Evaluation of the Genotoxicity in Fish Erythrocytes to Diagnose the Water Quality of Two Amazonian Estuaries Using the Micronucleus Test and Comet Assay

CLAUDIA ANTONIA CAMPOS RODRIGUES DE OLIVEIRA (  claudia.engpesca@yahoo.com.br ) Universidade Federal do Para https://orcid.org/0000-0002-1421-2085 Paulo Sergio dos Santos Souto Universidade Federal Rural da Amazônia: Universidade Federal Rural da Amazonia Dulcideia da Conceição Palheta Universidade Federal Rural da Amazônia: Universidade Federal Rural da Amazonia Marcelo de Oliveira Bahia Universidade Federal do Pará: Universidade Federal do Para Lorena Araújo da Cunha Universidade Federal do Pará: Universidade Federal do Para Maria de Lourdes Souza Santos Universidade Federal Rural da Amazônia: Universidade Federal Rural da Amazonia Tatiane do Nascimento Medeiros Rodrigues Universidade Federal do Pará: Universidade Federal do Para Bianca Bentes Universidade Federal do Pará: Universidade Federal do Para


Introduction
The hematological analysis of sh exposed to contaminants has been used increasingly for the monitoring of environmental quality, considering that these organisms respond to toxic compounds in a manner similar manner to other higher vertebrates, and may thus be used as bioindicators for the evaluation of chemical substances that are potentially teratogenic or carcinogenic in humans (Rivero, The Furo da Laura estuary (Fig.2) is the principal hydrographic feature of the municipality de Vigia, in north-eastern Pará, in the state's Salgado microregion. This ample estuary has a heavy daily tra c of both artisanal and industrial shing vessels . The margins of the estuary are dominated by Holocenic salt marshes and mangroves , with the vegetation cover being formed primarily by a mixture of mangrove forest and typical terra rme vegetation (Cardoso, 2009). The local climate is typical of the equatorial Amazon, being super-humid with relatively high temperatures, i.e., 26-39°C (Mesquita, 2020), with a marked division into rainy and dry (or less rainy) seasons, with monsoon-type rains during the six rst months of the year (January through June), that is, the rainy season, and much drier conditions during the second half of the year (July through December), which is de ned as the dry season ).

Collection and preparation of the samples
The specimens were collected with the assistance of local shers, who captured the animals in sh weirs of varying sizes (young and adults, considering the tamanho de primeira maturação sexual estimado por Lima et al., 2019), which contributed to the minimization of any selective effects in terms of specimen size, with stress being reduced using the approach of Ishikawa et al. (2010). The sh were placed immediately in a bucket containing water and 2% benzocaine (190mg/L) for approximately 3 minutes prior to biometry and the collection of blood samples. The standard sample was originally expected to be at least 20 specimens per season per site. Blood samples were collected from blood vessels in the caudal portion of each sh using 3 ml syringes containing the anticoagulant EDTA (ethylenediaminetetraacetic acid), following the method proposed by Ranzani-Paiva et al. (2013) and Ishikawa et al. (2010). Once the samples were collected, each individual was weighed (in grams) with a semi-analytic balance, and its total length (TL, in cm) was measured using an ichthyometer. All individuals were then released back into the water. The blood samples were stored in Eppendorf type micro-centrifugation tubes, which were kept on ice in a cooler at a temperature of 5-7ºC (Ishikawa et al., 2010) for transportation to the laboratory. The comet assays were based on the procedure of Singh et al. (1988), with minor modi cations (Hartmann; Speit, 1997).
The DNA damage was visualized at a magni cation of 1000x under an epi uorescence microscope equipped with a 515-560 nm excitation lter and 590 nm barrier lte. For the comet assays, being allocated to one of the ve damage classes de ned by Collins et al. (2001). Using a cell counter, 100 nucleoids were counted per animal, that is, 50 on each slide, using the visual classi cation approach based on the migration of the DNA fragments, with the damage being assigned to one of ve classes (Wachtel, 2017): 0 (no apparent damage), 1 (some damage), 2 (moderate damage), 3 (extensive damage), and 4 (maximum damage, apoptosis). These numerical values were considered to be the ranks of the damage classes (0-4) for the statistical analyses. The DNA damage index (DI) was calculated according to the following formula: DI (au): [(N1 * 1 + 2 * 2 + 3 * 3 + 4 * 4)] / 100 (total number of analyzed cells), where DI is the DNA damage index, au is the arbitrary unit, and N1-N4 are cells in classes on clean, labeled slides. The frequency of micronuclei was determined based on the method proposed by Carrasco et al. (1990) and Oliveira et al. (2020). Once stained, the slides were visualized under an immersion optical microscope at a magni cation of 1000x. A total of 2,000 cells were analyzed per individual, to determine the number of micronuclei and morphonuclear alterations (MNA) present in each cell, using a Zeiss Primo Star optical microscope at a magni cation of 1000x, with a manual blood cell counter. The data were recorded in Microsoft Excel ® spreadsheets.
In addition to the presence of micronuclei, the nuclear abnormalities present in the cells were described, counted, and classi ed following Carrasco et al. (1990) as: (1) blebbed (nucleus with a minor evagination of the nuclear membrane with euchromatin or heterochromatin); (2) lobed (nucleus with larger and more ample evagination than the blebbed cells); (3) vacuolated (nucleus containing vacuoles) or (4) notched (nucleus well de ned morphologically). The data on the quality of the water of the Murucupi River were obtained from the HIDROWEB site of the Brazilian National Water Agency (ANA), as well as monitoring by the Chemical Environmental Laboratory (LQA) of the Federal Rural University of Amazonia (UFRA) in Belém.

