Depuration time and sublethal effects of microcystins in a freshwater fish from water supply reservoir

Accepted Manuscript Depuration time and sublethal effects of microcystins in a freshwater fish from water supply reservoir Sabrina Loise de Morais Calado, Gustavo Souza Santos, Talitha Pires Borges Leite, Juliana Wojciechowski, Mário Nadaline, Junior, Deivyson Cattine Bozza, Valéria Freitas de Magalhães, Marta Margarete Cestari, Viviane Prodocimo, Helena Cristina Silva de Assis PII:

S0045-6535(18)31333-X

DOI:

10.1016/j.chemosphere.2018.07.075

Reference:

CHEM 21790

To appear in:

ECSN

Received Date: 31 March 2018 Revised Date:

13 July 2018

Accepted Date: 14 July 2018

Please cite this article as: de Morais Calado, S.L., Souza Santos, G., Borges Leite, T.P., Wojciechowski, J., Nadaline Junior., , Má., Bozza, D.C., de Magalhães, Valé.Freitas., Cestari, M.M., Prodocimo, V., de Assis, H.C.S., Depuration time and sublethal effects of microcystins in a freshwater fish from water supply reservoir, Chemosphere (2018), doi: 10.1016/j.chemosphere.2018.07.075. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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DEPURATION TIME AND SUBLETHAL EFFECTS OF MICROCYSTINS IN A FRESHWATER FISH FROM WATER SUPPLY RESERVOIR Sabrina Loise de Morais Caladoa, Gustavo Souza Santosb, Talitha Pires Borges Leitec,

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Juliana Wojciechowskia, Mário Junior Nadalineb, Deivyson Cattine Bozzad, Valéria

Freitas de Magalhãese, Marta Margarete Cestarib, Viviane Prodocimod, Helena Cristina Silva de Assisc*.

Ecology and Conservation Program Post-Graduation, Federal University of Paraná,

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Box 19031, 81530-990, Curitiba-PR, Brazil. [email protected];

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[email protected]

Department of Genetics, Federal University of Paraná, Box 19031, 81530-990,

Curitiba-PR, Brazil. [email protected]; [email protected]; [email protected] c

Department of Pharmacology, Federal University of Paraná (UFPR), Box 19031,

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81530-990, Curitiba, PR, Brazil. [email protected]; [email protected] Department of Physiology, Federal University of Paraná, Box 19031, 81530-990,

Curitiba-PR, Brazil. [email protected]; [email protected] Institute of Biophysics Carlos Chagas Filho 21941-902, Ilha do Fundão, Rio de Janeiro,

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Rio de Janeiro, Brazil

Corresponding author*:

Helena Cristina Silva de Assis Environmental Toxicology Laboratory, Department of Pharmacology, Federal University of Paraná (UFPR), Box 19031, 81530-990, Curitiba-PR, Brazil. E-mail:[email protected]

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DEPURATION TIME AND SUBLETHAL EFFECTS OF MICROCYSTINS IN A

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FRESHWATER FISH FROM WATER SUPPLY RESERVOIR

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Sabrina Loise de Morais Caladoa, Gustavo Souza Santosb, Talitha Pires Borges Leitec,

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Juliana Wojciechowskia, Mário Junior Nadalineb, Deivyson Cattine Bozzad, Valéria

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Freitas de Magalhãese, Marta Margarete Cestarib, Viviane Prodocimod, Helena Cristina

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Silva de Assisc*.

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Box 19031, 81530-990, Curitiba-PR, Brazil. [email protected];

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[email protected]

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Ecology and Conservation Program Post-Graduation, Federal University of Paraná,

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Curitiba-PR, Brazil. [email protected]; [email protected]; [email protected]

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81530-990, Curitiba, PR, Brazil. [email protected]; [email protected]

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Curitiba-PR, Brazil. [email protected]; [email protected]

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Rio de Janeiro, Brazil

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Department of Pharmacology, Federal University of Paraná (UFPR), Box 19031,

Department of Physiology, Federal University of Paraná, Box 19031, 81530-990,

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Institute of Biophysics Carlos Chagas Filho 21941-902, Ilha do Fundão, Rio de Janeiro,

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Department of Genetics, Federal University of Paraná, Box 19031, 81530-990,

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ABSTRACT Microcystins (MCs) are hepatotoxins that have been considered to be a worldwide

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problem due the effects that they can cause to environmental and human health systems.

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The Iraí Reservoir, located in the South of Brazil, is used as a water supply and MCs

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concentrations have been reported in this ecosystem. This study aims to determine the MCs

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concentrations in the Iraí Reservoir and to evaluate the MCs depuration time and the health

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of Geophagus brasiliensis using biomarkers. Water and fish samples were collected in the

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Iraí Reservoir from August 2015 to May 2016. Phytoplankton and chemical analyses were

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conducted using water samples and the fish were divided into two groups; the Immediate

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Group (IMM) and the Depuration Group (DEP). In the IMM group, the blood, liver,

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muscle, brain and gills were collected, in order to evaluate the genotoxic, biochemical and

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chemical biomarkers. The DEP group was used in the depuration experiment for 90 days,

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and after this period the fish were submitted to the same procedure as the IMM group. Our

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results suggested that fish accumulated MCs and it may have caused oxidative stress,

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neurotoxicity and molecular damage. Furthermore, MCs concentrations increased during

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the depuration time and it resulted in molecular damage over the first 30 days. After 90

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days, the recovery of the antioxidant system occurred. The depuration started on the 15th

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day, however, the toxins were still present in the samples. Therefore, the effects and the

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persistence of MCs are a risk to environmental systems and human health.

