4.1. Physicochemical and metal analysis in water
Previous studies, such as the one by Escobar (2015) and Gomes et al. (2019) reported that, due to anthropological activities on the banks, the Doce River was already polluted before the disaster. Despite this, Quadra et al. (2019) and Gomes et al (2019) reported increments of Fe, Mn, Al, Cu, and Pb in the Doce River shortly after the arrival of the mud from the collapse of the dam. Macêdo et al. (2020) found that the metals found in the highest concentrations in the Doce River’s water 32 months after the disaster were Al and Fe. The results obtained in the water analysis of the present study (32–44 months after the disaster) corroborate with what was obtained by Macêdo et al. (2020), since the most abundant metals in the assessment carried out in this study were also Al and Fe. Even four years after the disaster, the results show that Al remains above the maximum limits allowed by Brazilian law (National Environmental Council, Resolution 357/2005) and by USEPA (1998). The results obtained in the analysis of the gonads corroborated, partially, the results obtained in the water analysis, with Al being the metals with the highest concentrations.
4 .2. Biological Indices And Sexual Maturation
According to Vicentini and Araújo (2003), the sex ratio between individuals is basic information for the reproductive potential, providing important data about the dynamics and population structure of a species. Súarez et al. (2017), studying A. lacustris in the Pantanal, Brazil, reported higher proportions of females and attributed this to differences in the proportion of birth, mortality and growth; in addition to the need for rapid population growth, as a compensatory response to predation. According to Vicentini and Araújo (2003), another factor that can influence the proportion between males and females is the availability of food. Súarez et al. (2017) also reported that the imbalance in the proportion of males and females is unfavorable for natural selection in the environment. Contrary to the findings by Súarez et al. (2017) with A. lacustris, the present study revealed a balanced proportion between males and females in the population of the lower Doce River, which is a beneficial factor for the reproduction of this population.
The onset of sexual maturity is an important component in the population dynamics of fish (Köster et al. 2013, Súarez et al. 2017). This study was not designed to estimate the age of the first sexual maturation of the population of A. lacustris from the lower Doce River; but, when analyzing the data obtained, we found an alarming result (Table 10).
Necessary proportional growth (CN in %) for males and females of different species of the Astyanax genus, from 30–40% of mature individuals (L30 for males and L40 for females) to 50% of mature individuals (L50). TM = maximum population size
Doce River, Brazil
Paraguai River, Brazil
Súarez et al. (2017)
E. M. Atlântica Park, Brazil
Souza et al. (2015)
Iguaçu River, Brazil
Oliveira et al. (2019)
Dos Sinos River, Brazil
Dala-Corte e Azevedo (2010)
Studying a smaller population of A. lacustris (maximum size = 7 cm for males and females), Súarez et al (2017) reported male individuals with 30% gonadal maturation at 2.1 cm in length and 50% at 3.1 cm in length. The difference between 30 and 50% of matured individuals was only 1 cm (growth = 14.28%). On the other hand, the males in the present study needed to grow 6 cm (35.29% of the maximum size), so that 30% of individuals with gonads passed to 50%. Oliveira et al. (2019) reported that the population of A. bifasciatus in the lower Iguaçu River basin needed to grow 0.7 cm to pass from 30 to 50% of mature individuals, a growth of 5.83%. Dala-Corte and Azevedo (2010) reported an even lower percentage of growth (2.86%) in A. henseli, with a difference between 30 and 50% of individuals with gonads of only 0.4 cm.
For females, the results are even more alarming (Table 10). Other studies with species of the genus Astyanax observed a much lower need for proportional growth (1.11-5%) than that obtained in the present study, in order to increase from 40% of individuals with gonads to 50% (Souza et al. 2015, Dala-Corte and Azevedo 2010, and Oliveira et al. 2019, respectively). For the population of A. lacustris in the Pantanal, it was necessary to increase 0.3 cm (4.28% of the maximum size) to pass from 40% of individuals with gonads to 50% (Súarez et al., 2017). A. lacustris females from the lower Doce River needed to grow 22.22% to increase from 40% of individuals with gonads to 50%. Only one female was collected with a length between 14 and 15 cm, and it did not have evident gonads. This latency in the formation of gonads is a strong indication of a disruption in the reproductive biology of the population of A. lacustris from the lower Doce River and deserves to be the target of future investigations. Previous studies have reported that chronic exposure to metals, such as Al, can cause a delay in the sexual maturation of fish, as well as increased spermatogonia and spermatocytes and a significant decrease in spermatids and spermatozoa (Paschoalini et al., 2019). In fact, Chaube et al. (2010) demonstrated that metals are able to bind to androgen and progesterone receptors changing their functions.
