Geophagus brasiliensis (Teleostei: Cichlidae) as an indicator of toxicity of ornamental stone processing wastes

Journal Pre-proof Geophagus brasiliensis (Teleostei: Cichlidae) as an indicator of toxicity of ornamental stone processing wastes

Graciele Petarli Venturoti, Johara Boldrini-França, Aline Silva Gomes, Bárbara Chisté, Levy Carvalho Gomes PII:

S1532-0456(19)30355-2

DOI:

https://doi.org/10.1016/j.cbpc.2019.108639

Reference:

CBC 108639

To appear in:

Comparative Biochemistry and Physiology, Part C

Received date:

8 August 2019

Revised date:

19 September 2019

Accepted date:

8 October 2019

Please cite this article as: G.P. Venturoti, J. Boldrini-França, A.S. Gomes, et al., Geophagus brasiliensis (Teleostei: Cichlidae) as an indicator of toxicity of ornamental stone processing wastes, Comparative Biochemistry and Physiology, Part C(2019), https://doi.org/10.1016/j.cbpc.2019.108639

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© 2019 Published by Elsevier.

Journal Pre-proof Geophagus brasiliensis (Teleostei: Cichlidae) as an indicator of toxicity of ornamental stone processing wastes. Graciele Petarli Venturotia, Johara Boldrini-Françaa, Aline Silva Gomesa, Bárbara Chistéa, Levy Carvalho Gomesa* a

Laboratory of Applied Ichthyology, Universidade Vila Velha, Rua Comissário Jose

Dantas de Melo, 21. Vila Velha, Espírito Santo, Brazil

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* Address correspondence to [email protected]

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Abstract - Massive exploitation of geological resources may lead to serious

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environmental problems due to the inadequate disposal of the processing wastes, which are potentially hazard to terrestrial and aquatic environments. To evaluate the toxic

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effects of the residues generated in ornamental stones processing, Geophagus

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brasiliensis fish were contaminated with different concentrations of ornamental stone processing waste (OSPW) (250, 500, 750 and 1000 mg.L-1). The contaminated

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aquarium water showed increased total hardness and Ca, Na, K, Mg and Mn content,

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which lead to bioconcentration of Na+, K+ and Mg2+ in G. brasiliensis gills. The highest concentration of OSPW induced slight to moderate histopathological lesions in gills of exposed fish, such as structural detachment, hyperplasia of the lamellar epithelium and incomplete fusion of several lamellae. Micronucleus and comet assays revealed a dosedependent genotoxic damage in fish exposed to the contaminant. The biochemical analysis revealed a slight increase in catalase and reduction in superoxide dismutase activities in exposed fish, indicating that OSPW affects the oxidative stress of G. brasiliensis. The no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) parameters indicate that low concentrations of OSPW (even under 250 mg/L) may be detrimental to exposed organisms by causing oxidative damage. This study demonstrates the toxic potential of OSPW in G. brasiliensis, even 1

Journal Pre-proof in short-term exposure, revealing some morphologic and molecular parameters that may be used as biomarkers in monitoring aquatic ecosystems contaminated with this effluent. Keywords: ecotoxicology; genotoxicity; oxidative stress; morphological alterations; fish. 1. INTRODUCTION

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Countries in which there is extensive exploitation of geological resources, especially

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ornamental stones processing, such as Brazil (Montani 2017), have faced serious

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environmental problems due to accidents involving rejects or intentional allocation of their tailings in natural environments (Rana et al. 2016). According to Braga et al.

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(2010), ornamental stone processing wastes (OSPW) are rich in calcium (Ca2+),

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Magnesium (Mg2+), Sodium (Na+) and Potassium (K+). These ions, in high concentrations, can cause serious damage to the osmotic balance of fishes that live in

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impacted environments (Evans et al. 2011).