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Keywords:

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depuration; biomarkers.

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Iraí

Reservoir;

Microcystis aeruginosa; Microcystins;

accumulation;

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1. INTRODUCTION

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Intensive cyanobacterial blooms have been reported in aquatic ecosystems

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worldwide due to an increase of nutrients (Carmichael, 2012). The increase of nutrients is

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even greater when there are anthropogenic activities around water bodies (Chorus and

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Barthram, 1999). The Iraí Reservoir has cyanobacterial blooms dominated by Microcystis

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aeruginosa, which can occur during throughout the year (Fernandes et al., 2005).

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Microcystis aeruginosa can produce microcystins (MCs), a cyclic heptapepitide

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consisting of more than 100 structural variants as described in the literature (Carmichael,

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2012; Pham and Utsumi, 2018). The MC effects are primarily lead to liver damage and for

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this reason, they are known as hepatotoxins. The most common and toxic form is the

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microcystin-LR (MC-LR) (Gupta et al., 2003).

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The Iraí Reservoir is located in a humid subtropical region in Paraná state in the

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South of Brazil. It is used as a public water supply and cyanobacteral blooms have

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increased the costs of drinking water treatment. Moreover, previous studies reported a

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persistence of M. aeruginosa blooms and high concentrations of MC-LR in this

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environment (Bittencourt-Oliveira, 2003; Fernandes et al., 2005).

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One of the most serious cases of human poisonings from MCs contamination was in

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Caruarú, Brazil. In February 1996, at a hemodialysis clinic in Caruarú, 116 patients were

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intoxicated. One hundred developed acute liver failure and 52 died. According to the

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epidemiological investigation, patients at the clinic were treated with water containing MCs

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(Azevedo et al., 2002). Since 2000, the Brazilian Health Ministry incorporated

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cyanobacteria density and cyanotoxin concentration testing parameters that must be

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monitored in reservoirs utilized for drinking water.

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cyanobacteria in water supply reservoirs is 20.000 cells.mL-1 and the concentration limits of

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MCs for drinking water is 1.0 µg/L (Brazil, 2011).

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The maximum density limit of

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Several cases of animal poisonings by MCs have also been reported leading to a

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decrease in the reproduction and survival of cladocerans (Herrera et al., 2015); and

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hepatotoxicity, oxidative stress, osmoregulation imbalance and impairments in the gonadal

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development in fish (Malbrouck and Kestemont, 2006; Pavagadhi et al., 2012; Paulino et

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al., 2017; Hou et al., 2017). Hepatic damage and disordered hormone conversion in mice

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has also been reported (Zhong et al., 2017; Zhang et al., 2017).

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Due to exposure to MCs, mainly in aquatic organisms, MCs have been found to be

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accumulating in several groups of organisms; such as zooplankton (Ferrão-Filho et al.,

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2014), mussels (Preece et al., 2015) and fish (Hauser-Dars et al., 2015); and free

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microcystin concentrations reported have ranged from 0.42 µg/kg in crabs (De Pace et al.,

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2014) to 337.3 µg/kg in fish (Magalhães et al., 2001). Food web transfers and the

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persistence of these toxins have resulted in the intoxication of aquatic biota; as mussels,

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fish, crustaceans (Gibble et al., 2016; Pham and Utsumi, 2018; and humans; (Massey et al.,

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2018). In addition, the depuration experiments showed constant levels of MCs after 2

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weeks, and elimination, just after 60 days (Smith and Haney, 2006; Xie et al., 2004). Due to

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the MCs cyclic structure, they are resistant to physical and chemical factors. Therefore,

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MCs can persist longer in the environment and in the tissues of the organisms (Svrcek and

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Smith, 2004; Massey et al., 2018).

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Fish are biological models used in monitoring aquatic ecosystems because they are

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constantly exposed to alterations in environmental conditions. Geophagus brasiliensis is a

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freshwater fish, which are abundant in reservoirs and are frequently used as a source of

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animal protein for human consumption (Abelha and Goulart, 2004).

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In addition, another tool to monitor environmental quality are biomarkers, which

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can detect effects early. In this study the acetylcholinesterase enzyme was used as a 4

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neurotoxicity biomarker; and ion quantification and carbonic anhydrase enzyme as

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osmoregulation biomarkers in order to evaluate the environmental stress in aquatic

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environments (Mela et al., 2013a; Lécrivain et al., 2018). The environmental stress can also

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increase the reactive oxygen species (ROS) into cells, which results in oxidative stress and

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consequently molecular damage. The enzymes that are used as tools to evaluate the

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antioxidant system include catalase, superoxide dismutase, glutathione peroxidase and low-

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molecular-weight scavengers such as glutathione reduced (Oakes and Van der Kraag,

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2003). In addition, glutathione S-transferase is a useful biomarker for evaluating the

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biotransformation system, this enzyme catalyze the conjugation of compounds, as MCs,

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using GSH. This conjugation results in toxicity reduction and compounds excretion.

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Therefore, GST plays a role in defense against lipid and DNA damage (Van Der Oost and

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Vermeulen, 2003, Pham and Utsumi, 2018). In order to evaluate the DNA damage,

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genotoxic biomarkers can be used, including comet assays and the analysis of nuclear

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morphological alterations (Pavan da Silva et al., 2011; Hercog et al., 2017).

