Behavioral toxicity of tannery effluent in zebrafish (Danio rerio) used as model system

Science of the Total Environment 685 (2019) 923–933

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Behavioral toxicity of tannery effluent in zebrafish (Danio rerio) used as model system Thales Quintão Chagas a, Tenilce Gabriela da Silva Alvarez a, Mateus Flores Montalvão a, Carlos Mesak a, Thiago Lopes Rocha b,c, Amanda Pereira da Costa Araújo a, Guilherme Malafaia a,c,⁎ a b c

Biological Research Laboratory, Post-graduation Program in Conservation of Cerrado Natural Resources, Goiano Federal Institute, Urutaí Campus, Urutaí, GO, Brazil Laboratory of Environmental Biotechnology and Ecotoxicology, Institute of Tropical Pathology and Public Health, Federal University of Goiás, Goiânia, GO, Brazil Post-graduation Program in Genetics and Molecular Biology, Institute of Biological Sciences, Federal University of Goiás, Goiânia, GO, Brazil

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Antipredator response deficit is induced by tannery effluent (TE) in zebrafish. • Zebrafish does not recognize Oreochromis niloticus as a potential predator. • Danio rerio exposed to0020TE appeared to have co-specific preference.

a r t i c l e

i n f o

Article history: Received 9 May 2019 Received in revised form 11 June 2019 Accepted 16 June 2019 Available online 18 June 2019 Editor: Henner Hollert Keywords: Aquatic pollution Agroindustry effluents Fish Neurotoxicity biomarkers Animal behavior

a b s t r a c t The ecotoxicity of untreated tannery effluent (UTE) in several animal models has been reported; however, its effects on fish behavior, and neurotoxicity, remain unknown. Thus, the hypothesis that the chronic exposure to UTE can induce behavioral changes in adult zebrafish (Danio rerio) representatives, even when it is highly diluted in water, was tested. Animals exposed to 0.1% and 0.3% UTE for 30 days showed behavioral changes in visual social preference tests through their co-specific and antipredator defensive responses, which had indicated neurotoxic actions. Zebrafish exposed to UTE appeared to have not co-specific preference when it is paired with Poecilia sphrenops. In addition, only animals in the control group showed aversive behavior in the presence of the herein used predatory stimulus (Oreochromis niloticus). However, Cr, Na and Mg bioaccumulation was higher in zebrafish exposed to 0.1% and 0.3% UTE, although anxiogenic and anxiolytic effects were not observed in the models exposed to UTE in the novel tank diving or aggressiveness-increase-in-the-mirror tests. This outcome allowed associating the exposure to the pollutant and bioaccumulation with the observed behavioral changes. The present study is pioneer in scientifically evidencing the sublethal impact caused by chronic exposure to UTE in experimental environment simulating realistic aquatic pollution conditions. Accordingly, results in the current research should motivate further investigations to broaden the knowledge about the real magnitude of UTE biological impacts on the aquatic biota. © 2019 Elsevier B.V. All rights reserved.

⁎ Corresponding author at: Biological Research Laboratory, Goiano Federal Institute – Urutaí Campus (Urutaí, GO, Brazil), Rodovia Geraldo Silva Nascimento, 2,5 km, Zona Rural, Urutaí, GO 75790-000, Brazil. E-mail address: [email protected] (G. Malafaia).

https://doi.org/10.1016/j.scitotenv.2019.06.253 0048-9697/© 2019 Elsevier B.V. All rights reserved.