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To assess the impacts of pollutants on the environment, it is necessary to measure the effects that these substances cause in organisms that inhabit these ecosystems (Wells et al. 2001; Nikinmaa 2014). In this context, an appropriate group of biological responses, called biological markers or biomarkers, provide a connection between exposure rates to xenobiotics and levels of tissue contamination, and are useful to determine the degree of impact of a pollutant on the biota of an ecosystem (Handy et al. 2003, Hook et al. 2014; Hernández et al. 2019). Biomarkers can be identified at various levels of the biological organization, encompassing from molecular and cellular responses to morphological alterations (Van der Oost et al. 2003). Among biomarkers, those based on molecular and cellular response may indicate the first signs of environmental disturbance and have been more 2

Journal Pre-proof commonly used in biomonitoring programs (Nigro et al. 2006; Nikinmaa 2014). Enzymatic activities, such as Glutathione S-transferase (GST), superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT), as well as genotoxicity (micronucleus and comet assays), are cellular and molecular parameters frequently used in biomonitoring (Stegeman et al. 1992; Ayllon And Garcia-Vazquez 2000; Pacheco and Santos 2001; Çavas and Ergene-Gözükara 2003; Van der Oost et al. 2003). In addition, histopathological changes are also considered sensitive tools for evaluation of

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the effects of pollutants on exposed animals, allowing the detection of direct toxic

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effects of chemical compounds on target organs (Camargo and Martinez 2007). Gills

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and liver are the targets of several types of contaminants and, for this reason,

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morphological changes in these organs are broadly used as indicators of contamination (Mazon et al. 2002).

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Geophagus brasiliensis (popularly known as Cará) is a native fish of South and Central

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America, with a wide geographic distribution, with preference for lentic environments, such as lakes, lagoons, flood plains and low flow streams, inhabiting predominantly

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backwater regions (Almeida et al. 2001, 2002; Ventura et al. 2008; Carvalho et al. 2010; Fragoso-Moura et al. 2016; Morais et al. 2016; Guzmán-Guillén et al. 2017). Since these fish naturally occur near to extractive regions of ornamental rocks, the species is susceptible to OSPWs, therefore should be used as indicator of this source of contamination. So, the objective of this study was to evaluate the genotoxic, enzymatic and morphological effects of acute exposure to OSPW in G. brasiliensis to assess the risks involved in the ornamental stones processing industry.

2. MATERIAL AND METHODS 2.1 Fish obtaining and acclimatization 3

Journal Pre-proof Fish were purchased from a hatchery and were acclimatized for 30 days in polyethylene tanks with capacity of 250 L, located in Laboratory of Applied Ictiology from University of Vila Velha (Vilha Velha, ES, Brazil), with constant aeration and daily feeding (Propescado commercial feed composed of 45% crude protein of high performance). The water from the acclimation tanks was partially changed (70%) once a week, and the bottom water was siphoned every day. After acclimatization, fish were submitted to the experiments, following animal experimentation welfare guidelines

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described by Nickum and colleagues (2004). 2.2 Contaminant

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OSPW samples were obtained from five stone processing companies (marble and

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granite) of high production capacity. OSPWs were collected after at least 48 h of marble

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and granites sawing, cutting and polishing to achieve a realistic waste composition. The samples were kept in closed containers and, for the experiments, a fraction of 200 g of

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each industry waste was collected. The collected samples were placed in an incubator at

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60 ºC for 24 hours for moisture reduction, being weighed at the end of the process. Samples were incubated again in the same conditions and weighed every hour until achievement of constant weight. Fractions were then grounded and homogenized, and aliquots of same weight of each sample were pooled. Aliquots were withdrawn from the pooled sample for fish treatment in the moment of the experiments. 2.3 Experiment with G. brasiliensis Fish (5.11±1.83g and 6.68±0.73cm) were individually transferred to six litters aquaria and again acclimatized in filtered and dechlorinated water for 48 hours with constant aeration and without feeding. After animal acclimation, water was totally exchanged to contaminated water (concentrations of 0, 250, 500, 750 and 1000 mg of OSPW per liter of water). Each concentration was tested in six replicates, totaling 30 aquaria. Once all 4