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Furthermore, the use of the biomarkers in ecotoxicological studies are more

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advantageous when an index of biomarker integration that shows the general patterns of

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response are used; such as the Integrated Biomarker response index (IBRv2) (Beliaeff and

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Burgeot, 2002).

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The aims of the present study were to use chemical analyses to determine the

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Microcystin-LR (MC-LR), YR (MC-YR), RR (MC-RR) and LA (MC-LA) concentrations

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in the water samples, and in a particular freshwater species, the Geophagus brasiliensis

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from the Iraí Reservoir. The biochemical and genotoxic biomarkers were used in order to

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evaluate the health of the G. brasiliensis, and to understand the persistence of these toxins

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in the fish, using a 90-day depuration experiment. 5

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2. MATERIAL AND METHODS

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2.1 Sampling Water and fish samples were collected in the Iraí Reservoir (-25° 23’17’’S -49°

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6’20’’W) from August 2015 to May 2016. The water was sampled in August, November,

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February and May, totaling 4 sampling periods. The samples were taken at a depth of 50 cm

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below the water surface and stored in dark bottles for phytoplankton and chemical analysis.

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These samples were then transported at 4 °C to the laboratory and stored at -20 °C until the

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chemical analysis was conducted. Three replicates per sample were carried out.

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The fish sample (Geophagus brasiliensis) was collected in August. The total of 50

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fish were divided into two groups. The first group was called the “Immediate group”

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(IMM) and the second group was called the “Depuration group” (DEP). The IMM was the

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group that we collected the tissues on the same day of the sampling. These fish were

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anesthetized with benzocaine 0.0001%, followed by blood sampling taken (500 µL) from

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caudal vein for genotoxic and biochemical biomarker analyses. After that, the fish were

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measured, weighed and euthanized by medullar section. Muscle samples were used for

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chemical analyses and to measure the acetylcholinesterase activity (AChE). The brain

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samples were removed to measure the AChE and for genotoxic biomarkers. The liver was

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collected for biochemical and genotoxic biomarkers, and the gills were used for

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biochemical biomarkers.

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The DEP was used to evaluate the depuration time of the contaminants, mainly

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cyanotoxins. The fish were collected on the same day as the IMM and transported to the

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laboratory. In the laboratory, the fish were kept in tanks with filtered and dechlorinated tap

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water in controlled conditions for 90 days. The water used for the experiment was also

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chemically analyzed. 6

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2.2 Depuration experiment The experiment was divided into 4 groups, according to the days: 7 days (DEP7),

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15 days (DEP15), 30 days (DEP30) and 90 days (DEP90) (Figure 1). During each period,

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we collected tissues from 10 fish. The fish were subjected to the same procedure as

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performed with the IMM group (See 2.1). Every 72 hours, the water was replaced and the

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water and feces samples were collected for the chemical analysis of cyanotoxins. All tanks

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were maintained under controlled conditions; including aeration, a temperature of (27°C),

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feeding with a balanced commercial fish food supply (once/day ad libitum), population

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density (1g/L: fish/water), ammonium, nitrate and the pH.

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Figure 1: Depuration experiment for 90 days. IMM: immediate group; DEP7: group 7 days; DEP15: group 15 days; DEP30: group 30 days and DEP90: group 90 days.

2.3 Phytoplankton analyses

Water samples for the qualitative analysis of the phytoplankton were collected in

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dark bottles and preserved in a Transeau solution. The species were identified according to

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Komárek and Anagnostidis (1986); and Anagnostidis and Komárek (1988). Water samples

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for the quantitative analysis of the phytoplankton (cell/mL) were collected in dark bottles

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and preserved in acetic Lugol, and the cells were counted by random fields using an

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inverted microscope Olympus IX70 (Utermöhl, 1958; Venrick, 1978; Chorus and Bartram,

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1999). We used 10 mL Utermöhl chambers and the samples remained in sedimentation for

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24 hours before enumeration. All the species, including Microcystis aeruginosa, were

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present in at least 75 fields counted or 100 cells of the most abundant individual, thus

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reducing the counting error to 20% (Lund et al., 1958).

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2.4 Chemical analyses

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Quantifications of cyanotoxins were carried out with water samples; and the muscle

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and feces of Geophagus brasiliensis by using a liquid chromatography tandem mass

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spectrometer (LC-MS/MS) according to the method described by Spoof et. al. (2003) and

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Sampaio et al. (2015). The cyanotoxins analyzed were Microcistin-LR (MC-LR),

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Microcistin-YR (MC-YR), Microcistin-RR (MC-RR) and Microcistin-LA (MC-LA). These

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toxins were chosen to be the most commonly found in aquatic environments according to

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the literature (Preece et al., 2017). MCs analysis was performed by LC-MS/MS (API 3200

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Q-Trap) using a C18 (250=4.6 mm, 100A, 5 mm) reverse phase analytic column. The

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chromatography was carried out under a linear gradient from 1 to 60% methanol over 5

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min, with a final isocratic stage holding at 60% methanol for 1 min. The mobile phases

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were: A (water; ammonium acetate 5 mM; formic acid 0.1%) and B (Methanol; ammonium

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acetate 5 mM; formic acid 0.1%); and the injected volume was 20 µL with a flow rate of

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1mL/min. The transitions were analyzed for the MCs and the linearity of this assay was 1–

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600 mg/L. Using a single point standard curve, the total coefficients of variation were 26.4,

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10.5, 12.6, and 10.7% at 0.78, 5.2, 104, and 1040 mg/ L.

