**2. Methodology**

**1. Introduction**

86 National Parks - Management and Conservation

According to Ramelow et al. [1] and Schulz et al. [2], fishes are excellent tools to the aquatic environment biomonitoring because they are used for assessing a lot of environmental disturbing factors such as changes in the rate of growth and sexual maturation. Besides, changes in fish community structure, such as species abundance and diversity, may also reflect the

In this way, erythrocytes of fish have been shown to be a safe tool for the micronucleus test [4, 5]. The micronucleus test is considered an advantageous technique whose analysis is relatively simple. In addition, the simplicity and speed of obtaining peripheral fish blood make the technique even more suitable for the evaluation of environmental contamination [6].

In this context, the selection of species that can reflect the environmental situation of PNCM becomes of great relevance to monitor the interferences that this Conservation Unit has been suffering over the years. Fishes are excellent bioindicators because they are at the top of the trophic chain and reflect the impacts in a given ecosystem through their normal and/or

The development and standardization of methodologies capable of predicting the effects of contamination on aquatic organisms are extremely relevant for biomonitoring studies in a Conservation Unit. Among these methodologies, the use of biomarkers of aquatic contamination in fish is particularly important because it shows initial biological responses and may be useful to subsidize monitoring and environmental management actions [8]. Biomarkers are biological responses to stress caused by pollutants and/or physical stressors and can be used

Research indicates that when aquatic ecosystems are polluted with organic and inorganic contaminants, fish will inevitably be contaminated [10, 11]. The possible effects of such contaminants on fish can be assessed by using several types of biomarkers, which are defined as the biological responses, as well as the effects caused by the pollutants and which identify signs of initial damage in organisms [12]. Livingstone [13] considers as biomarkers the bodily fluids, cells or tissues, as well as the responses of the exposed organisms, in physiological as well as behavioral or energetic levels, being, therefore, molecular biomarkers, cellular or organisms, being some of them are specific pollutants. The genetic material of eukaryotic cells of fish species can also be altered by exposure to dissolved chemicals in the water, resulting in the formation of micronuclei, which can be used as biomarkers to assess the degree of contamination in the environment [14]. The micronuclei are derived from chromosomal fragments resulting from breaks that are not incorporated into the main nucleus of the daughter cells after mitosis due to damage introduced into the parental cells [15]. Micronuclei tests have not yet been performed in PNCM and in the water ecosystems of southern Maranhão. Thus, these data may serve to obtain more complete clues of contaminants that may be inducing. In this way, recognizing the need to provide the sustainable use of natural resources and the environmental quality of the PNCM and to the local communities, as well as the need to know the effect of the possible impacts on the fish of the region, the aim was to contribute with the scientific knowledge related to biomarkers genotoxic in two species of sweet fish (*Hypostomus pusarum* and *Mylossoma duriventre*) in order to subsidize biomonitoring and management programs in the protected areas.

effects of various stressors on the biotic integrity of a river [3].

organic composition in the medium or long term [7].

to identify early signs of damage to aquatic organisms [9].

#### **2.1. License and statement of ethics committee**

Fish collection was done through the research authorization of the Chico Mendes Institute for Biodiversity Conservation—ICMBio (SISBIO, 55361/2017). The protocol of the ethics committee was approved by the State University of Maranhão (13/2017 CRMV-MA) through the Ethics and Animal Experimentation Commission (CEEA).

#### **2.2. Study area**

The Chapada das Mesas National Park (PNCM) (**Figure 1**) is a protected area, which is located in the South of Maranhão, between the following cities: Riachão, Estreito and Carolina [16].

The PNCM climate is tropical: humid and hot, characterized by having two defined seasons, one being dry and the other rainy. The rainy season is in the period from November to March, with rainfall concentrated in February [17]. This region contains an extensive and rich hydrographic network with approximately 400 springs and the main water courses that supply the city of Carolina. In addition, the PNCM protects numerous watercourses and springs from several rivers, such as the Farinha River (with numerous waterfalls), Itapecuru, Urupuchete, Corrente

**Figure 1.** Map of the Chapada das Mesas National Park, Brazil showing the sampling sites. CRS = São Romão Waterfall; CP = Prata Waterfall.

and Lajinha. The hydrographic basin of the Farinha River is one of the main tributaries of the Tocantins river basin, being the most explored from the ecotourism and local point of view.