Journal Pre-proof fish were placed in individual aquariums, the dissolved oxygen (DO) concentration was measured and the aeration was adjusted to achieve similar DO concentrations. Exposure of fish to the contaminant were carried out during 96 h, without feeding, with constant aeration and temperature. At the end of the experiment, some parameters, such as DO (mg/L), pH, temperature (T, ºC) and electrical conductivity (EC, μs/cm), were measured using a YSI Environment multiparameter apparatus directly in the aquaria. The total water hardness was measured by titration with EDTA (Federation and Association

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2005), with no dilution. A sample of aquaria water was acidified (pH < 2.0) for further

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determination of Cd, Pb, Mn and Cr using an atomic absorption spectrometer (AAS)

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ICE 3500, coupled to a graphite furnace GFS35(Z) (Thermo Fisher Scientific, Waltham,

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MA, USA). Mg, Ca, Na and K concentrations in water samples were measured by flame

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mode of ICE-3500 AAS. 2.4 Tissue collection and preparation

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After exposition, fish were individually removed from the aquaria and euthanized with

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Benzocaine solution (0.2 g/L). The blood was collected by caudal vein puncture for micronucleus and comet assays. After blood collection the following tissues were extracted: i) left gill arches for histopathological analysis; ii) right gill arcs for analysis of ion concentration (Mg2+, Ca2+, Na+ and K+); and iii) liver for analysis of oxidative stress. The livers were packed in microtubes and stored at -80 °C until analysis. The left gill arches were placed in individual cassettes, immersed in 2.5% glutaraldehyde (GTA) for 24 h and then transferred to 0.5% GTA solution, where they were maintained until preparation of the histological section. The right gill arches were weighed, placed in 15 mL falcon tubes and kept in ice until processing. 2.5 Genotoxic damage

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Journal Pre-proof For micronucleus test, the blood collected from each animal was immediately dripped and smeared onto two slides. Erythrocytes were fixed for 30 min in methanol (100%) and stained with Giemsa (5%), as described by Grisolia et al. (2005). Cells were counted on a microscope Nikon Eclipse E200LED, with a 100x objective, with the aid of immersion oil. To determine the number of micronuclei, the frequencies were calculated and expressed per 1000 cells in each slide, obtaining an average from the blind count of two slides per fish analyzed.

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DNA fragment analysis was performed using comet alkaline assay and silver nitrate

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staining, as described by Tice et al. (2000) and Andrade et al. (2004), with some

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modifications. Blood samples were diluted 20-fold in phosphate buffer and thereafter,

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10 μl of the diluted blood was mixed with 100 μL of low melting agarose (0.75%) at 40 °C. The blood was then placed on slides pre-coated with agarose (1.5%) and covered

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with coverslips. The slides were kept on ice until solidification. After removal of the

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coverslips, the slides were placed into a lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton X-100 and 10% DMSO, pH 13) for 4 h, with light protection. After

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that, the slides underwent an electrophoretic run at 25 V and 300 mA for 15 min at low temperature. Thereafter, the slides were neutralized with Tris buffer (0.4 M) and fixed with fixative solution (15% trichloroacetic acid, 5% zinc sulfate heptahydrate, 5% glycerol) for 10 min. Slides were stained with silver nitrate and the images of 100 randomly selected nuclei (50 nuclei of each slide) were visually analyzed. Each image was classified according to the comet tail length (class 0 for no damage up to class 4 for maximum damage), adapted from Kobayashi et al. (1995). The damage index (ID) was calculated with the sum of the number of nucleoids observed in each damage class, multiplied by the value of their respective class of damages.

