Fish have been used as environmental biondicators in a number of genotoxic studies (Grisólia and Cordeiro, 2000; Souto, 2004; Arias et al., 2007; Costa et al., 2008; Delunardo et al., 2013; Carrola et al., 2014; Lima et al., 2015; Rocha et al., 2016; Bueno et al., 2017; Hussain et al., 2018; AnvariFar et al., 2018; Delunardo et al., 2020; Santana et al., 2020). These vertebrates are appropriate models for this type of research due to their capacity for the metabolization and accumulation of pollutants that also pose a risk to humans (Ranzane-Paiva, 2013; Ventura, 2015; Lima et al., 2015). Plagioscion squamosissimus is an important fishery resource in the Amazon region, providing subsistence and a source of income for many local populations (Chao et al., 2015; Barbosa et al., 2021), although this species is also an important animal model for the biomonitoring of aquatic environments (Viana et al., 2013; Rocha et al., 2016), given its sensitivity to toxic substances, as well as its relative abundance in most areas (Jonsson and Castro, 2005; Santos et al., 2020). In the present study, significantly higher levels of damage were recorded in the P. squamosissimus treatment (contaminated) group in comparison with the control in both the comet assays and the micronucleus test.
In the present study, the fish collected in the municipality of Barcarena were shorter (TL), on average, than those from Vigia, but heavier, with higher frequencies of micronuclei, nuclear alterations, and indices of comet damage in comparison with Vigia, which point to the influence of the differences in the quality of the water in the two estuaries. In particular, while the maximum damage class (4) was the most frequent in the Barcarena samples, with 28% of the nucleoids analyzed, it was the least frequent class (1.8%) in the Vigia samples, where a majority of the nucleoids (59%) were undamaged (class 0). The results of the micronucleus test were similar to the findings of Santos et al. (2015), who analyzed the weight-length ratio and the frequency of micronuclei in the tambaqui, Colossoma macropomum, exposed to agricultural toxins and herbicides in northern Brazil. As in the present study, these fish had gained relatively more weight than length, and the correlation between the weight-length ratio and the micronucleus test may be a useful parameter for the biomonitoring of contaminated environments (Santos et al., 2015), as well as providing important insights into the physiological condition of fish exposed to pollution, given that animals from more impacted environments tend to have a lower condition factor (K) than those from unpolluted areas (Oliveira-Ribeiro et al., 2013).
As in the present study, descriptive indicators of size classes have been used in a number of studies to evaluate the seasonal and spatial variation in fish communities, which may be related to shifts in the behavior of the species and their physiological response to pollution (Arias et al., 2007; Costa et al., 2008; Copatti & Copatti, 2011. Santos et al., 2015). In Senegal, for example, a reduction of the maximum length of the fish of a community was observed after 20 years of anthropogenic impact (Ecoutin et al., 2010). However, the fish collected from the Murucupi River in the present study were similar in size to those collected from this same river by Oliveira et al. (2019).
This study showed that the physical and chemical parameters of the water are complemented by the toxicity tests, given that these tests evaluate the potential effects of the substances found in the water on the local biological system. In the present study, the dissolved oxygen concentrations recorded in the Murucupi River were lower than those found in the control area, but higher than those recorded by Pereira et al. (2007), that is, 2.6 mg/L, which is well below the concentration recommended in resolution 357 of the Brazilian National Environment Council, CONAMA (DO > 5.0 mg/L). These authors associated the low dissolved oxygen concentration recorded in the Murucupi River with a significant increase, above natural levels, in the organic matter in the water, which was derived from the effluents discharged into the river. The relatively acid pH recorded in both study areas in both seasons is typical of Amazonian watercourses, which tend to be relatively rich in kaolinite and humic acid derived from the decomposition of plant material (Pereira et al., 2007; Medeiros et al., 2017).
The endogenous occurrence of micronuclei is well documented, although it rarely exceeds one micronucleus per thousand cells, i.e., 0.1% (Thomé et al., 2016). There is no strict consensus on the number of cells that need to be analyzed per animal to provide a reliable estimate of the frequency of micronuclei, with published studies being based on samples of between 1,000 and 10,000 cells. Ghisi et al. (2010) investigated the optimum sample size with the aim of standardizing procedures, and compared counts of 1,000, 2,000, 3,000, and 4,000 cells, eventually reaching the conclusion that a sample of only 1,000 cells provided satisfactory results. In the present study, the counts of 2,000 erythrocytes per animal proved to be more than adequate for the identification of micronucleated cells and nuclear alterations in the erythrocytes of P. squamosissimus from the two study estuaries. The numbers of micronuclei and morphological nuclear alterations found in both Barcarena and Vigia were similar to the findings of Rocha et al. (2016), who also used P. squamosissimus as a bioindicator in the Marajó Archipelago of northern Brazil, although the values were much higher than those recorded in the Nile tilapia, Oreochromis niloticus (Cichlidae), by Bueno et al. (2017) from a reservoir in southeastern Brazil during both the dry and rainy seasons. The high genotoxic concentrations observed in the water from the impacted area during the rainy season may be linked to the more intense rains, given that Silva et al. (2014) found that more intense rainfall provokes a greater lixiviation of the chemical substances into the river from the surrounding soil.