Statistical analyses
The number of micronuclei and morphonuclear alterations were analyzed per specimen, considering the site (natural or anthropogenic), and the relative size of the specimens, as well as all possible combinations of these variables. Prior to the analyses, the data were tested for normality and the homoscedasticity of variances, to ensure the application of one-way and factorial Analyses of Variance (ANOVA), considering an error of 5%. The analyses were all run in the Statistica 10.0 software.
Finally, a Redundancy Analysis (RDA) of all the effects and factors analyzed in the study was run in CANOCO 4.5 (Software for Canonical Community Ordination), with the signi cance of the contribution of the independent variables (study areas and specimen size) to the variability in the data being assessed by 9999 permutations (Monte Carlo Method). This analysis included only the signi cant (p<0.05) variables identi ed in the analysis. The dependent variables (the percentage of micronuclei and the categories of DNA damage) were allocated to separate presence/absence matrices, in which each line represents an individual, with each matrix being related to a second ('treatment') matrix, to which the independent variables were added one by one.

Results
A total of 54 specimens were collected during the present study, in the two estuaries. The sh collected in Vigia were signi cantly larger (TL) on average than those from Barcarena (F = 17.69; p < 0.01), although the specimens from Barcarena were heavier than those from Vigia (Table 1), but the difference was not signi cant (F = 0.085; p > 0.05).
No signi cant variation was found in the pH, although the dissolved oxygen concentrations were lower in the Murucupi River than Furo da Laura. The highest nitrate concentrations were recorded in Barcarena in both seasons (Table 2).

Comet assays
The DNA Damage Index varied signi cantly between the study sites (F=64.96, p<0.01), and was higher in Barcarena than in Vigia (Fig. 3). Table 3 shows the distribution of the different nucleoid classes observed in the samples from the two study sites. On average, 28.25±14.4% of the nucleoids analyzed in the samples from Barcarena were assigned to the highest damage class (4), whereas only 1.8% of the cells in the samples from Vigia were assigned to this class (Table 3).

Micronuclei and morphonuclear alterations
A total of 107,990 erythrocytes were analyzed in the present study. Overall, the largest number of abnormal cells was observed in the specimens from Barcarena ( Table 5). All the nuclei observed with abnormalities were of the evagination type.

Uni-and multi-variate analyses
The sh collected in the municipality of Vigia were larger (TL), on average, than those collected in Barcarena, although the latter were heavier. Signi cant differences were recorded between the Vigia and Barcarena samples in the mean number of cells in damage classes 0, 2, 3, and 4, with the largest numbers in all classes except 0 being recorded at Barcarena, peaking in class 4 (in absolute and relative terms), with a mean of 28.25±14.43 in comparison with 1.8±2.20 at Vigia (Table 5; Fig. 3). The difference between estuaries in class 1 was not signi cant. The morphonuclear alterations recorded at Vigia were not signi cantly different from those recorded at Barcarena, although the presence of micronuclei at Barcarena was much greater than that recorded at Vigia (Table 5; Fig. 3).
In the Redundancy Analysis, the highest nitrate concentrations were recorded in quadrants 1 and 2 ( Fig.  4), which were associated with the impacted area (Barcarena), while the highest pH and dissolved oxygen concentrations were associated with Furo da Laura, where the lowest levels of genomic damage and morphonuclear alterations were recorded, and the lowest number of erythrocytes with micronuclei. Overall, then, the cellular effects analyzed in the present study are associated with the quality of the water in the two study estuaries, given that the factors tested accounted for 76.31% of the variability in the data (Table 6). In the present study, the sh 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 in uence 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 ndings 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 sh 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 , as well as providing important insights into the physiological condition of sh 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).

Discussion
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 sh communities, which may be related to shifts in the behavior of the species and their physiological response to pollution ( 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 signi cant increase, above natural levels, in the organic matter in the water, which was derived from the e uents 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  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 con rm 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 sh 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 ve 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 e uents 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 sh, which indicated that the water of the river should not be used even for irrigation. 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 e ciency 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 shery 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 identi cation 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 shery resources as a source of income and subsistence for the traditional local populations. This is the rst 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 rst detailed data on genotoxicity in the sh populations of the study area.

Declarations
Ethics approval and consent to participate (The collection of the sh specimens analyzed in the present study was authorized by the Ethics Committee for the Use of Animals in Research (CEUA) of the Federal University of Pará in Belém, Brazil, through license number 727721082 (Appendix 1). Specimen collection was also authorized by the federal Biodiversity Information and Authorization System (SISBIO), through license number 15080-9 ) Consent for publication (All authors are aware of the publication of data in this article) Availability of data and materials (Not applicable)

Competing interests (Not applicable)
Funding (This study was nanced in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brasil (CAPES) -Finance Code 001).
Authors' contributions (Not applicable)

Author contribution statement
All authors contributed to the study conception and design.
CACRO "collected and captured the animals in the eld and analyzed the data of the comet and micronucleus assay in the laboratory" PSSS "performed the collection in the eld" DCP "analyzed the micronucleus in the laboratory" MOB "interpreted the data from the comet assay" LAC "performed the comet test procedures" MLSS "performed the water collections and analysis" TNMR "assistance in writing and reviewing the manuscript" BB "assistance in writing and reviewing the manuscript" após a exposição à fração de óleo diesel acomodada em água.