2.6 Enzymatic analysis 6

Journal Pre-proof For enzymatic analysis, animals' livers were thawed, homogenized with phosphate buffer (pH 7.0) and centrifuged (18000 x g) for 30 min at 4 °C. The supernatant was used for the enzymatic analyzes. Total protein content in the supernatant was determined by Bradford method (Bradford, 1976). The enzymatic activity of Glutathione S-Transferase (GST; E.C. 2.5.1.18) was determined using phosphate buffer (pH 7.0), 1 mM GSH and 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The enzyme kinetics was determined by absorbance at 340 nm in a Spectramax 190

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spectrophotometer (Molecular Devices, San José, CA, USA), and the absolute activity

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was estimated using the extinction coefficient of CDNB (Habig et al. 1974; Habig and

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Jakoby 1981). The enzymatic activity of Catalase (CAT; EC 1.11.1.6) was estimated by

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continuous evaluation of the decrease of hydrogen peroxide (H2O2), according to Aebi (1984). Samples were evaluated at 240 nm in a spectrophotometer SP-220 (Bioespectro,

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Curitiba, PR, BR). The enzymatic activity of Glutathione Peroxidase (GPX; EC

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1.11.1.9) was determined as described by Hopkins and Tudhope (1973). The kinetic activity of GPX was calculated by absorbance at 340 nm on a Spectramax 190

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microplate reader (Molecular Devices, San José, CA, USA). Absolute activity was estimated using the NADPH reduction coefficient. The enzymatic activity of Superoxide Dismutase (SOD; EC 1.15.1.1) was determined as described by Flohé and Ötting (1984). The kinetic activity of SOD was calculated by absorbance at 550 nm, measured in a SP-220 spectrophotometer (Bioespectro, Curitiba, PR, BR). Absolute activity was estimated using the cytochrome C reduction coefficient.

2.7 Determination of ions in the gills Gill samples were digested using concentrated nitric acid. After 24 hours, the digested material was diluted for the analyzes. The concentration of Na+, K+, Ca2+ and Mg2+ was measured by Flame Atomic Absorption Spectrophotometry (FAAS) on an ICE 3500 7

Journal Pre-proof equipment (Thermo Fisher Scientific, Waltham, MA, USA). Standard solutions were made with concentrated analytical reagents (Merck, Darmstadt, DE) and each standard curve was performed with at least five different concentrations. 2.8 Histopathology Gill samples was dehydrated by increasing alcohol concentration every hour until reaching 100%, in which it was incubated overnight. Tissues were infiltrated with

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historesin solution (Historesin kit, Leica Biosystems, Wetzlar, DE) and placed in

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histomold wells with a mixture of 1:15 (v/v) of embedding and hardening solution, respectively. For each gill, eight semiseriate longitudinal cuts of 0.5 to 0.8 μm were

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made using a Lupetec (MRP09) microtome. Cuts were mounted into glass slides and

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staining with toluidine blue. Slides were analyzed in a Nikon Eclipse E200LED

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microscope with a 4, 10 and 40x objective.

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2.9 Description and classification of histopathological alterations

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The occurrence of histopathological changes in the organs was semi-quantitatively evaluated by two ways: a) calculation of the Mean Alteration Value (MAV), according to Schwaiger et al. (1997), which assigns a numerical scale for damage incidence (1 absence of histopathological alterations; 2 - occurrence of localized lesions and 3 lesions widely distributed in the tissue); and b) Determination of the Histopathological Alteration Index (HAI), based on the severity of each damage, according to Poleksic and Mitrovic-Tutundzic (1994). For HAI determination, tissue were initially classified into progressive stages of damage: SI for alterations that do not compromise the tissue functions (structure detachment, lamellar epithelial hyperplasia, lamellar disarrangement, incomplete fusion of several lamellae, complete lamellar fusion, dilation of the blood sinuses, telangiectasia, vascular congestion and mononuclear 8

Journal Pre-proof infiltrate); SII for more severe alterations that impair tissue functions (rupture of the lamellar epithelium without hemorrhage, rupture of the lamellar epithelium with hemorrhage and lamellar aneurysm) and SIII for very severe and irreversible alterations (necrosis). HAI was then calculated for each gill by the formula HAI = (1 X SI) + (10 X SII) + (100 X SIII). Tissue alterations were indicated by HAI as following: 0 to 10 for normal tissue function; 11 to 20 for slight damage; 21 to 50 for moderate damage; 51 to 100 for severe damage and values greater than 100 for irreparable damage (Poleksic and

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Mitrovic-Tutundžic 1994). 2.10 Statistics

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Water parameters were analyzed by one-way ANOVA, followed by Tukey's test

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(p<0.05). The remained results were fitted to a polynomial regression with the OSPW

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concentration as independent variable and the response of biomarkers as the dependent variable. Data were presented as mean and standard error in graphs.