The comet assay is not used to detect mutations per se, but rather, genomic lesions that can, in fact, be corrected, and can thus also be used to study DNA repair mechanisms, providing important insights into the kinetics and the type of lesion that has undergone repair, although it cannot confirm the adequacy of the repair process itself (Albertini et al., 2000). The frequencies of the nucleoid classes analyzed in Barcarena were similar to those recorded by Rocha (2009) in the peripheral blood of C. macropomum and the saddle cichlid, Aequidens tetramerus (Cichlidae), exposed to different concentrations of methylmercury. This study showed that the damage levels detected by the comet assay were greater than those observed in the micronucleus test, a pattern similar to the results of the present study, which can be considered to be the normal pattern, given that the comet assay evaluates primary DNA damage, which is rarely passed down to future generations of cells.
The high percentages of micronuclei, nuclear alterations, and comet damage observed in the P. squamosissimus specimens from Barcarena may be related, in part, to the piscivorous feeding habits of this species. Porto et al. (2005) evaluated the genotoxic effects of mercury pollution in three fish species of the order Characiformes with distinct feeding adaptations, using the micronucleus test, and found that the mean frequency of micronuclei in the piscivorous species was approximately five times higher than those recorded in the detritivorous and omnivorous species. Hussain et al. (2018) also used the micronucleus test, nuclear alterations, and comet assay to investigate the effects of exposure to industrial and domestic effluents in the rohu, Labeo rohita (Cyprinidae), in the Chenab River in Faisalabad, Pakistan. This study recorded acute levels of toxicity and high contamination rates, which contributed to an increase in the mortality of the fish, which indicated that the water of the river should not be used even for irrigation.
The smelting plant in Barcarena belongs to one of the world’s largest corporations in the aluminum sector. Over the past 20 years, a number of environmental accidents related to the processing of bauxite have been recorded in the municipality, in particular the 2018 incident, when toxic red mud leaked from one of the tailing ponds, which was considered to have been one of the most significant environmental disasters in the history of the Amazon region (Steinbrenner et al., 2020). Following this incident, the company received an official warning, and was required to implement a number of mitigatory measures (Lemos and Pimentel, 2021).
Given the potential impact of the smelting plant, a number of studies have investigated the damage to the Murucupi River, in particular the concentrations of heavy metals present in the water and the local soils (Pereira et al., 2007; IEC, 2009, 2018; Lima et al., 2015; Medeiros et al., 2016 and 2017; Almeida-Junior et al., 2019), as well as the water-borne diseases transmitted to the local population (Marinho, 2016). Pereira et al. (2007) recorded aluminum concentrations 13.2 times higher than the recommended level in the water of the Murucupi River. In high concentrations, aluminum may provoke neurological disorders in human beings, such as Alzheimer’s and Parkinson’s diseases (SILVA JÚNIOR, 2013). In fish, the aluminum dissolved in acidic water, which comes into contact with the gills, provokes an increase in the pH and the formation of insoluble Al2(OH)3, which damages the DNA (Muniz and Oliveira-Filho, 2006; Sultana et al., 2020) and eventually suffocates the animal. This has raised a number of concerns in the population of Barcarena in terms of the quality of the fish available for human consumption in the region. Barros et al. (2010) and Foran (1990) concluded that the health risks of the consumption of contaminated fish are 20–40 times higher than those associated with the ingestion of contaminated water, given that aquatic organisms are capable of concentrating trace elements to up to 105 times the concentrations observed in the environment. Marinho et al. (2016) evaluated the profile of the morbidity of the residents of the municipality of Barcarena and the surrounding area, and concluded that a number of different infirmities caused by parasites, as well as infectious and respiratory diseases, may be maximized by the poor quality of the water and other natural resources used as a source of food by this population. This highlights the need for a more detailed quantitative and qualitative investigation of possible environmental contamination in the region.
The analyses employed in the present study permitted the application of a holistic approach to the investigation of the environmental variables that affect the biological parameters of P. squamosissum, providing, with multivariate analyses, a powerful tool for the interpretation of complex data on water quality. Shrestha and Kazama, (2007) evaluated the efficiency of statistical models for the analysis of water quality data, based on the standard parameters most frequently employed, and concluded that the data on river discharge, temperature, the biochemical demand for oxygen, pH, electrical conductivity, nitrate, and ammoniated nitrogen are the parameters that best correlate with the quality of the water of aquatic environments, and should be employed in this type of analysis. In the present study, the temperature, pH, and dissolved oxygen were employed, with clear results in terms of the deleterious effects on the watercourses and one of the principal local fishery resources, i.e., P. squamosissimus.
Considering the ecological and economic importance of the Amazonian es-tuaries that were the focus of the present study, the identification of the possible impacts caused by local bauxite smelting operations would provide important insights for the implementation of measures designed to minimize the deleterious effects of these impacts on the local biota and, ultimately, the human populations in the area. Adequate mitigatory measures should guarantee the productivity of these environments for future generations, especially considering the importance of their fishery resources as a source of income and subsistence for the traditional local populations. This is the first study of its type in this region, and it provides important insights into the physiological response of the species to the environmental alterations evaluated in the analyses. Direct and immediate action is necessary from the public authorities to ensure the highest level of care with the treatment and storage of the residues in the tailing ponds to avoid potentially major impacts on the local rivers. The present study also provides the first detailed data on genotoxicity in the fish populations of the study area.