Environmental parameters in PNCM waterfalls
The average values of the abiotic variables recorded in the PNCM during the entire period sampled (rainy and dry) are shown in table 1. All the analyzed variables presented similar values and were in accordance with the recommendation of CONAMA Resolution No. 430 (Brazil 2011).
Tab. 1 Environmental parameters measured at Cachoeira de São Romão-SRW and Cachoeira da Prata-PW, Chapada das Mesas National Park- MA
Parâmetros
|
SRW
|
PW
|
Valores Recomendados
|
|
Chuvoso
|
Estiagem
|
Chuvoso
|
Estiagem
|
|
O2 Dissolvido (ppm)
|
8,01
|
8,10
|
8,01
|
8,11
|
>5 mg/Lb
|
pH
|
7,8
|
8,9
|
7,0
|
8,5
|
6,5-8,0b
|
Temperatura (°C)
|
28,8
|
29,1
|
28,9
|
29
|
28 - 32°Cb
|
Condutividade
|
30,5
|
32,6
|
30,1
|
32,9
|
--
|
Sólidos Totais Dissolvidos (TDS)
|
29,6
|
20,1
|
29,8
|
20,8
|
500 mg/L
|
bCONAMA Resolution No. 430 of 05/13/2011
The water quality of an aquatic ecosystem is a reflection of the use and occupation of the land around it (Shrestha and Kazama 2007). To understand the possible impacts caused by human action in these environments and the organisms present, limnological studies are essential, as they help in control and monitoring (Tundisi and Matsumura-Tundisi 2008). In our research, no differences were observed between the abiotic factors evaluated at the collection points in both seasonal periods, with values according to the Brazilian resolution. However, a more in-depth study of chemical analyzes of water quality is recommended, since, according to Zhou et al. (2012), water bodies end up becoming recipients of pollutants from adjacent landscapes, acting as integrators of land-water interactions, in addition to the influence of adjacent biophysical and socioeconomic factors. These answers may help to make more accurate decisions about the frequent presence of micronucleus damage found in the erythrocytes of fish collected at the SRW point (potentially impacted area), which will be discussed later.
Biometrics of species
The results of the statistical analysis of the biometric data of L. taeniofasciatus and H. punctatus collected in Cachoeira de São Romão and Cachoeira da Prata, can be seen in tables 2 and 3, respectively.
Tab. 2 Biometric data of Leporinus taeniofasciatus collected at Cachoeira de São Romão-SRW and Cachoeira da Prata-PW, Chapada das Mesas National Park, in the rainy and dry season
Parameters
|
Mean±Standard Deviation
|
SRW
|
PW
|
|
Rainy
|
Dry
|
Rainy
|
Dry
|
LT (cm)
|
13,87 ±1,55a
|
11,99±1,74
|
18,23±1,12b
|
11,72±2,00a
|
FL (cm)
|
14,74±1,55
|
10,62±1,65a
|
16,60±8,40b
|
11,52±1,70
|
SL (cm)
|
12,35±2,49
|
9,53±1,39
|
15,30±0,34
|
10,64±1,66
|
WT (g)
|
15,12±9,66b
|
17,15±10,66a
|
51,33±28,58b
|
18,75±11,61a
|
a,b indicates statistical difference (p<0.05). Total individuals sampled: 25. Number of species in Cachoeira de São Romão: 10. Number of species in Cachoeira da Prata: 15. Biometric data: CT (total length); CF (furcal length); CP (standard length); EN (total weight).
Tab. 3 Biometric data of Hypostomus punctatus collected at Cachoeira de São Romão-SRW and Cachoeira da Prata-PW, Chapada das Mesas National Park, in the rainy and dry season
Parameters
|
Mean±Standard Deviation
|
SRW
|
PW
|
|
Rainy
|
Dry
|
Rainy
|
Dry
|
LT (cm)
|
15,17±0,77
|
22,95±4,63a
|
14,20±6,42
|
25,75±5,63a
|
FL (cm)
|
14,10±0,01a
|
19,65±2,77
|
12,83±5,39b
|
20,36±3,74
|
SL (cm)
|
13,05±0,35
|
18,00±1,70a
|
11,76±4,57
|
19,49±1,50b
|
WT (g)
|
26,00±2,82b
|
32,00±28,28a
|
13,66±12,66b
|
90,13±23,81a
|
a,b indicates statistical difference (p<0.05). Total individuals sampled: 32 Number of species in Cachoeira de São Romão: 13. Number of species in Cachoeira da Prata: 19. Biometric data: CT (total length); CF (furcal length); CP (standard length); EN (total weight).
The biometric data submitted to the normality test for L. taeniofasciatus showed that there was a statistical difference about the biometry of the taxa during the sampling periods (rainy and dry). However, biometrics were higher for the rainy season than for the dry season. For the species H. punctatus, the data indicate a significant difference between treatments for the São Romão and Prata waterfalls. However, taxa of H. punctatus captured in the dry season showed higher values of total length (LT) and weight (WT), for the two sampling areas.
The biometric difference for L. taeniofasciatus may be related to its reproductive period, since species of this genus have total spawning and their reproductive season normally occurs from December to March (Tavares and Godinho 1994); on the other hand, the captured during the dry season (June and November) led to the sampling of smaller individuals in the São Romão and Prata waterfalls.
When comparing between species, H. punctatus showed higher biometric data than L. taeniofasciatus. Representatives of the Locaridae family have split spawning, which provides an advantage to the species, by reducing intraspecific competition between them (Agostinho et al. 1991). Thus, it is likely that the species H. punctatus was captured in all reproductive cycles for the sampling points of the PNCM, which conferred greater biometry about L. taeniofasciatus. With this, it is possible to explain the variation of the total length (LT) and total weight (WT) between the different sampling periods (dry and rainy seasons).