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The calibrations were linear (R2=0.99), the detection limit (LOD) used was 0.1

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ng/mL and the quantification limits (LOQ) ranged from 0.5 to 40.0 ng/mL. The parent

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compound and its fragmented ions followed a mass-to-charge ratio (m/z): MC-LR: 995.5

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>135.1, MC-YR: 1045.5>135.1, MC-RR: 519.7>135.1 and MC-LA: 910.7>135.1.

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2.4.1 Water 8

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The 500 ml water samples were stored at -20°C and lyophilized. The lyophilized

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samples were weighed and extracted three times with a methanol:buthanol:water solution

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(20:5:75 v/v). In each extraction the samples were centrifuged and the supernatants were

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collected; in accordance with the procedure described by Krishnamurthy (1986). The

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supernatants were dried, resuspended in MilliQ water, purified in C18 Strata X

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phenomenex and eluted with 100% methanol/TFA. The fraction obtained containing the

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toxins was dried and resuspended in methanol (HPLC 100%) in order to analyze. The

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analyses were performed using a liquid chromatography tandem mass spectrometer (LC-

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MS/MS).

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2.4.2 Fish

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All fish collected from each time interval had their muscles removed. Muscles were

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weighed (5g) and extracted two times with methanol (100%). In each extraction the

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samples were homogenized, centrifuged and the supernatants were collected. The

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supernatants were dried, resuspended in Milli-Q water, purified in C18 Strata X

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phenomenex and eluted with a 100% methanol/TFA solution. The fraction obtained

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containing the toxins was dried and resuspended in methanol (HPLC 100%) for analysis.

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The analyses were performed using a liquid chromatography tandem mass spectrometer

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(LC-MS/MS). The procedures for the fish feces samples were the same as the water

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samples (See 2.4.1). After the MCs quantification in the muscle samples the values were

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compared with the Tolerable Daily Intake (TDI) (0.04 µg/Kg of body weight/day) for MC-

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LR (WHO, 1998). For the calculation of the values we used the MCs concentrations in the

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muscle samples (immediate group), the estimated fish meal size (0.227 kg for adults and

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0.114 kg for children) and the estimated human body weight (70 kg for adults and 16 kg for

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children) (WHO, 1998; Preece et al., 2015).

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2.5 Biochemical Biomarkers The muscle and brain samples were homogenized in a phosphate buffer (0.1 M) at

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pH 7.5, and each of the samples was centrifuged at 12000 g for 30 min at 4°C. The

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supernatants were used to measure acetylcholinesterase (AChE) (Ellman et al., 1961

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modified for microplates by Silva de Assis, 1998).

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The liver was homogenized in a phosphate buffer (0.1 M) at pH 7.0, and each of the

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samples was centrifuged at 15000g for 30 min at 4°C. The supernatants were used to

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measure the activity of the superoxide dismutase (SOD) (Gao et al. (1998), catalase (CAT)

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(Aebi, 1984), glutathione peroxidase (GPx) (Paglia and Valentine, 1967), glutathione S-

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transferase (GST) (Keen et al., 1976), and the concentrations of the non-protein

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thiol/glutathione (GSH) (Sedlak and Lindsay, 1968) with modifications (Guiloski et al.,

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2017) and lipoperoxidation (LPO) (Jiang et al., 1992).

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The gills (0.016 ± 0.0007g, n = 30; mean and SEM) were weighed and

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homogenized at 10% (weight/ volume, in g/mL) with a 10 mM phosphate buffer (10 mM

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tris-phosphate, 225mM mannitol and 75mM sucrose) at pH 7.4, using an Ultrasonic Fisher

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Scientific Homogenizer, Model FB120 (10 s, at 4 pulse/s, in 50% amplitude). The

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homogenate was then centrifuged (~2000 g for 5 min at room temperature), and the

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supernatant was separated for the protein and Carbonic anhydrase (CAA) assays. The CAA

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was assayed according to the method described by Vitale et al. (1999) based on Henry

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(1991), and described in Prodocimo et al. (2015). A pH reduction followed for 20 s, with

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readings every 4 s, after the addition of cold water (~4°C) saturated with CO2 to the

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phosphate buffer containing the tissue homogenate. The slope of the regression line of a pH

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reduction over a long period, resulted in the catalyzed rate of activity of the carbonic

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anhydrase present in the sample (Henry, 1991; Vitale et al., 1999).

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The plasma sodium (Na+) concentration was determined through flame photometry

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(Digimed DM-63, Brazil) and the plasma magnesium (Mg++) concentration was determined

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using a commercial kit (Labtest, Brazil) with an absorbance reading of 505 nm.

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2.6 Protein concentration

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In order to calculate the specific activity and the levels of the biochemical

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biomarkers, the total protein concentration was quantified using bovine serum albumin as a

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standard (Bradford, 1976).

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2.7 Genotoxic biomarkers

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The micronucleus, nuclear morphological alterations and comet assay analysis were

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performed with blood samples. The comet assay was used to evaluate the liver and brain

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tissues. The micronucleus analysis was analyzed by a method described by Heddle (1973),

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and Schmid (1975). The frequency of the nuclear morphological alterations blebbed (B),

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lobed (L), notched (N) and vacuolated (V) were analyzed through the methodology

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proposed by Carrasco et al. (1990). The comet assay was carried out using a method

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described by Singh et al. (1988), and modified by Ferraro et al. (2004).