Another factor observed is that A. lacustris individuals from the lower Doce River showed a size greater than 60% of the maximum size for 50% of the population to have gonads (male = 64% and female = 61%). The population of A. lacustris of the Paraguay River has 50% of individuals with gonads with 44.28% of the maximum size, for both males and females.
The reproduction peak found in this study was in Sep Oct for females and Jan Feb for males during the rainy season in the region. Species of the Astyanax genus usually have a seasonal breeding strategy, increasing in the rainy season, between spring and summer (Dala-Corte and Azevedo 2010). Gurgel et al. (2004) and Araújo et al. (2019) reported the same reproductive peak in the summer, coinciding with rainy peaks in the region. These results corroborate the results obtained in the present study. Súarez et al. (2017) also found this increase in the periods from Nov Dec to Jan Feb, and reported that both the increase in rain and temperature create more favorable conditions for the development and feeding of juveniles (Oliveira et al. 2010). In addition, the high volume and high waterflow found at UHE Mascarenhas may influence the increase in reproduction during the rainy season as a strategy to maximize the fitness of A. lacustris.
Although the present study found a higher percentage of gonads with reproductive capacity in the rainy season, the other periods also showed matured gonads in a lower percentage, which indicates multiple reproduction. Souza et al. (2015) suggest that this fragmented reproduction strategy may be associated with the unpredictable nature of the region's conditions, a reality observed at UHE Mascarenhas due to the high volume of water released in the region. Súarez et al. (2017) raise the hypothesis that the hydrological unpredictability of the breeding regions of Astyanax species leads to the evolution of different reproductive strategies for the species of the genus. Some populations use total reproduction as a strategy to maximize fitness, such as the population of A. lacustris in the upper Paraná River basin. Other populations –such as A. bimaculatus, studied by Araújo et al. (2019)– presented results, similar to the present study, in a reservoir in Paraíba do Sul in southeastern Brazil. Araújo et al. (2019) justify this behavior as a way of reducing juvenile predation and competition for food and shelter. With multiple reproduction, different niches are occupied and this leads to less competition among adults for spawning sites and, among larvae, for food resources.
4.3. Concentration of metals in the gonads
Through a study done with Barbus grypus, Barbus sharpeyi and Cyprinus carpio, Alhashemi et al. (2012) concluded that sex is an important factor and that it can interfere with the bioaccumulation of metals in fish. In B. grypus and B. sharpeyi, a greater bioaccumulation of metals was observed in females when compared to males. However, in C. carpio, males showed greater bioaccumulation of metals when compared to females. This result corroborates with the ones observed in the present study since, in relation to Al, Cr, Fe and Pb, males bioaccumulated significantly higher concentrations than females. The other metals showed no significant difference in relation to the sex of the individuals.
According to Passos et al. (2020), the lower Doce River is a region that has frequent resuspension events that can make elements, previously sedimented, available again in the water column. Silva et al. (2010) stated that the period from October to March had the highest rainfall. Thus, the metals considered the most abundant in the lower Doce River region (Al and Fe) would become available and toxic during such resuspension events. Passos et al. (2020) reported high concentrations of Fe in the elutriate and sediment of the Doce River in November 2018, with an average of 590 µg.L− 1 and 26.04 mg.kg− 1, respectively. This corroborates the result found in the present study, which showed a higher concentration of Fe in the gonads of males in Sep Oct.
In freshwater fish, Al is known to affect their reproduction, as in the case of Oncorhhynchus mykiss (Hwang et al. 2000), due to a directly proportional reduction in vitellogenesis. In addition, acidified water, result of high concentrations of Al (Hwang et al. 2000 and Correia et al. 2010), is known to impair fish reproduction by affecting fertility, egg viability, spawning success, gonadal development, and production of gametes. These effects have serious consequences for fish populations exposed to Al (Correia et al. 2010). Passos et al. (2020) state that, in addition to Al, Fe also causes deleterious effects in fish exposed to the metal, which can lead to increased mortality and histopathological changes in liver cells. When compared to the results of the present study, it is concluded that, in addition to histopathologies in liver cells, Fe may also be responsible for histopathologies in gonadal cells.
