Ecotoxicological evaluation of water from the Sorocaba River using an integrated analysis of biochemical and morphological biomarkers in bullfrog tadpoles, Lithobates catesbeianus

Avaliação ecotoxicológica da água do Rio Sorocaba utilizando análise integrada de biomarcadores bioquímicos e morfológicos em girinos de rã-touro, Lithobates catesbeianus

ABSTRACT

Lithobates catesbeianus tadpoles were exposed for 96 h to water from two sites of the Sorocaba River (summer and winter), Ibiúna (PI) and Itupararanga reservoir (PIR) that contained metals. In the liver, in PI, the glutathione peroxidase (GPx) decreased, and the glutathione S-transferase (GST) and carbonyl proteins (PCO) increased.

In PIR, the glutathione reduced (GSH) increased, while there was a decrease in catalase (CAT), GPx, GST, PCO, and superoxide dismutase (SOD). In winter, GPx and GST increased in both points. Regarding the kidneys, lipoperoxidation (LPO) levels and GST decreased, while GSH increased in the summer. In the winter, LPO increased in PI.

In the muscle, in the summer, there was an increase in GSH and GST and change in PCO. In the winter, the levels of PCO increased and CAT decreased in PIR. The area and volume of the hepatocyte and nucleus area increased in the summer and decreased in the winter. Hepatic melanin decreased in the summer after exposure to PIR water.

There were the systemic effects of Sorocaba River water exposure at different times of the year with alterations in biomarkers at different levels, in which kidney shows highest Integrated Response of Biomarkers (IBR) value followed by liver and muscle. Biochemical biomarkers were more sensitive than morphological ones. The more sensitive biochemical markers were MT, PCO, GST and LPO. These effects confirm the hypothesis of metabolic alteration in bullfrog tadpoles by the Sorocaba River water.

Keywords: Contaminants, Amphibians, Morphology, Oxidative stress, Enzymes

RESUMO

Girinos de Lithobates catesbeianus foram expostos por 96 h à água de dois locais do Rio Sorocaba (verão e inverno), Ibiúna (PI) e reservatório de Itupararanga (PIR) que continham metais. No fígado, em PI, a glutationa peroxidase (GPx) diminuiu, e a glutationa catalase (CAT), GPx, GST, PCO e superóxido dismutase (SOD). No inverno, GPx e GST aumentaram em ambos os pontos. Em relação aos rins, os níveis de lipoperoxidação (LPO) e GST diminuíram, enquanto GSH aumentou no verão. No inverno, LPO aumentou em PI. No músculo, no verão, houve aumento de GSH e GST e alteração em PCO. No inverno, os níveis de PCO aumentaram e CAT diminuiu em PIR.

A área e o volume do hepatócito e a área do núcleo aumentaram no verão e diminuíram no inverno. A melanina hepática diminuiu no verão após a exposição à água PIR . Houve efeitos sistêmicos da exposição à água do Rio Sorocaba em diferentes épocas do ano com alterações em biomarcadores em diferentes níveis, nos quais o rim mostra o maior valor de Resposta Integrada de Biomarcadores (IBR) seguido pelo fígado e músculo.

Os biomarcadores bioquímicos foram mais sensíveis do que os morfológicos. Os marcadores bioquímicos mais sensíveis foram MT , PCO, GST e LPO. Esses efeitos confirmam a hipótese de alteração metabólica em girinos de rã-touro pela água do Rio Sorocaba . A S-transferase (GST) e as proteínas carbonílicas (PCO) aumentaram. No PIR, a glutationa reduzida (GSH) aumentou, enquanto houve uma diminuição na…

Introduction

The presence of metals in aquatic ecosystems increases at an alarming rate and is considered to be a worldwide problem. Although some metals are trace elements necessary for various biological processes of all species (Uriu-Adams and Kleen, 2005), they become toxic at high levels due to an interference with various metabolic processes (De Boeck et al., 2003; Authman et al., 2012).