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To provide information concerning risk assessment, the no observed effect

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concentration (NOEC) and the lowest observed effect concentration (LOEC) were determined. NOEC was considered as the highest OSPW concentration in which there was not significant alteration compared to control by an ANOVA e Dunnett´s test (p<0.05), while LOEC was considered as the lowest concentration in which it was observed significant effect in relation to control (p<0.05). All statistical analyzes were performed in SigmaPlot, version 12.5 (Systat Software, San Jose, CA).

3. RESULTS The mean water temperature during the experiment was 27.03 ºC ± 0.11 and the mean DO concentration was 7.19 mg/L ± 0.21, with no significant differences among treatments. The values of monitoring parameters (pH, electrical conductivity, total 9

Journal Pre-proof hardness and ions) of aquarium water are indicated in Table 1. The results show a significantly rise of all the physical-chemical parameters, such as pH and conductivity and hardness, as well in Mg, Ca, Na, K and Mn concentration in response to the increase in OSPW concentration. The concentration of Cd, Pb and Cr were below the detection limit of the equipment (data not shown). The micronucleus and DNA damage (comet assay) frequencies are shown in Figure 1, while the activities of catalase, glutathione S-transferase, glutathione peroxidase and

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superoxide dismutase enzymes are shown in Figure 2. A crescent genotoxic activity was

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observed in fish in response to the increase in OSPW concentration (Fig. 1). Regarding

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the enzymatic activity, different results were observed for each enzyme. Catalase and

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superoxide dismutase activities were positively correlated with OSPW concentration,

response to OSPW (Fig. 2).

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while glutathione S-transferase and glutathione peroxidase enzymes had no dose-

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The concentrations of Na+, K+, Ca2+ and Mg2+ found in the gills of the animals exposed to OSPW are shown in figure 3. There is a positive correlation between Na+, K+ and

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Mg2+ in gills and OSPW concentration, but no correlation was observed for Ca2+ in response to OSPW. The histological analysis of the gills of G. brasiliensis showed that the alterations are characterized by localized damages in this organ, since MAV was 1 for the control and exposed group at the concentration of 250 mg/L, 1.33 at 500 mg/L, 1.83 at 750 mg/L and 2 at 1000 mg/L. The histopathological alteration index (HAI) of the gills showed a positive correlation with OSPW concentration (Fig. 4). The histopathological analysis showed crescent frequencies of structural detachment and hyperplasia of the lamellar epithelium in gills of fish exposed to 750 and 1000 mg/L of OSPW, while an increase in frequency of incomplete fusion of several lamellae was observed at concentrations greater than 500 mg/L (Fig. 5).

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Journal Pre-proof Table 2 presents the NOEC and LOEC values for each analyzed parameter. NOEC concentration for Micronucleus, DNA damage, SOD and HAI was 250 mg/L of OSPW, while the first toxic effects to the contaminant (LOEC) were observed in the dose of 500 mg/L. LOEC concentration for CAT activity was 250 mg/L, however the tested OSPW concentrations did not allow the determination of NOEC for this enzyme.

