In the present study, there was an increase in the hepatic GST activity at the highest concentrations of metals. Furthermore, genotoxic damages were also detected in the two tested concentrations of Fe + Mn (T2 and T3). Similar results are reported by Coppo et al. (2018) and Passos et al. (2020) that found changes in biochemical, genetic, and physiological functions in O. niloticus exposed to high concentrations of Mn and in Astyanax lacustris (Characidae) exposed to the Doce River water. The exposure of aquatic organisms to metals, in addition to causing ionic and osmotic disturbances in some species, can alter aerobic and energetic metabolisms and induce the generation of reactive oxygen species (ROS), causing important oxidative damage to biomolecules, such as lipids, proteins, and DNA (Jijie et al. 2020). O. niloticus is a species of worldwide importance in aquaculture and, because of that, it is possible to understand not only the possible damage to the biota but possible risks to human health as well. Several authors have studied the effects caused by various contaminants in tilapias (Alkaladi et al. 2020; Ayyat et al. 2020; Chen et al. 2020; Lopes et al., 2020; Mahboob et al. 2020; Mohamed et al. 2020).
The exposure to Fe + Mn induced a significant increase in the frequency of micronucleated erythrocytes. The formation of micronuclei in the exposed tilapia reflects structural problems or chromosomal changes during mitosis; therefore, it is possible to identify the genotoxic potential of chemicals such as metals, even those essential to metabolism (Kample et al. 2018). The same result was found in acute exposure to Mn at concentrations of 3.88 and 7.52 mg/L in the fish species C. auratus (Valbona et al. 2018), as well as in a study with exposure to iron oxide (0.3 mg/L) in guppy fish (Poecilia reticulate) (Qualhato et al. 2017). Both studies detected the genotoxic potential of isolated metals, and our research, in this way, has been complementing the effects of these metals together.
We detected high levels of DNA damage, with the formation of class 3 and 4 comets in treatments T2 and T3, which are the highest DNA damage levels that can be found. The T3 fishes were the most affected, differing significantly from the other groups. Coppo et al. (2018) found that isolated Mn harms the replication of genetic material in O. niloticus. A study on the exposure of a guppy fish to iron oxide identified comet formation from short experimental exposures (3 and 7 days) to longer ones (14 and 21 days) (Qualhato et al. 2017). With these extents of damage, consequently, there is interference in the accuracy of the genetic material replication in the fish organism. Therefore, the comet assay is an important biomarker for checking acute DNA changes in the presence of Fe and Mn (Hariri et al. 2020). Both analyzes, micronucleus test and comet assay, proved to be efficient to evaluate the effects of these two metals together in O. niloticus, showing good biomarkers for this purpose.
GST is an enzyme of fundamental importance in protecting organisms from environmental stressors. Its activity may increase or decrease when exposed to metals, depending on the concentration and the period of exposure (Guilherme et al. 2008). The increase in the hepatic GST activity may indicate the onset of the organism's detoxification process against the metal, since, this enzyme participates in the biotransformation and conjugation of xenobiotic, and the liver plays an important role in metabolizing contaminants (Landi 2000; Moniruzzaman et al. 2020). Studies corroborate our results, through the detection of changes in aquatic organisms exposed to metals (i.e., Fe and Mn). Valbona et al. (2018), found a significant increase in GST activity in specimens of Carassius auratus, as well as Veronez et al. (2018) reported an increase in GST in liver tissues of tadpoles exposed to Fe, Mn, and iron ore. Thus, GST is a good biomarker to assess the degree of impact and the effects caused by Fe + Mn in fish and may contribute to the understanding of the mechanisms of action of these compounds in the face of environmental variation. On the other hand, CAT activity did not change in any treatment for both organs and hence cannot be considered a contamination biomarker for these associated metals in the gills and liver of O. niloticus. Other metabolic routes may have been activated (Pandey et al. 2003).
Even at low concentrations, associated Fe + Mn was potentially dangerous to fish specimens in the present study. Despite the importance of studying the effect of Fe and Mn together, especially due to the composition of iron ore, few studies portray their synergistic effects on fish. The rupture of the iron mining tailings dam in Mariana, Brazil released several toxic elements in the environment, such as Fe and Mn. According to Queiroz et al. (2018), seven days after the Fundão dam burst (in 2015), the concentration of Fe and Mn found in the sediment of the Doce River estuary were 34,900 and 586 mg/kg, respectively. In 2018, three years after the disaster, metal concentrations remained high, with 26,450 mg of Fe/kg and 1075 mg of Mn/kg (Passos et al. 2020). Thus, with resuspension events in the sediment, which are frequent in rivers, the metals associated with the sediment particles can become bioavailable again in the water column, contaminating the biota present in the river (Queiroz et al. 2018). Hence, understanding how these metals work together is extremely necessary. In general, mining activities are very damaging to ecosystems and the biota present, and in an accident, there is a very high risk of altering the food chain, with persistent damage to local biodiversity in the long term (Espindola et al. 2016).