The toxic effects of metals on aquatic organisms often depend on their ability to increase cell levels of oxygen-reactive species (ROS) (Van der Oost et al., 2003; Viarengo et al., 2007; Atli and Canli, 2010; Barhoumi et al., 2012), as demonstrated in fish (Ruas et al., 2008; Franco et al., 2009; Monteiro et al., 2010; Barhoumi et al., 2012; Carvalho et al., 2012, 2015; Sakuragui et al., 2013) and in amphibians (Veronez et al., 2016; Jayawardena et al., 2017; Boiarski et al., 2020; Carvalho et al., 2020).

ROS and the reactive nitrogen species (RNS) are able to alter biomolecules, changing their structures and functions. In lipids, they can cause peroxidation or lipoperoxidation (LPO) (Palmer et al., 1987; Van der Oost et al., 2003; Vasconcelos et al., 2007) and in proteins they can cause oxidation and provide carbonyl derivatives which can be introduced into these molecules through reaction with aldehydes derived from LPO (Parvez and Raisuddin, 2005; Vasconcelos et al., 2007; Cattaneo et al., 2011; Machado et al., 2014). In nucleic acids, they can cause chromosomal fragmentation or damage to the mitotic apparatus (Heddle et al., 1983; Mouchet et al., 2006).

The main antioxidant and redox regulator in cells to combat oxidation of cellular constituents is reduced glutathione tripeptide (GSH). GSH plays a key role in detoxifying peroxides, RNS, and xenobiotic compounds (such as electrophilic reactive molecules) in cells (Han et al., 2006).

Regarding antioxidant enzymes involved in combating ROS, we have catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR) in addition to the biotransformation enzyme of xenobiotics, glutathione S-transferase (GST) which have been used as biomarkers in biomonitoring programs around the world, using aquatic organisms as bioindicators (Carvalho et al., 2012; Abdel Rahman et al., 2019; Hinojosa-Garroa et al., 2020). These enzymes convert reactive radicals into non-reactive molecules, neutralizing ROS and maintaining the redox state in the tissues (Halliwell and Gutteridge, 2007).

Metals also alter the levels of metallothionein (MT) as demonstrated in fish (Hashemi et al., 2008; Werner et al., 2008; Barhoumi et al., 2012; Sakuragui et al., 2013; Yologlu and Ozmen, 2015) and in amphibians (Carvalho et al., 2017). This molecule binds strongly to metals because it contains about 30% cysteine (Berntssen et al., 2001) and is used as a biomarker for exposure to metals. Its cellular expression is induced by zinc (Zn), copper (Cu), cadmium (Cd), mercury (Hg), cobalt (Co), bismuth (Bi), nickel (Ni) and silver (Ag) (Werner et al., 2008; Barhoumi et al., 2012; Sakuragui et al., 2013; Yologlu and Ozmen, 2015).

Evaluated biomarkers at different levels are an important tool to elucidate systemic effects of aquatic contaminants in anurans (Pérez-Iglesias et al., 2019).

Morphological biomarkers in the liver are used to test effects of xenobiotics in systemic physiology and metabolism. An important biomarker present in the liver is the melanomacrophages (MMs). These cells are widely used to describe effects of environmental stressors (De Oliveira et al., 2017). MMs are melanina-pigmented cells and possess catabolic functions (De Oliveira et al., 2017).

Due to melanin presence in cytoplasm, MMs act as a non-enzymatic antioxidant system (Fenoglio et al., 2005). In addition, liver is an important organ for biotransformation of xenobiotics. This process involves hepatocytes and MM functions (Fenoglio et al., 2005). Therefore, liver alterations are an important response to test aquatic contamination exposure in anurans. Exposure to metals in aquatic environments is particularly harmful to amphibian species and can become an important factor in the decline of their population (Pérez-Iglesias et al., 2015; Soloneski et al., 2016; Carvalho et al., 2017; Carlsson and Tydén, 2018; Boiarski et al., 2020).

Amphibians are vulnerable to the presence of environmental contaminants because they have low mobility, larval stage with periods of their lives in aquatic and terrestrial ecosystems, and permeable skin. Regarding tropical or subtropical species, there is little knowledge of the biology, and some of them are at risk of extinction.