4. DISCUSSION

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The growing concentration of Mg, Na and K in the water contaminated with OSPW was

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proportional to the concentration of these ions in the animal’s gills, indicating their crescent absorption by the tissue as they accumulate in the water. Wurts and

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Perschbacher (1994) demonstrated that Ca2+ can be toxic to fish in high concentrations;

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however, this ion was not bioconcentrated in the gill of G. brasiliensis, indicating that

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Ca2+ did not contribute to the toxic effects induced by OSPW. The mitigative effect of magnesium on calcium absorption by G. brasiliensis should be considered, since a

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protective effect of magnesium against metal ion bioavailability and toxicity has already

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been reported (Schamphelaere and Janssen 2002). In G. brasiliensis, as in most teleosts, gills play a fundamental role in the processes of osmoregulation, acid-base balance and excretion of nitrogen compounds, besides being the main organ responsible for gas exchange (Fontaínhas-Fernandes et al. 2008; Evans et al. 2011). The gill epithelium is the main contact surface of the animal with the environment and, in the presence of stressors, the gills may undergo modifications as defense responses. Some of these modifications, such as hyperplasia, aims to increase the diffusion barrier to the pollutant or stressor, while in lamellar fusion, the goal is to widen the distance between external and internal environments, decreasing the surface area in contact with the medium (Erkmen and Kolankaya 2000). However, increased respiratory distress may induce vasodilation, edema and lifting of the gill epithelium 11

Journal Pre-proof (Barišić et al. 2015). Among the damages observed in the histological sections of G. brasiliensis gills, there are structural alterations of the epithelium, epithelial detachment and hyperplasia, all aggravated with the increase in contaminant concentration. Several studies have shown that the exposure of fish to environmental pollutants produces changes in the gill filaments, such as thickening filamentous epithelium, which may arise from the proliferation of chlorine cells and undifferentiated epithelial cells (Schwaiger et al. 1997; Garcia- Santos et al. 2007; Fontaínhas-Fernandes et al.

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2008). Another lesion observed only in the group exposed to the highest concentration

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of OSPW (1000 mg/L), is the rupture of the gill epithelium. According to Mallatt

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(1985), this epithelial rupture reflects the direct effect of the pollutants and occurs in

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situation of marked toxicity.

The micronucleus (MN) test is based the occurrence of chromosomal breaks and losses,

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being one of the most employed methods in the evaluation of the mutagenic effects

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caused by xenobiotics (Udroiu 2006; Farag and Alagawany 2018). In this study, a significant augment in MN occurrence was observed with increasing concentrations of

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OSPW. Similarly, Aguiar et al. (2016) observed an increase in MN frequency when treating Oreochromis niloticus individuals with different concentrations of OSPW for 72 h. Despite the significant differences among groups, MN occurrence was low (maximum of 5 per 1000 cells), possibly due to the reduced hematopoietic rate of fishes, considering that only cells in division has the potential to generate micronuclei (Fenech 2000). Fish erythrocytes may remain in circulation for up to 160 days, depending on the species (Tavares-Dias and Moraes 2004); therefore, acute exposure experiments may result in relatively low number of suitable erythrocytes for the test, which reinforces the importance of performing at least one complementary genotoxic assay in this case (Ghisi and Cestari 2013). The need of additional tests to evaluate

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Journal Pre-proof DNA damage is supported by several studies that reported no significant increase in MN frequencies in acute exposure of fishes to xenobiotics (Bücker et al. 2006; Winter et al. 2007, Ghisi and Cestari 2013; Disner 2017). In this context, a comet alkaline assay was employed of an additional test to evaluate DNA damage in G. brasiliensis exposed to crescent concentrations of OSPW. Intact cells with no DNA damage were predominant in the control group, with few occurrences of DNA breaks, while in contaminated groups, especially in the

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concentration of 1000 mg/L, cells with extensive DNA damage and presenting

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apoptosis signals were the most frequent ones, although intact cells could still be

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observed, as also reported in other studies (Benassi 2004; Bruchchen et al. 2013).

