Genetic and biochemical effects induced by iron ore, Fe and Mn exposure in tadpoles of the bullfrog Lithobates catesbeianus

Highlights

• Iron ore, Fe and Mn provoke toxicity during metamorphosis of Lithobates catesbeianus.

• Exposure to Fe and Mn delays development of tadpoles.

• Fe and Mn accumulate in body of L. catesbeianus after exposure to iron ore, Fe and Mn.

• Plasma ferritin concentration increased in tadpoles exposed to iron ore.

• Iron ore, Fe and Mn cause morphological, genotoxic and biochemical effects in tadpole.

Abstract

For decades, the extraction of minerals has intensified in order to meet the demand of industry. Iron ore deposits are important sources of metals, such as iron (Fe) and manganese (Mn). The particulate ores can be dispersed during extraction, transport and storage, with potential to induce biological impacts. Amphibians are very sensitive to environmental stressors. Therefore, the present study aimed to assess the effects of iron ore, Fe and Mn exposure during the metamorphosis of Lithobates catesbeianus. Endpoints analyzed included morphological (biometrical and developmental analyses), whole body Fe and Mn concentration in, plasma ferritin concentration, erythrocyte DNA damage (measured through comet assay and micronucleus test) and liver activity of enzymes involved in oxidative status [glutathione S-transferase (GST) and catalase (CAT)]. Tadpoles were kept under control condition (no contaminant addition) or exposed to iron ore (3.79 mg/L as fine particulate matter); Fe (nominal concentration: 0.51 mg/L Fe as C₁₀H₁₂FeN₂NaO₈; Fe-EDTA); and Mn (nominal concentration: 5.23 mg/L Mn as 4H₂O.MnCl₂) for 30 days. Virtually, no mortality was observed, except for one tadpole found dead in the iron ore treatment. However, tadpoles exposed to iron ore had longer tail than those kept under control conditions while tadpoles exposed to manganese chloride showed higher body length than control ones. Exposure to Fe and Mn induced a delay in tadpole metamorphosis, especially when these metals are presented not as a mixture (iron ore). Tadpoles exposed to iron ore had increased whole body Fe and Mn while those exposed to Fe and Mn accumulated each metal individually. Tadpoles exposed to any of the contaminants tested showed a significant increase in erythrocyte DNA damage and frequency of micronuclei. In addition, they showed higher liver GST activity respect with those kept under control conditions. Plasma ferritin concentration and liver CAT activity were higher only in tadpoles exposed to iron ore. These findings indicated that tadpoles accumulated Fe and Mn at the whole body level after exposure to the single metals or to their mixture as iron ore. In addition, they indicate that Fe and Mn accumulation can induce oxidative stress with consequent significant developmental, genotoxic and biochemical effects in L. catesbeianus tadpoles.

Introduction

Iron ore deposits are associated with the environmental availability of metals such as copper (Cu), zinc (Zn) and magnesium (Mg). They are also important sources of iron (Fe) and manganese (Mn). Indeed, these are the main metals present in iron ore. Thus, iron ore mining and metal casting can influence the cycle of Fe and Mn (Lima and Pedrozo, 2001). In temperate environments, residues from iron ore mining have increased the levels of dissolved ions and particles in suspension, changing the water chemistry and metals bioavailability (Pereira et al., 2008).

For decades, the extraction of iron ore has been intensified in order to meet the industrial demand for steel production (Luz and Lins, 2004). Brazil is the second largest producer of iron ore in the world, while the Espírito Santo state (southeastern Brazil) is one of the largest iron ore exporters in the world (Lima and Pedrozo, 2001, Luz and Lins, 2004, IBRAM, 2012, Carvalho et al., 2014). Activities associated with this production involve iron ore beneficiation and transfer to ports. The iron ore is mined and carried out from the Minas Gerais state (southeastern Brazil), and further beneficiated in the Espírito Santo state. Therefore, the whole environment may be subjected to the impact of the dispersal of fine particulate iron ore resulting from this activity.

Iron is one of the most abundant elements on Earth, after oxygen, silicon and Mg (Cox, 1997). It is easily oxidized, being rarely found in its elemental form (Fe). It is primarily oxidized to the ferrous form (Fe²⁺) and thereafter to the ferric form (Fe³⁺) (Huebers, 1991, O’Neil, 1994, Bury et al., 2011). Fe³⁺ is prone to form iron hydroxides, which are insoluble (Lima and Pedrozo, 2001).

Iron speciation occurs by the oxidation of this element in the water column under the influence and in association with microorganisms and macroorganisms. In the aquatic environment, Fe²⁺ is oxidized to Fe³⁺, with the resulting ferric hydroxide form being precipitated and deposited. However, there are benthic organisms favoring the conversion of Fe³⁺ into Fe²⁺ through redox reactions. Thus, Fe can diffuse back into the water column (Bury et al., 2011). The diffusion/return of Fe²⁺ in the water surface can occur by biodisturbance, physical resuspension and upwelling (Lima and Pedrozo, 2001, Bury et al., 2011).

Iron is essential for life, being important for oxygen transfer, immune function, and DNA synthesis (Berg et al., 2008, Pan et al., 2009). However, the excess of Fe can be harmful, triggering a suite of free radical reactions, which can induce damage to proteins, lipids and nucleic acids (Berg et al., 2008). Animals have developed sophisticated systems for safely storing the excess of Fe. This metal is transported in the blood serum associated with transferrin and binds to specific receptors that are located on the cell membrane. Cellular uptake of Fe into the cell cytosol occurs when this metal is combined with apoferritin to form ferritin, which is a protein that acts as a cellular Fe store (Umbelino and Rossi, 2006). This protein is found mainly in the liver and kidneys (Berg et al., 2008).

Manganese can also be present in the aquatic environment under the oxidized state, thus forming Mn²⁺ and Mn⁴⁺ (Pereira et al., 2008). The latter is the predominant and more soluble form in water. The involvement of this metal in redox processes enables the release of the soluble part present in the sediment into the water column (Takeda, 2003).

As Fe, Mn is a trace metal essential for animals' life, acting primarily in the brain, where it is involved in the synaptic neurotransmission. This metal can be acquired through the diet or by absorption through the lungs. Its deficiency in the diet affects Mn homeostasis and neural activity. However, excess of Mn acts as a toxic agent to the brain, as this metal has pro-oxidant activity. Abnormal concentrations of Mn in the brain, especially in the basal ganglia, are associated with neural disturbances like the Parkinson’s disease (Takeda, 2003, Vieira et al., 2012).

Factors influencing water quality can be harmful and affect aquatic animals' health under natural conditions. In aquaculture, including frog cultivation, changes in water quality can induce mortality of cultivated organisms and important economical losses for the producer. Frog production in captivity for commercial purposes has been a very common practice; the most used species is the bullfrog Lithobates catesbeianus. This species is found living in permanent water bodies, where it feeds and reproduces at a high rate.

In light of the above, the aim of the present study was to evaluate the effects of iron ore, Fe and Mn during the metamorphosis of the bullfrog L. catesbeianus. Endpoints analyzed included biometrical, developmental, genetic, and biochemical analyses. Whole body metal (Fe and Mn) concentrations were also considered.