Although Al and Fe showed higher concentrations in the gonads of the organisms, Cr presented a greater bioaccumulative effect in relation to other metals when BCF was calculated. Despite being an essential metal, Cr – in its hexavalent form, which normally corresponds to a fraction of the total Cr found in water (Campagna et al. 2013)– is considered mutagenic, carcinogenic and has several harmful impacts on biota (Bakshi and Panigrahi 2018). Bakshi and Panigrahi (2018) revealed that Cr can affect the behavioral, histological, biochemical, genetic, and immunological conditions of organisms; thus, it can be concluded that, although the concentration of Cr in the gonads was low, its potential for bioconcentration and contamination can lead to changes in the fish exposed to it.
4.4. Histological damage to the gonads
Among the histopathologies observed in the testicles of Clarias gariepinus exposed to xenobiotics, there is the release of immature cells into the lumen (Agbohessi et al. 2015). Da Cuña et al. (2013) also reported similar changes in Cichlasoma dimerus males exposed to an insecticide. The release of immature cells into the lumen is indicative of deficiency in spermatogenesis and, possibly, testicular functionality (Da Cuña et al 2011). Both the studies by Agbohessi et al. (2015) and Da Cuña et al. (2011; 2013) corroborate the results obtained in this study, which observed the release of immature cells into the lumen in the testicles of A. lacustris in greater quantity (47.36%) when compared to other histological damages found. The presence of testicular oocytes, found in a smaller quantity, is of great concern in the reproductive biology of A. lacustris. In dioecious fish, the presence of intersex is considered a signature of exposure to xenobiotics (Luzio et al. 2016). Although the genotype of intersex fish had not been determined, the macro and microscopic aspects of their gonads indicated that the individuals were male (Prado et al. 2011). Intersex gonads have been found in several studies carried out in rivers contaminated with xenobiotics in North America (Fossi et al. 2004), England (Hinck et al. 2009) and in the Mediterranean Sea (Jobling et al. 2009). Prado et al. (2011) conducted a study with Astyanax fasciatus in a reservoir in southeastern Brazil and, comparing the results obtained, this study found few individuals with the presence of gonads in intersex. Prado et al. (2011) reported a high prevalence of intersex in the studied organisms and suggested a high risk of contamination by xenobiotics at the collection sites. Although the prevalence in this study was lower, the presence of fish with intersex can directly impact the development and reproduction of offspring and, consequently, the population of A. lacustris in the lower Doce River can be affected in the long term (Prado et al. 2011).
According to Dyer (2007), metals can act as xenobiotics in exposed organisms through specific high-affinity pathways, such as, for example, the interaction with estrogen receptors. In a study by Luzio et al. (2016) with Danio rerio, the authors emphasize that exposure to xenobiotics can delay the development of oocytes, inducing them to death and potentially reducing the individual's reproductive success. Luzio et al. (2016) found a significant number of atretic oocytes and considered it the major responsible for the increase in the damage rate in the collected female gonads. Atresia is a degenerative and resorption process that occurs, both naturally and under conditions of environmental contamination (Weber et al. 2003). The results obtained by Luzio et al. (2016) corroborate the results found in the present study. Oocyte atresia was the most frequently damage found in the female organisms studied, with a prevalence of 39.64% in relation to the other histological damages analyzed. Studies with Pimephales promelas (Kidd et al. 2007) and Chalcalburnus tarichi (Kaptaner and Ünal 2011) also obtained similar results, with a high rate of atresia in organisms exposed to xenobiotics.
Cardoso et al. (2018) performed an experiment with Danio rerio females in order to confirm the applicability of the semi-quantitative method of counting in female gonads. Despite correctly establishing the stage of gonadal maturation, Cardoso et al (2017, 2018) concluded that, if the objective was to obtain a general classification of maturation or some differences between treatments, the semi-quantitative method is considered appropriate; however, if the objective of the work is to explore in detail the structural components of the gonads (such as primary, cortical, vitellogenic, and previtellogenic oocytes), the recommended method is the quantitative one. The present study confirms this statement since the stages of gonadal maturation of males and females were successfully determined by the semi-quantitative method. Nonetheless, when a more detailed analysis of female gonads was made using the quantitative method, the correlation between histological damage and the concentration Al and Fe was significantly positive. The qualitative method did not obtain any significant results in relation to metals. Moreover, when the results were classified into levels of histological damage, the semi-quantitative method classified females classified as Class 2 of histological damage with moderate changes. On the other hand, the quantitative methodology classified females as Class 3 of histological damage with clear alterations in the tissue of the organ. Thus, it is concluded that the method of quantitative determination of histological damage in female gonads from A. lacustris is more recommended than the semi-quantitative method.