#### **2.3. Sampling sites and fishes in the PNCM**

In total, 32 fishes were sampled in PNCM: [1] São Romão Waterfall (n = 12) and [2] Prata Waterfall (n = 20). The stations were georeferenced by Global Positioning System (GPS). The fishes were collected in the rainy period (March 2017) and in the dry period (June 2017) with fixed nets 22 in the upstream and downstream of the waterfalls. The genera selected for analysis of the biomarkers were *H. pusarum* and *M. duriventre*. The selection of species is related to their habit and their frequency throughout the years in PNCM rivers and waterfalls.

These data indicate that all the abiotic factors of PNCM waterfalls are within the values accepted by the National Environmental Council [19]. CONAMA Resolution No. of 17 May 2011, which complements and amends Resolution No. 357/2005 of 17 March 2005, presents specific values that classify freshwater bodies (lentic and lotic) and shows that below recommended levels, these values may cause adaptive changes in the morphology of erythrocytes of bioindicator species (such as fish) and, consequently, a decrease in hematocrit

**Table 1.** Environmental parameters analyzed at each sampling location in Chapada das Mesas National Park, Brazil.

**Parameters. São Romão Waterfall Prata Waterfall Recommended values**

Genotoxic Biomarkers in Fishes of the Chapada Das Mesas National Park, Brazil

http://dx.doi.org/10.5772/intechopen.72558

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(ppm)a 11 12.5 >5 mg/L<sup>b</sup>

(°C) 28.0 28.5 28–32°C<sup>b</sup>

pHa 7.32 7.45 6.5–8.0<sup>b</sup>

The results from statistical analysis of the *M. duriventre* biometric in São Romão and Prata

The biometric data submitted to the normality test for *M. duriventre* indicated that there is not a significant difference between the treatments for the São Romão and Prata Waterfall. According to Pinheiro-Sousa [8], the statistical difference can be related to the environmental

In addition, the biometrics data were higher for fish in the dry period in the São Romão Waterfall and in the rainy period for Cachoeira da Prata. This difference between the size

**São Romão Waterfall Prata Waterfall**

Total number of species sampled = 19; number of species in São Romão Waterfall = 8 and number of species in Prata

**Table 2.** Biometric data of *M. duriventre* sampled in the São Romão and Prata Waterfall, Chapada das Mesas National

Waterfall = 11. Biometric data: TL (total length); FL (fork length); SL (standard length) and TW (total weight).

TL (cm) 7.63 ± 1.02 14.06 ± 5.25 16.5 ± 4.02 13.1 ± 4.62 FL (cm) 6.9 ± 1.08 13.14 ± 5.17 12.25 ± 5.58 13.12 ± 4.78 SL (cm) 6.16 ± 0.90 11.4 ± 4.67 14.2 ± 3.46 11.16 ± 4.01 TW (g) 6.66 ± 4.61 36.4 ± 37.40 93.66 ± 58.73 61.6 ± 59.21

**Rainy season Dry season Rainy season Dry season**

conditions of the available resources in two distinct points of a protected area.

values [16, 20].

Dissolved O<sup>2</sup>

Temperature<sup>a</sup>

Mean value during the dry and rainy seasons.

Resolution No. 357, CONAMA (Brazilian Legislation) 15 March 2005.

a

b

**3.2. Biometric data**

*3.2.1. M. duriventre*

Park, Brazil.

Waterfall from PCNM can be observed in **Table 2**.

**Parameter (mean ± SD) Means ± Standard deviations (SD)**

#### **2.4. Environmental parameters**

Physicochemical parameters—temperature, pH and dissolved oxygen—were measured at each site during the dry and rainy season when fishes were sampled. The parameters were analyzed using the ASKO multiparameter.

#### **2.5. Micronuclei, morphological nuclear abnormalities and biometric data**

Specimens of *H. pusarum* and *M. duriventre* were sampled, transferred to a plastic vat with water and then anesthetized for 5 min in clove solution. Blood was collected from the gills of individual *H. pusarum* and *M. duriventre* (n = 32 from the two sampling sites) using heparinized syringes. A drop of blood from each fish was placed on two microscope slides and smeared. The slides were left to dry at room temperature for 24 h and then fixed in absolute ethanol for 30 min. One set of slides (n = 32) was stained with 10% Giemsa diluted in phosphate buffer (pH 6.8) and analyzed using a light microscope. A total of 2000 cells per slide were analyzed. Micronuclei and morphological nuclear abnormalities in the erythrocytes were deemed indicative of genotoxicity [18].

For each fish specimen, biometric data—total length (TL), fork length (FL), standard length (SL) and total weight (TW)—were recorded.

#### **2.6. Statistical analysis of data**

The obtained data were submitted to the normality test, and the obtained results were compared by Student's t-test. For the differences in location between the means obtained for the two collection sites and the biometric data, the multiple comparison test (P < 0.05) was used.