Lithobates catesbeianus is a species whose biology is well known among amphibians, it has great adaptive capacity to any environment, and it is easy to obtain specimens for laboratory tests. Several studies propose the use of L. catesbeianus as a model species with high potential in the evaluation of harmful effects of contaminated water (Boone et al., 2007; Ossana et al., 2013; Paetow et al., 2013; Veronez et al., 2016; Boiarski et al., 2020; Carvalho et al., 2020; Motta et al., 2020) and the results obtained may, to a certain extent, be considered as a subsidy for creating standards for the protection of endemic species in Brazil.

In the present study, we assumed that the Sorocaba River contains metals (Al, Cd, Cu, Mn, and Zn) and that these vary seasonally and cause changes in the different levels of biochemical, morphological and morphometric biomarkers in bullfrog tadpoles, L. catesbeianus. To test our hypotheses, lipoperoxidation biomarkers (LPO), reduced glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione S-transferase (GST) and the concentrations of metallothionein (MT) and carbonyl proteins (PCO) and morphological alterations in the liver, kidney and muscle were selected as stress indicators in tadpoles after exposure to Sorocaba River water.

Section snippets

Study area

The area belongs to one of the six sub-basins that make up the Sorocaba Médio Tietê basin (SMT), it has an area of 929 km² and is located in the southeastern portion of São Paulo State. This hydrographic basin is formed by the Una, Sorocabuçu and Sorocamirim rivers, whose headwaters are located in the municipalities of Ibiúna, Cotia, Vargem Grande Paulista and São Roque that form the Sorocaba River, a tributary of the Itupararanga Reservoir (Fig. 1). The choice of sampling points was based on

Metals in water and sediment

The analyses of the water and sediment samples, from both periods, are shown in Table 1, Table 2, respectively. The water temperature and pH did not show significant changes during the exposure period; however, the temperature was higher in the summer (22–26 °C) and with a lower pH (6.7) compared to the winter (13–18 °C and pH 8.0). The physical-chemical parameters were within the values provided by the Brazilian Environmental Council Resolution (CONAMA, 357/2005) at the beginning of the

Discussion

The evaluation of the physical and chemical parameters of the Sorocaba River revealed a growing condition of anthropic interference and the changes in these factors can influence the bioavailability of metals and, therefore, their toxicity. The available forms of nitrogen in water (for example, nitrate or nitrite), have consequences for the environment and living beings since the occurrence of diseases or toxicity of free ammonia such as the reduction of dissolved oxygen. Thus, according to 

Conclusion

The tadpoles were highly susceptible to water from the Sorocaba River, in both places (PI and PIR) and periods (i.e summer and winter) even in low concentrations of metals, thus could be a pollution sensitive bioindicator. Antioxidant enzyme and molecule measurements, as well as oxidative damage and morphology, showed that these responses were triggered after tadpoles’ exposure to contaminated water. Finally, determining the extent and severity of water contamination by pollutants is difficult, 

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This study was supported by grants from the Brazilian research funding institution FAPESP (no. 2017/23781-9). Fernandes, I.F. would like to thank CAPES (Coordination for the Improvement of Higher Level – or Education- Personnel) for the scholarship for Social Demand. Valverde, B.S.L. received a grant from CAPES and de Oliveira, C. was supported by CNPq (304552/2019-4). The authors were involved in the research oversight and the preparation of the manuscript. There was no conflict of interest

References (115)