Micronucleus (MN) and Nuclear Morphological Changes (AMN)
Table 4 shows the incidence of micronucleus (MN) and nuclear morphological changes (AMN) in erythrocytes of L. taeniofasciatus and H. punctatus collected at different sampling points in the PNCM.
Tab. 4 Quantitative number of micronuclei (MN) and nuclear morphological changes (AMN) in Leporinus taeniofasciatus and Hypostomus punctatus from Chapada das Mesas National Park- MA
|
|
Rainy
|
|
|
SRW
|
PW
|
Species
|
N
|
MN
|
NV
|
NB
|
NE
|
MN
|
NV
|
NB
|
Leporinus taeniofasciatus
|
15
|
8
|
10
|
15
|
2
|
12
|
0
|
0
|
Hypostomus punctatus
|
10
|
4
|
1
|
5
|
0
|
2
|
0
|
7
|
|
|
Dry
|
|
|
SRW
|
PW
|
Species
|
N
|
MN
|
NV
|
NB
|
NE
|
MN
|
NV
|
NB
|
Leporinus taeniofasciatus
|
10
|
4
|
0
|
45
|
36
|
22
|
4
|
52
|
Hypostomus punctatus
|
22
|
4
|
4
|
26
|
10
|
12
|
0
|
0
|
N = total sampled individuals; MN =Micronucleus; NB =Binucleated Nuclei and NV=Vacuolized Nuclei
The so-called genotoxic agents have the potential to cause damage to the genetic material of organisms, such as breaks in the DNA strand, changes in nucleotides, and mutations (Frenzilli et al. 2004). The micronucleus test has been a widely used biomonitoring tool for analyzing the increase in the frequency of micronucleated cells, with this it is possible to verify the quality of the waters for the populations that exist there and help in the prevention of diseases caused by these alterations (Sommer et al. 2020). In this study, it was possible to observe that the incidence of MN was low about AMN in the sampling periods, indicating that the environment still does not present a significant risk to the health of the species. However, the genotoxic alterations found were greater for L. taeniofasciatus about H. punctatus, especially in the dry season. According to the bioecology of the taxa, the species H. punctatus has a dermesal/benthic habit and is probably more resistant to environmental conditions and, consequently, to the possible environmental impacts of the PNCM, about L. taeniofasciatus. Similar to our results, Castro et al. (2019) evaluating genotoxic damage in the species Colossoma macropomum also found a higher rate of changes in the dry season, probably due to greater bioaccumulation of xenobiotics in this period. These data are important and highlight H. punctatus as a more appropriate bioindicator species for biomarker studies in the PNCM.
Figure 2 shows a photomicrograph of the alterations found in L. taeniofasciatus and H. punctatus for the two areas sampled at different points in the PNCM. Changes such as micronucleus (MN), vacuolated nuclei (NV), and binucleated nuclei (NB) were found.
The diverse genotoxic alterations found in the blood cells of aquatic organisms, such as fish, may indicate a compromised physiological and biochemical state (Adeyemo et al. 2009). According to Castro et al. (2019), the abnormalities found in fish erythrocytes may be related to cytotoxicity processes, influences on cell Division, and genotoxicity or mutagenicity. The micronucleus test has the function of detecting structural and numerical chromosomal alterations, an important phase that helps in the study of mutagenic substances (Heddle et al. 1983). Although studies carried out with micronuclei have been more used in mammals, in aquatic environments they allow the observation of the existence of genotoxic properties in substances present in the environment (Udroiu 2006; Pinheiro-Sousa 2015).
However, despite the occurrence of MN and AMN in the fish sampled in the São Romão and Prata waterfalls, it was possible to understand that the amount of alterations was not high compared to the values found for other Conservation Units in the State of Maranhão (Carvalho Neta et al. 2015; Castro et al. 2018). Thus, despite the low frequency of AMN and MN, it is suggested that the São Romão and Prata Waterfalls still do not suffer from point sources of pollution. In addition, in most of the analyzed slides, cells of the defense system, such as lymphocytes, eosinophils, and monocytes were found (Fig. 3). Leukocytes are defense cells and their group corresponds to neutrophils, eosinophils, and basophils, differentiation occurs morphologically based on granulation, they are used to monitor infections or tissue damage (Satake et al. 2009).
Lymphocytes vary in size and are mostly rounded cells, as well as their nucleus, which has dense chromatin. Their cytoplasmic projection helps to differentiate them from thrombocytes in the bloodstream (Matos and Matos 1988). In contrast, monocytes are large and larger cells than lymphocytes, with basophilic cytoplasm, spherical shape with irregularities, a small nucleus with dense chromatin, and absence of nucleolus, they are called cells in transit in peripheral blood (Tavares-Dias and Moraes 2004; Thrall et al. 2014). In addition, monocytes have phagocytic activity, they occur when there is an inflammatory reaction consisting of an immune response that increases their importance for defense mechanisms, in addition, they have the cytotoxic ability, that is, they can change cellular metabolism, thus accelerating the phagocytic activity of bacterial antigens (Thrall et al. 2014)
Eosinophils are smaller and have different sizes that depend on the size of the granules in the cytoplasm and the amount; the cytoplasm is rich in coarser granules present throughout the cytoplasm, with a pink color – orange makes its identification easier; the nucleus is rounded with compacted chromatin (Ranzani-Paiva and Silva-Souza 2004). These cells are more present in the hematopoietic, mesenteric, intestinal submucosa, peritoneal fluid, and gills tissue (Tavares-Dias et al. 2002). These data indicate that, probably, the degree of stress in the two fish species evaluated is considered low, due to the high frequency of defense cells in their blood tissue. Thus, the evaluation of hematology is an important tool for understanding the health of fish and the natural resources available in the PNCM.