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2.8 Integrated biomarker response index (IBR)

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The Integrated biomarker response index (IBRv2) integrates the biomarker results

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and was used in this study to evaluate the deviation of the biomarker responses among the

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groups (Beliaeff and Burgeot, 2002; modified by Sanchez et al., 2013). This index was

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evaluated among the immediate group (IMM) and the depuration groups (DEP7, DEP15,

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DEP30 and DEP90). The IMM was used for the baseline values (reference value) for each

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biomarker (T0). The ratio between the mean (T0) and the mean of each depuration group for

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each biomarker was log-transformed (Yi); and the standard deviation (s) and the general

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mean (µ) were calculated. The (Yi) values were standardized: Zi=(Yi - µ) / s. The

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difference among the (Zi) of the IMM and (Zi) of the depuration groups were determined as

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(A). The IBRv2 was determined using (A) value, and the higher the IBRv2 value means the

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greater the difference among the groups. The (A) value for each biomarker was represented

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in a plot to show the deviation of each biomarker response.

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2.9 Statistical analyses

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The Levene’s homogeneity test and the Shapiro-Wilk normality test preceded data

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analyses. ANOVA one-way was followed by the Tukey test and the Kruskall Wallis was

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followed by the Dunn’s test; were used to analyze the differences among the groups. The

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biochemical and genotoxic biomarkers were also analyzed using the Principal Coordinates

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Analysis (PCoA; Gower, 1966) and Permutational Multivariate Analysis of Variance

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procedure (PERMANOVA; Anderson, 2001). These were used to summarize and show

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general patterns. The correlation between the PCoA axes and the biomarkers were analyzed

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by Pearson and Spearman. The analyses were carried out using software R 3.2.2 (R Core

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Team 2015). The significant level was P<0.05.

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3. RESULTS

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3.1 Phytoplankton densities

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We identified 23 cynobacteria species, and Microcystis aeruginosa was the

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dominant species in the Iraí Reservoir during the August collection. In addition, the M.

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aeruginosa blooms were present in all collections (Table 1).

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3.2 Microcystin concentration in water and in G. brasiliensis

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We identified MC-LR, MC-YR and MC-RR in water samples from the Iraí

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Reservoir and in water samples from the depuration experiment. The cyanobacteria density

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and the MCs concentrations were not correlated. Therefore, periods with a higher density of

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cyanobacteria were not in the period with higher concentrations of MCs. MC-LR, MC-YR 12

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and MC-RR were also identified in the feces and muscle samples in G. brasiliensis (Figure

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2). No MCs concentrations were found in the filtered and dechlorinated water used during

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the depuration experiment. May 2016

Dens 52088 353632 203 4883 19737 0 0 1424 0 32962 249251 0 1221 15871 0 0 9360 0 0 814 12208 814 407 0

% 6.2 42.0 0.0 0.6 2.3 0.0 0.0 0.2 0.0 3.9 29.6 0.0 0.1 1.9 0.0 0.0 1.1 0.0 0.0 0.1 1.5 0.1 0.0 0.0

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Table 1: Cyanobacterial densities from August/2015 to May/2016 in Iraí Reservoir August November February 2015 2015 2016 Species Dens % Dens % Dens % Aphanocapsa delicatissima 0 0.0 0 0.0 194925 50.1 Aphanocapsa grevillei 0 0.0 184446 47.5 0 0.0 Arthrospira sp. 0 0.0 0 0.0 0 0.0 Cylindrospermopsis raciborskii 0 0.0 3357 0.9 0 0.0 Dolichospermum planctonicum 0 0.0 13327 3.4 305 0.1 Geitlerinema amphibium 712 0.3 203 0.1 0 0.0 Limnothrix sp. 610 0.2 0 0.0 610 0.2 Limnothrix sp2 0 0.0 3764 1.0 0 0.0 Merismopedia tenuissima 6918 2.7 0 0.0 0 0.0 Merismopedia sp. 0 0.0 27469 7.1 53106 13.7 Microcystis aeruginosa* 104075 41.2 88408 22.7 61753 15.9 Oscillatoria sp. 2136 0.8 2238 0.6 0 0.0 Planktolyngbya limnetica 0 0.0 0 0.0 0 0.0 Pseudanabaena muscicola 0 0.0 0 0.0 7935 2.0 Pseudanabaena sp. 1017 0.4 407 0.1 2849 0.7 Pseudanabaena sp1 0 0.0 0 0.0 1628 0.4 Pseudanabaena sp2 36319 14.4 0 0.0 8444 2.2 Radiocystis sp. 0 0.0 0 0.0 8444 2.2 Rhabdoderma lineare 1729 0.7 305 0.1 0 0.0 Rhabdoderma sp. 712 0.3 0 0.0 0 0.0 Rhabdoderma sp1 0 0.0 0 0.0 0 0.0 Rhabdoderma sp2 0 0.0 0 0.0 0 0.0 Rhabdoderma sp3 0 0.0 0 0.0 0 0.0 Woronichinia sp. 0 0.0 18312 4.7 5595 1.4 Dens: Cyanobacterial densities (cell/mL) %: Percentage of Cyanobacteria to total phytoplankton

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AC C

Figure 2: MCs concentration in water from Iraí Reservoir (A) August/2015 until May/2016; water from depuration experiment (B) 90 days; feces from G. brasiliensis (C); and muscle from G. brasiliensis (D) Mean±SD.

MC-LR and MC-YR concentrations oscillated throughout the depuration time in the

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water, feces and muscle samples. However, MC-RR concentrations decreased during the

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depuration experiment in the muscle samples. When we compared the toxin concentrations

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in water, feces and muscle, they decreased throughout the 90 days but the toxins were still

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present in the fish muscles. These results showed the dynamics of the toxins depuration.