• A.M. Abdel-Moneim et al.Histopathological biomarkers in gills and liver of Oreochromis niloticus from polluted wetland environments, Saudi ArabiaChemosphere(2012)
• I. Ahmad et al.Anguilla anguilla L. oxidative stress biomarkers responses to copper exposure with or without bnaphthoflavone pre-exposureChemosphere(2005)
• B.C. Almroth et al.Oxidative damage in eelpout (Zoarces viviparus), measured as protein carbonyls and TBARS, as biomarkersAquat. Toxicol.(2005)
• B.C. Almroth et al.Protein carbonyls and antioxidant defenses in corkwing wrasse (Symphodus melops) from a heavy metal polluted and a PAH polluted siteMar. Environ. Res.(2008)
• A.P.C. Araújo et al.Hepatotoxicity of pristine polyethylene microplastics in neotropical Physalaemus cuvieri tadpoles (Fitzinger, 1826)J. Hazard Mater.(2020)
• G. Atli et al.Response of antioxidant system of freshwater fish Oreochromis niloticus to acute and chronic metal (Cd, Cu, Cr, Zn, Fe) exposuresEcotoxicol. Environ. Saf.(2010)
• G. Atli et al.Response of catalase activity to Ag⁺, Cd²⁺, Cr⁶⁺, Cu²⁺ and Zn²⁺ in five tissues of freshwater fish Oreochromis niloticusComp. Biochem. Physiol., C(2006)
• M.M.N. Authman et al.Metals concentrations in Nile tilapia Oreochromis niloticus (Linnaeus, 1758) from illegal fish farm in Al-Minufiya Province, Egypt, and their effects on some tissues structuresEcotoxicol. Environ. Saf.(2012)
• S. Barhoumi et al.Spatial and seasonal variability of some biomarkers in Salaria basilisca (Pisces: Blennidae): implication for biomonitoring in Tunisian coastsEcol. Indicat.(2012)
• M.H.G. Berntssen et al.Tissue metallothionein, apoptosis and cell proliferation responses in atlantic salmon (Salmo salar L.) parr fed elevated dietary cadmiumComp. Biochem. Physiol., C(2001)
• M.M. BradfordA rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingAnal. Biochem.(1976)
• M.M.P. Camargo et al.How aluminum exposure promotes osmoregulatory disturbances in the neotropical freshwater fish, Prochilodus lineatusAquat. Toxicol.(2009)
• C.S. Carvalho et al.Biomarker responses as indication of contaminant effects in Oreochromis niloticusChemosphere(2012)
• C.S. Carvalho et al.Copper levels and changes in pH induce oxidative stress in the tissue of curimbata (Prochilodus lineatus)Aquat. Toxicol.(2015)
• C.S. Carvalho et al.Blood cell responses and metallothionein in the liver, kidney and muscles of bullfrog tadpoles, Lithobates catesbeianus, following exposure to different metalsEnviron. Pollut.(2017)
• C.S. Carvalho et al.Biomarkers of the oxidative stress and neurotoxicity in tissues of the bullfrog, Lithobates catesbeianus to assess exposure to metalsEcotoxicol. Environ. Saf.(2020)
• B.R.C. Chagas et al.Metabolic responses in bullfrog, Lithobates catesbeianus after exposure to zinc, copper and cadmiumComp. Biochem. Physiol., C(2020)
• G. De Boeck et al.Differential metallothionein induction pattern in three freshwater fish during sublethal copper exposureAquat. Toxicol.(2003)
• G.L. EllmanTissue sulfhydryl groupsArch. Biochem. Biophys.(1959)
• H.I. Falfushynska et al.Responses of biochemical markers in carp Cyprinus carpio from two field sites in Western UkraineEcotoxicol. Environ. Saf.(2009)
• C. Fenoglio et al.Effects of environmental pollution on the liver parenchymal cells and Kupffer-melanomacrophagic cells of the frog Rana esculentaEcotoxicol. Environ. Saf.(2005)
• J.L. Franco et al.Zinc reverses malathion-induced impairment in antioxidant defensesToxicol. Lett.(2009)
• T. Grune et al.Selective degradation of oxidatively modified protein substrates by the proteasomeBiochem. Biophys. Res. Commun.(2003)
• J.A. Heddle et al.The induction of micronuclei as a measure of genotoxicity: a report of the U.S. Environmental Protection Agency Gene-Tox ProgramMutat. Res.(1983)
• M. Javed et al.Studies on biomarkers of oxidative stress and associated genotoxicity and histopathology in Channa punctatus from heavy metal polluted canalChemosphere(2016)