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A slight increase of CAT and reduction of SOD enzyme activities were observed with the increase of contaminant concentration in the water. However, the decrease in SOD

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activity may be a result of pH rise in the medium, since the activity of this enzyme

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decreases in alkaline pH (Halliwell and Gutteridge 2007). The enzymes analyzed in this work act on different metabolic pathways in the

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organism: i) CAT, SOD and GPx act on the primary line of defense of organisms against free radicals by stabilizing superoxide radicals and hydrogen peroxide formed in the respiratory metabolism, avoiding the formation of more toxic radicals (Ighodaro and Akinloye 2017); and ii) GST acts directly on organism’s detoxication in an attempt to transform xenobiotics into less toxic substances (Conrad and Angeli 2017). The increase in CAT activity is possibly a direct indicative of stress caused by the contaminant, since the activity of this enzyme depends only on H2O2 present in the medium (H2O2 + H2O2  2H2O + O2). Some environmental contaminants cause an increase in fatty acids oxidation, which can result in oxidative stress and greater CAT activity in liver (Das et al. 2018; Olivares-Rubio and Vega-Lopez 2016). Furthermore,

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Journal Pre-proof the increase of CAT activity in G. brasiliensis contaminated with OSPW corroborates with the results observed in the histopathological analysis, micronucleus test and comet assay, evidencing the toxic effect of the contaminants in these animals. Although the reduction of GST activity is a direct indicative of exposure of animals to chemical pollutants, no dose-response effect was observed for this enzyme in the testes OSPW dose. Similarly, exposure to OSPW did not influence GPx activity in G. brasiliensis. The efficiency of the hydroperoxide GPx depends on the availability of

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glutathione in its reduced form (GSH), which is also used in the GST catalytic cycle.

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Furthermore, according to Halliwell and Gutteridge (2007), when hydrogen peroxide is

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present at low concentrations (normal physiological conditions), glutathione peroxidase

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is responsible to transform it into water; otherwise, at high concentrations, there is an increase in catalase activity.

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In dose-response toxicological studies, determination of NOEC and LOEC has been

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considered a standard parameter to provide information concerning risk assessment and decision-making of environmental contaminants (Wieczerzak et al. 2016). According to

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NOEC and LOEC obtained in this study, most effective damages occur from the concentration of 500 mg/L of OSPW, although CAT activity was already determined in the lowest tested concentration (250 mg/L). This indicate that low concentrations of OSPW (even under 250 mg/L) may already be detrimental to exposed organisms by causing oxidative damage and should, therefore, be considered in aquatic environmental risk assessment. 5. CONCLUSION The results obtained in this study demonstrate the dose-dependent genotoxicity potential of OSPW in G. brasiliensis individuals. Moreover, the bioconcentration of sodium, potassium and magnesium ions in the animals’ gills lead to slight to mild 14

Journal Pre-proof histopathologic damage in this organ, which could be used as a morphological biomarker of contamination monitoring. Beyond these morphological alterations, OSPW also affected the activity of catalase and superoxide dismutase enzymes, indicating that this contaminant may also affect oxidative stress in exposed fish. Furthermore, the first toxic signs of OSPW in G. brasiliensis were observed even in the lowest dose tested, which is a warning remark for environmental risk assessment in aquatic environments affected with effluents from ornamental stone processing

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activities and reinforces the potential of this species as an indicator in monitoring

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OSPW contamination.

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6. ACKNOWLEDGMENTS

The authors acknowledge the financial support from Fundação de Amparo À Pesquisa e

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Inovação do Espírito Santo (FAPES, Foundation of Support to Research and Innovation

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from Espírito Santo), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Coordination for the Improvement of Higher Education Personnel) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, The National Council for Scientific and Technological Development).