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The MCs concentrations were below the legislation limits (0.04 µg/kg of body weight/day).

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Assuming a consumption of 0.227 kg fish meal per adult and 0.114 kg for a child, the MC-

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LR average uptake could be 0.0039 µg/kg/day for adults and 0.009 µg/kg/day for children;

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MC-YR could be 0.0104 µg/kg/day for adults and 0.0192 µg/kg/day for children; and MC-

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RR could be 0.0031 µg/kg/day for adults and 0.0057 µg/kg/day for children.

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3.3 Biomarkers

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The AChE activity in the brain was lower in the IMM group than DEP15, DEP30

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and DEP90 (P = 0.0069; P = 0.0003; P = 0.0016). DEP30 and DEP90 were higher than

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DEP7 (P = 0.0094; P = 0.0336). In the muscle, the activity was higher only in DEP30

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when compared with the IMM group (P = 0.0384).

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The GST activity was higher in DEP30 than the IMM and DEP7 groups (P = 0.0486; P = 0.0243).

In the stress oxidative biomarkers, the SOD activity was lower in DEP15, DEP30

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and DEP90 when compared with the IMM group (P = 0.0361, P = 0.0285, P = 0.0072). The

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CAT activity had no significant difference among the groups compared to the IMM group.

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The GPx activity was higher in DEP30 and DEP90 than the IMM group (P = 0.0289, P =

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0.0001), and DEP90 was higher than DEP7 (P = 0.0259). The GSH levels increased in

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DEP90 compared with the IMM group (P = 0.0285).

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groups (P = 0.0355, P = 0.0023, P = 0.0016) (Figure 3).

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AChE Muscle (nmol/mgprotein/min)

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Figure 3: Biochemical biomarkers in G. brasiliensis. Mean±SD P<0.05. A: Acetylcholinesterase (brain); B: Acetylcholinestesase (muscle); C: Superoxide dismutase; D: Catalase; E: Glutathione; F: Glutathione S-transferase and H: Hidroperoxides. 0: Immediate group (IMM); 7: 7 days (DEP7); 15: 15 days (DEP15); 30: 30 days (DEP30) and 90: 90 days (DEP90).

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The plasma Na+ had no significant difference among the depuration groups and the

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IMM group. However, in the DEP30 group the plasma Na+ decreased when compared to

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DEP15 and DEP90 (F=4.8741; P = 0.0211; P = 0.0121). The plasma Mg++ was higher in

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DEP30 than the IMM group (F=4.6696; P = 0.0047), and the specific activity of the

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branchial carbonic anhydrase (CAA) was lower in the DEP30 and DEP90 groups

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(F=22.706; P = <0.001) compared to the IMM group (Figure 4).

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The micronucleus and morphological nuclear alterations were present in some cells

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but they were not different among the groups (supplementary material 1). The blood score

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of the comet assay was lower in the DEP90 group compared to the IMM group (F= 9.9435;

319

P = 0.0338). In liver, the DEP15 and DEP30 scores was higher than the IMM group

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(F=6.594; P = 0.0011; P = 0.0107). In the brain, significant differences were not observed

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among the groups (Figure 4).

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The Permanova results showed that the groups are different (F= 5.3706; R2=

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0.3384, P = 0.001). The DEP7 group presented smaller differences in relation to the IMM

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group. Most of the differences began in the DEP15 and DEP30 groups. The greatest

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difference was shown in the DEP90 group. The first PCoA axis explains 22.4% and the

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second axis 18.1% (Figure 5). Brain AChE, GPx, GST, GSH, comet assay liver and

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plasmatic Mg++ were positively related and SOD, LPO and CAA were negatively related

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with the first axis. Muscle AChE, CAT, LPO, comet assay liver, comet assay blood and

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brachial CAA were positively related with the second axis (supplementary material 2).

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The IBR values were DEP7 (11.19), DEP15 (17.87), DEP30 (21.62) and DEP90

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(23.19) in comparison with the IMM group. These results were in agreement with the

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Permanova and PCoA results. Thus, indicating a difference among the groups, and in

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support of the highest difference being at 90 days (supplementary material 3). 17

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Figure 4: Biochemical biomarkers of osmoregulation and comet assay in G. brasiliensis. Mean±SD P<0.05. A: Na+ Plasmatic; B: Mg++ Plasmatic; C: Carbonic anhydrase; D: Comet assay (blood); E: Comet assay (liver); and F: Comet assay (brain). 0: Immediate group (IMM); 7: 7 days (DEP7); 15: 15 days (DEP15); 30: 30 days (DEP30) and 90: 90 days (DEP90). Comet assay: % damage in the tail.

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Figure 5: Multivariate analysis of biomarkers in G. brasiliensis. 0: Immediate group (IMM); 7: 7 days (DEP7); 15: 15 days (DEP15); 30: 30 days (DEP30) and 90: 90 days (DEP90).

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4. DISCUSSION Microcystis aeruginosa was the dominant cyanobacteria in the Iraí Reservoir in

338

August and its density throughout the year was above the recommended limit according to

339

the legislation (20.000 cells/mL). Based on the legislation, when the densities are high it is

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necessary to monitor cyanobacteria and cyanotoxins on a weekly basis. Others studies have

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reported the persistent blooms of the M. aeruginosa in this water body (Bittencourt-

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Oliveira, 2003; Fernandes et al., 2005), which can produce microcystins. In the present

343

study, we found MCs concentrations below the legislation limits (1 µg/L) (Brazil, 2011).