• J.H. Keen et al.Mechanism for the several activities of the glutathione S-transferasesJ. Biol. Chem.(1976)
• R.L. Levine et al.Carbonyl assays for determination of oxidatively modified proteinsMethods Enzymol.(1994)
• C. Machado et al.Effect of temperature acclimation on the liver antioxidant defence system of the Antarctic nototheniids Notothenia coriiceps and Notothenia rossiiComp. Biochem. Physiol., B(2014)
• A.K. Mishra et al.Acute toxicity impacts of hexavalent chromium on behavior and histopathology of gill, kidney and liver of the freshwater fish, Channa punctatus (Bloch)Environ. Toxicol. Pharmacol.(2008)
• F. Mouchet et al.Genotoxic and stress inductive potential of cadmium in Xenopus laevis larvaeAquat. Toxicol.(2006)
• S. Parvez et al.Protein carbonyls: novel biomarkers of exposure to oxidative stress-inducing pesticides in freshwater fish Channa punctata (Bloch)Environ. Toxicol. Pharmacol.(2005)
• P.M. Peltzer et al.Effect of exposure to contaminated pond sediments on survival, development, and enzyme and blood biomarkers in veined treefrog (Trachycephalus typhonius) tadpolesEcotoxicol. Environ. Saf.(2013)
• J.M. Pérez-Iglesias et al.Toxic and genotoxic effects of the imazethapyr-based herbicide formulation Pivot H® on Montevideo tree frog Hypsiboas pulchellus tadpoles (Anura, Hylidae)Ecotoxicol. Environ. Saf.(2015)
• J.M. Pérez-Iglesias et al.Biomarkers at different levels of organisation after atrazine formulation (SIPTRAN 500SC®) exposure in Rhinellas chineideri (Anura: Bufonidae) Neotropical tadpolesEnviron. Pollut.(2019)
• C.B.G. Ruas et al.Oxidative stress biomarkers of exposure in the blood of three cichlid species from a polluted riverEcotoxicol. Environ. Saf.(2008)
• M.M. Sakuragui et al.Integrated use of antioxidant enzymes and oxidative damage in two fish species to assess pollution in man-made hydroelectric reservoirsEnviron. Pollut.(2013)
• A.N. Abdel Rahmana et al.Alleviative effects of dietary Indian lotus leaves on heavy metals-induced hepato-renal toxicity, oxidative stress, and histopathological alterations in Nile tilapiaOreochromis niloticus (L.). Aquaculture(2019)
• H. AebiCatalase
• Standard guide for conducting acute toxicity tests on test materials with fishes, macroinvertebrates, and amphibians. (E 729-96)
• Report of the AVMA panel on euthanasiaJ. Am. Viola Soc.(2001)
• I.S.A. Anni et al.Life-time exposure to waterborne copper III: effects on the energy metabolism of the killifish Poecilia vivíparaChemosphere(2019)
• Standard Methods for the Examination of Water and Wastewater(1992)
• B. Beliaeff et al.Integrated biomarker response: a useful tool for ecological risk assessmentEnviron. Toxicol. Chem.(2002)
• E. Beutler et al.Improved method for the determination of blood glutathioneJ. Lab. Clin. Med.(1963)
• D.R. Boiarski et al.Assessment of antioxidant system, cholinesterase activity and histopathology in Lithobates catesbeianus tadpoles exposed to water from an urban streamEcotoxicology(2020)
• N.C. Bonai et al.Distribution of metals in the sediment of the Itá Reservoir, BrazilActa Limnol. Bras.(2009)
• M.D. Boone et al.Multiple stressors in amphibian communities: effects of chemical contamination, bullfrogs, and fishEcol. Appl.(2007)
• G. Carlsson et al.Development and evaluation of gene expression biomarkers for chemical pollution in common frog (Rana temporaria) tadpolesEnviron. Sci. Pollut. Res.(2018)
• D.C.P. Casarini et al.Relatório de estabelecimento de valores orientadores para solos e águas subterrâneas no Estado de São Paulo(2001)
• R. Cattaneo et al.Tissue biochemical alterations of Cyprinus carpio exposed to commercial herbicide containing clomazone under rice-field conditionsArch. Environ. Contam. Toxicol.(2011)

There are more references available in the full text version of this article.