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Table 1. pH, electrical conductivity, total hardness, ions concentration (Mg, Ca, Na and K) and Mn in the water of Geophagus brasiliensis exposed to different concentrations of ornamental stone processing waste (OSPW). EC, electrical conductivity; TH, total hardness. Lowercase letters in row indicate significant statistical differences by ANOVA followed by Tukey´s test (p<0.05). Data correspond to mean ± SE (n = 6 for each treatment). OSPW (mg/L)

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pH

EC (µS/cm)

TH (mg/L)

Na (mg/L)

K (mg/L)

Ca (mg/L)

Mn (mg/L)

0

7.09±0.01a

50.65±0.41a

59.89±4.42a

9.60±0.11ª

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Mg (mg/L)

7.19±0.30a

16.34±1.39a

2.77±0.02a

2.41±2.09a

250

8.42±0.03b 118.91±0.19ac 108.17±7.34ac

8.07±0.21b 21.03±2.73b

3.10±0.09a

8.35±0.86ab

500

8.44±0.02b 126.51±0.55ac 114.05±8.22ac 10.61±0.25c

9.73±0.36ab 24.49±1.08bd

3.48±0.08a

10.91±0.91ab

750

8.51±0.02c 132.04±0.16bc 122.49±9.11bc 11.61±0.29b 10.56±1.25ab 28.12±1.81cd

3.65±0.13b

13.54±0.90b

1000

8.92±0.02d

3.67±0.26ab

15.39±2.81b

= 0.011

=0.006

p-value

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p e

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9.75±0.35a

rn

< 0.001

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148.99±0.64b 136.10±9.30b < 0.001

< 0.001

11.99±0.32b < 0.001

12.31±0.42b 30.41±0.40c < 0.001

< 0.001

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Journal Pre-proof Table 2. The no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) of some parameters induced by ornamental stone processing waste (OSPW) in Geophagus brasiliensis. LOEC and NOEC for GST and GPX can’t be calculated due the lack of significant difference among OSPW concentrations.

NOEC (mg/L)

Micronucleus

500

250

DNA damage

500

CAT

250

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LOEC (mg/L)

SOD

-

500

250 250

500

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HAI

250

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Parameter

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Journal Pre-proof FIGURE CAPTIONS Figure 1. Correlation among (A) Micronuclei and (B) DNA damage (comet assay) frequencies in Geophagus brasiliensis and different ornamental stone processing waste (OSPW) concentrations. Data is expressed as mean ± SE (n = 6). The frequencies of micronuclei were expressed per 1000 cells (‰) from each fish. DNA Damage index was calculated after measurement of 100 randomly selected nuclei from each fish.

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Figure 2. Correlation among specific activity of (A) catalase (CAT), (B) glutathione S-

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transferase (GST), (C) glutathione peroxidase (GPX) and (D) superoxide dismutase (SOD) in Geophagus brasiliensis and different ornamental stone processing waste

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(OSPW) concentrations. Data is expressed as mean ± SE (n = 6).

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Figure 3. Correlation among (A) Na+, (B) K+, (C) Ca2+ and (D) Mg2+ ions in the gills of

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Geophagus brasiliensis and different ornamental stone processing waste (OSPW)

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concentrations. Data is expressed as mean ± SE (n = 6). Figure 4. Correlation among (A) Histopathological alteration index (HAI) and (B)

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mean alteration value (MAV) of gills of Geophagus brasiliensis and different ornamental stone processing waste (OSPW) concentrations. Data is expressed as mean ± SE (n = 6).

Figure 5. Histological alterations of Geophagus brasiliensis gills induced by ornamental stone processing waste (OSPW). (A) Gills with no alteration (control); (B) occurrence of lamellar epithelium hyperplasia (HE), incomplete lamellar fusion (LE), and venouns sinuses (VS); (C) Occurrence of structure detachment (SD) with interstitial edema; (D) Occurrence of incomplete lamellar fusion, (E and F) incomplete lamellar fusion, lamellar aneurysm (LA) and vascular congestion (VC). Scale barr: 50 µm.

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Figure 2

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Figure 5

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Graphical abstract

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Journal Pre-proof Highlights - Ornamental stone processing industry wastes (OSPWs) even at low concentration induce mutagenicity, genotoxicity and oxidative stress in Geophagus brasiliensis; - G. brasiliensis individuals exposed to OSPW also presented histopathological alterations in gills;

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- G. brasiliensis is a suitable indicator of OSPW toxical effects.

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