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However, there is a possibility of the accumulation of these toxins in sediment and aquatic

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organisms. In addition, human intoxication can occur through recreational activities such as

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sports and fishing, and during the consumption of contaminated organisms (Zegura et al.,

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2011). It is important to note that during the spring season (November) MC-RR and YR

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toxin concentrations were higher than MC-LR. It shows the need to analyze several MCs

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instead of evaluating only the MC-LR toxin.

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The cell numbers of M. aeruginosa and the MCs concentrations were not correlated

351

suggesting that the cyanobacteria strain compositions can vary considerably (Willis et al.,

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2016; Orr et al., 2017). It is well known that the production is higher during early phase

353

growth and decreases with increasing cell density. Therefore, cell counts can over or

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underestimated the cyanotoxin concentrations, and it can be a threat to the environment and

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human health when only cyanobacteria specifications and enumerations are used to

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determine the alert levels in water samples (Galet et al., 2017).

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MCs concentrations in fish samples showed that the aquatic organisms were

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bioaccumulating these toxins which can be transferred throughout the food web.

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Geophagus brasiliensis is an omnivorous species, which can ingest the toxins directly or 19

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via vectors, consequently increasing exposure to these toxins (Hauser-Davis et al., 2010).

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Moreover, this fish is consumed by the local human population, but the concentrations

362

found were below that of the Tolerable Daily Intake (TDI) (0.04 µg/kg of body weight/day)

363

established by the World Health Organization (WHO, 1998).

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Several studies also have reported a bioaccumulation of microcystins and an

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incomplete depuration at long term exposure in different aquatic species (Lance et al.,

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2014; Paldavičienė et al., 2015; Preece et al., 2015). Interestingly, in this present study,

367

after 90 days in clean water, the fish still had toxins in their tissues and they were still

368

eliminating them in the water. Others studies reported MCs concentrations increased in

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tissues of aquatic organisms in the depuration period (Soares et al., 2004; Mohamed &

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Hussein, 2006). Moreover, these results showed that the depuration of MCs is not fast and

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after a bloom, they can persist in water and tissues. The problem is even more serious when

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blooms are present all year and organisms are constantly exposed, such as in the Iraí

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Reservoir.

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We also observed in the present study, that during the depuration time the MCs

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concentrations increased in the water and fish samples. In 30 days, GST activity and DNA

376

damage in liver samples also increased. As GST is a biotransformation enzyme, our results

377

suggested that the activation of this biotransformation system started between 15 to 30

378

days. When MCs enter into cells they can be bound to phosphatase proteins and GSH. As a

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result of depuration process our results suggested that the toxins were metabolized and

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released. Thus, after the toxins were released, DNA damage was apparent in the liver and

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the MCs concentrations were higher in the muscles. In the present study only free toxins

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could be detected; it was not possible to measure the bound toxins with protein

383

phosphatases and glutathione. Amorim and Vasconcelos (1999) also suggested that the

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increase of MCs could be due to the metabolization of protein phosphatases, which leads to

385

the increase of toxins in fish. The release of these toxins via feces and urine, consequently

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leads to the increase of these toxins in the water. The increase of these concentrations in fish can also be due to feces reingestion.

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Some studies reported a reingestion of feces, such as a secondary mechanism of

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contamination, which leads to the main excretion of MCs through the feces (Amorim &

390

Vasconcelos, 1999; Lie et al., 2018).

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Our biomarkers results showed a decrease in AChE activity suggesting

392

neurotoxicity in the IMM group. The decrease of acetylcholinesterase activity can be

393

caused by contaminants such as pesticides, metals, pharmaceuticals and cyanotoxins

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(Molica et al., 2005; Araújo et al., 2016; Guerreño et al., 2016; Aguirre-Martinez et al.,

395

2016). MCs are known to be hepatotoxic, but some studies have suggested neurotoxic

396

effects and a decrease in AChE activity (Kist et al., 2012; Wu et al., 2016). In addition, we

397

cannot disregard the presence of other anticholinesterase agents, which may be present in

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this environment. There are many anthropogenic activities around the Iraí Reservoir such as

399

agriculture, burial grounds, industries, a hospital and human settlements.

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SOD activity was lower in the depuration groups. This result suggests that there is

401

recovery of the antioxidant system during the depuration time. Studies also showed that

402

SOD activity increased when the fish were exposed to microcystins (Puerto et al., 2009;

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Isibor, 2017)

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The GSH levels and GPx activity were lower in the IMM group. These lower levels

405

of intracellular GSH could be explained by the transport of GSH (outside) and MCs

406

(inside) via OATPs (Amado and Monserrat, 2010). GPx uses GSH as a substrate, so its

407

activity was limited to the IMM group. Consequently, reactive oxidative species (ROS) 21

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increase and it can cause molecular damage. After 30 and 90 days of the depuration time,

409

the GSH levels and GPx activity increased and the LPO and DNA damage decreased. A

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previous study showed the same relationship between the GPx activity and molecular

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damage in G. brasiliensis (Calado et al., 2017).

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In addition, to the important role in the protection of the cell membranes, GPx also

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degrades hydrogen peroxide. In the present study, it could have played a more important

414

role than the CAT enzyme, which also degrades hydrogen peroxide showing that there was

415

no difference among the groups.

