias latipes* were exposed to estrogen

Currently, there is growing concern about the presence of pharmaceuticals in aquatic envi‐ ronments and their possible environmental impacts. Ternes et al. (1999) identified the pres‐ ence of various estrogens in sewage effluent and wastwater treatment plant (WWTP) in

These compounds are characterized by mimicking the structure and function of natural sex steroids leading to endocrine disorders, which can result in abnormal sexual and reproduc‐

Deltamethrin, a pyrethroid pesticide widely used, is listed by the USEPA as endocrine dis‐ ruptor, interfering with the reproductive system (Tramujas et al., 2006). Concentrations of 0.01 and 0.1 mg/L of deltamethrin presented toxic to fertilization of eggs of silver catfish (*Rhamdia quelen*). Since the concentrations 0.1, 0.5 and 1.0 mg/L proved to be toxic in the hatching and survival rates of 12 and 24 hours for eggs and larvae of silver catfish (Monta‐ nha, 2010). Tramujas et al. (2006) reported histological changes in levels in the gonads of

In recent years, monitoring the impacts of pesticides and other pollutants on sperm produc‐ tion of various kinds of research has been aimed (Mabilia et al., 2008). According to Moore and Waring (2001), even at low levels, cypermethrin (synthetic pyrethroid) in water caused a negative effect on reproductive functions in Atlantic salmon (*Salmo salar*) (Begum, 2005; Mabilia et al., 2008; Montanha, 2010; Montanha; Pimpão, 2012), causing a reduction in fertili‐ zation rate (Moore; Waring, 2001). Tramujas et al. (2006) reported a reduction in egg hatch‐ ing rate of zebrafish (*Danio rerio*) exposed to deltamethrin. Rodrigues (2007) reported hatching in early zebrafish (*Danio rerio*) exposed to dichlorodiphenyltrichloroethane (DDT), with consequent reduction in survival rate. Aydin et al. (2005) and Rodrigues (2007) report‐ ed mortality of larvae of carp (*Cyprinus carpio*) exposed to cypermethrin and zebrafish (*Dan‐*

DDT at concentrations of 50 μg/L, and cause premature death hatching of larvae of *Danio rerio*, as well as parathion, from which 20 μg/L cause a decrease in the growth rates of fish of

the endocrine system (Lara et al., 2011).

210 New Advances and Contributions to Fish Biology

17 β-estradiol (Bila; Dezotti, 2003).

Germany, Brazil and Canada (Bila; Dezotti, 2003).

tive function in animals and humans (LARA et al., 2011).

aquatic organisms subjected to different concentrations of deltamethrin.

*io rerio*) exposed to DDT, respectively after 12 hours of exposure.

the same species (Rodrigues, 2007).

Some studies have reported the influence of heavy metals in fish breeding, referring to the influences of contaminants on the quality of the gametes on the fertilization of the oocytes and the quality of the larvae (Hilbig et al., 2008).

The water contamination by lead, the moment of fertilization to the first 8 hours of incuba‐ tion eggs artificially catfish gray (quelen) at a concentration greater than 0.25 mg/L cause a deleterious effect on the percentage of fertilized eggs (Hilbig et al., 2008).

Water contamination fertilization with cadmus at levels up to 28.65 mg/L does not reduce rates of fertilization and hatching. However, concentrations above 20 mg/L cause reduction in sperm motility catfish (quelen) (Witeck et al., 2011).

In the study by Routledge et al. (1998), two fish species, *Oncorhynchus mykiss* and *Rutilus ru‐ tilus*, were exposed for 21 days at concentrations of 17 β-estradiol and estrone environmen‐ tally relevant. According to these researchers, the results confirmed that estrogens identified in effluents are present in sufficient amounts to cause the synthesis of vitellogenin, a protein that plays an important role in the reproductive system of female oviparous vertebrates, in fish species. Kang et al. (2002) clearly show that exposure to estrogen concentration of 17 βestradiol environmentally relevant (in the range of 30-500 ng/L), for three weeks, induces high levels of vitellogenin hermaphrodite and the incidence of male fish species *Oryzias lat‐ ipes* (Bila; Dezotti, 2003).

According to studies by Panter et al. (1998), low concentrations of 17 β-estradiol and estrone caused effects in male fish species *Pimephales promelas*, as vitellogenin synthesis and testicu‐ lar inhibition (Bila; Dezotti, 2003).

Exposure to pesticides and other toxic substances during the prenatal and perinatal, can al‐ ter the reproductive system of fish without compromising growth and viability of offspring, but cause functional changes that become apparent later in adulthood (Tramujas et al., 2006; Montanha; Pimpão2012). Many xenobiotics are known to affect reproduction in many or‐ ganisms (Mayonet al., 2006; Montanha, 2010).