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Fish osmoregulation may be affected by the chronic exposure to MCs (Malbrouck

417

and Kestemont, 2006). Sodium plasma (~140 mM) and magnesium (~1,5 mM)

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concentrations of G. brasiliensis were similar to those previously reported for freshwater

419

fish (Evans and Claiborne 2009). Freshwater fishes present sodium plasma levels regulated

420

by the chloride gill cells and the concentrations were not different among the DEP groups,

421

when compared to the IMM group, indicating a maintenance of plasma homeostasis

422

(Marshall and Grosell, 2006; Evans and Claiborne 2009). However, plasma Na+ decreased

423

during the first 30 days when compared to the 15 and 90 day periods, followed by an

424

increase in MCs; observed at 30 days. MCs can lead to a decrease in plasma Na+ due to

425

their inhibitory action on gill chloride cell ionic pumps (mainly Na+/K+ -ATPase)

426

(Malbrouck and Kestemont, 2006). Na+/K+-ATPase inhibition reduces the electrochemical

427

gradient for the sodium absorption from water (Marshall and Grosell, 2006; Evans and

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Claiborne 2009). On the other hand, rainbow trout, Oncorhynchus mykiss exposed to 3%

429

lyophilized Cyanobacterial biomass for 30 days presented no disturbance in plasma sodium

430

(Kopp et al., 2014).

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through glomerular filtration followed by tubular absorption (Beyenbach, et al., 1993;

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The plasma magnesium concentration is maintained by the kidney

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Freire, et al., 1996; Marshall and Grosell, 2006). After 30 days of depuration, plasma

433

magnesium levels increased compared to the IMM group. It could be a compensatory

434

response indicating an increase of tubular reabsorption or a degenerative alteration of renal

435

tissue caused by an elevation of MCs concentrations at the same time, as observed in

436

rainbow trout (Malbrouck and Kestemont, 2006). The maintenance of plasma levels of

437

sodium and magnesium at the end of the 90 days of depuration indicates that a longer

438

period of depuration in the laboratory may lead to the stabilization in plasmatic parameters.

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Carbonic anhydrase activity is considered to be an important biomarker of toxicant

440

exposure in fish gills from freshwater fishes (ArasHisar, et al., 2004; de Polo & Scrimshaw,

441

2012; Lionetto, et al., 2012; Mela, et al., 2013a,b). This plays an important role in

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respiration, ion and acid-base balance through the reversible reaction of CO2 hydration,

443

which results in hydrogen ion (H+) and bicarbonate (HCO3−) (Marshall and Grosell, 2006;

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Evans and Claiborne, 2009). Branchial carbonic anhydrase activity decreased after 30 and

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90 days of depuration when compare to the IMM group, thus showing a downregulation of

446

enzyme activity after this period of depuration. The CAA reduction observed in 30 days (in

447

association with possible inhibition of Na+/K+-ATPase by MCs) may be responsible by a

448

reduction in plasma Na+ (Marshall and Grosell 2006; Evans and Claiborne, 2009). A CAA

449

reduction in gills was also observed in zebrafish exposed to aphantoxin in the laboratory for

450

3-12 hours (5.3 and 7.61g STX eq/kg body weight) (Zhang et al., 2016). The higher CAA

451

activity in IMM group, 7 and 15 days could be a compensation for osmoregulatory,

452

respiration, and/or acid-base (prevention of acidosis) disturbances (Randall and Brauner

453

1998; Henry and Swenson 2000, Perry and Gilmour 2006) caused by contaminants in water

454

(Freire et al., 2015).

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biomarkers used were efficient in achieving the objectives of the present study and the

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The

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multivariate analysis and integrated biomarker response index, which showed differences

457

among the groups. The results suggested that the removal of the contamination in this water

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body is able to improve the health of the fish. In addition, these analyses and the integrated

459

index showed that the depuration started on the 15th day with greater differences at the end

460

of the experiment. Therefore, after 90 days the toxin was still present, showing the

461

persistence of the MCs.

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5. CONCLUSION

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The Iraí Reservoir has persistent blooms of M. aeruginosa and MCs contamination.

464

Even at concentrations below the limit established by legislation, MCs caused toxic effects

465

to the organisms such as oxidative stress, lipid and DNA damage. We cannot discard the

466

possibility of other contaminants that may be causing the toxic effects. Fish were

467

bioaccumulating these toxins and after 90 days they were still present in the organisms. G.

468

brasiliensis can transfer them to other aquatic organisms and to human populations that use

469

this fish species as a source of animal protein. The MCs depuration started on day 15 and

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the toxins were eliminated by feces. Due to the persistence of the MCs in aquatic

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ecosystems, it is necessary to further monitor and conduct more studies in order to prevent

472

these blooms in water bodies.

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ACKNOWLEDGEMENTS

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We would like to thank Professor Marina for helping us with the water maintenance

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and fish sampling. In addition, we would also like to thank Mr. R. Guedes, Ms. L. Brandão

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and Mr. R. Rogers for their excellent assistance in the laboratory, G.A. Torres for help in

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the analysis of physiological data; and CNPq and CAPES for their financial support.

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Amorim, A., Vasconcelos, V. 1999. Dynamics of microcystins in the mussel Mytilus

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quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-

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ACCEPTED MANUSCRIPT HIGHLIGHTS - The Iraí Reservoir is contaminated by Microcystins (MCs). - Fish have accumulated MCs above the established limit for human consumption. - Neurotoxicity, oxidative stress, molecular and liver damage were observed in fish.

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- A recovery of the antioxidant system and molecular damage was present 90 days after.

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- After the depuration time, MCs were still detected in the muscles of the fish.