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1. Introduction Tannery stands out as one of the most profitable raw-material supply activities, since it supports upholstery, footwear and clothing industries; therefore, such market boosts the economy in several countries, mainly in Asia and Latin America (Hu et al., 2011). According to Qasim et al. (2015), billions of dollars are generated every year by bovine skin import and export, but its processing requires large amounts of water, as well as inorganic and organic compounds with high polluting potential (Suresh et al., 2001). Heavy metals, such as Cr, Ni, Cd and Pb, and organic compounds like nonylphenol, hexachlorobenzene, 4aminobiphenyl, benzidine and dibutyl phthalate are just few components found in tannery effluent (TE), which enters aquatic ecosystems and causes several ecotoxicological impacts (Kumar et al., 2008; Guimarães et al., 2016a, 2016b). Many studies have been dedicated to develop new techniques and methods to treat this waste before its disposal in aquatic environments (Sharma and Simsek, 2019; Moradi and Moussavi, 2019; Chandrasekaran et al., 2019; Fernandez et al., 2019). However, the application of these technologies in local contexts, mainly in developing countries, remains very distant from reality, since the direct clandestine TE discharge into aquatic environments is a common practice in rudimentary tannery facilities (Ahamed and Kashif, 2014; Kumar et al., 2014). Therefore, the dissipation of TE chemical constituents can have different impacts on the trophic levels of aquatic ecosystems when they enter the water courses. Among the ecotoxicological effects of TE, we can highlight the biological changes observed in different plant and animal species exposed to it (Oral et al., 2007; Nicola et al., 2007; Souza et al., 2016). Vertebrates appear to be very vulnerable to TE, mainly aquatic species such as fish (Borgia et al., 2018), ephemeral aquatic life species such as amphibians (Amaral et al., 2018a; Montalvão et al., 2018), terrestrial species such as birds (Souza et al., 2017a, 2017b) and mammals (Guimarães et al., 2016a, 2016b; Guimarães et al., 2017; Estrela et al., 2017; Quintão et al., 2018; Rabelo et al., 2018; Oliveira et al., 2018; Guimarães et al., 2019). Fish species have been widely used as biomonitors since they are considered the most suitable models to monitor pollution in aquatic systems (Linde-Arias et al., 2008). These vertebrates are found in different aquatic habitats, in different biomes and world domains; therefore, they play an important ecological role in food chains, mainly because they carry energy from lower to higher trophic levels (Beyer, 1996), fact that justifies their wide use as experimental models in ecotoxicological studies (Norrgren, 2012). Therefore, understanding TE toxicant uptake, behavior change and responses in fish has high ecological relevance (van der Oost et al., 2003). Previous studies have reported the negative influence of exposure to TE on the metabolic processes of Poecilia reticulata (Aich et al., 2015), on developmental impairments in Danio rerio embryos (Rocha and Oliveira, 2017), on the increased expression of metallothionein in the hepatic tissue of P. reticulata (Aich et al., 2017); on micronuclei induction in peripheral erythrocytes, on exfoliated gill and kidney cells of Oreochromis niloticus (Weldetinsae et al., 2017); on immunotoxicity in Cyprinus carpio (Murugesan et al., 2012; Borgia et al., 2018), and on mutagenicity and genotoxicity in Labeo calbasu, Puntius sophore and Mystus vittatus (Nagpure et al., 2016) and Labeo rohita (Walia et al., 2015). TE also induced cytotoxic effects on Etroplus suratensis (Taju et al., 2012), as well as biochemical and histopathological changes in Oreochromis mossambicus (Navaraj and Yasmin, 2012) and hematological changes in Channa punctatus (Parveen et al., 2017). All these toxic effects reflect scientists and governmental authorities' concern with environmental issues induced by inadequate TE disposal; the aforementioned studies substantiate pollution remediation and aquatic biodiversity conservation strategies. From an experimental viewpoint, it is important using several biomarkers to minimize misinterpretations in complex pollution cases (Galloway et al., 2004; Barreto et al., 2018; Rodrigues et al., 2018; Maulvault et al., 2018).

However, some important biomarkers were not the focus of the studies referred above, as well as of many other investigations focused on the impact of pollutants/contaminants on the biota, such as the behavioral responses in fish exposed to TE. Although behavioral categories have been used as tools to characterize and identify the toxicity of aquatic pollutants, the above-mentioned studies did not pay attention to behavioral biomarkers in fish exposed to TE. According to Melvin and Wilson (2013) and Legradi et al. (2018), behavioral responses observed in fish are important biomarkers to assess environmental risks resulting from aquatic pollution if one links different physiological functions to ecological processes. However, small behavioral changes, unnoticeable in histological, biochemical, genetic and molecular analyses, among others, can affect individuals' fitness, as well as the dynamics of their populations (Melvin and Wilson, 2013). Thus, the aim of the current study was to evaluate the neurotoxic potential of TE in adult zebrafish (D. rerio) representatives who were used as model system – the models were exposed to different environmentally relevant TE dilutions in water. Based on previous reports on the ecotoxicity of these pollutants, the hypothesis that TE can induce important behavioral changes in fish inhabiting polluted aquatic ecosystems was tested. Assumingly, studies such as the present one broaden the possibility of using sensitive biomarkers to evaluate ecotoxicology in fish, as well as to provide scientific subsidies for the adoption and planning of pollution remediation and prevention actions. 2. Materials and methods 2.1. Animals and experimental design Adult zebrafish (D. rerio) representatives of both sexes (6–8 months; 0.2–0.4 g) were used as model system, based on recommendations by Barba-Escobedo and Gould (2012) and Acosta et al. (2016). They were purchased in a commercial nursery (Goiânia, GO, Brazil) and kept under conventional sanitary conditions for laboratory fish breeding (Dammski et al., 2011) in the aquatic organism laboratory of the Biological Research Laboratory of Goiano Federal Institute - Urutaí Campus (GO, Brazil). D. rerio is a tropical freshwater fish natural to rivers in Southern Asia, mainly in Northern India, Pakistan, Bhutan and Nepal (Grunwald and Eisen, 2002; Spence et al., 2006; Engeszer et al., 2007). This species has been used as model organism in neurobehavioral studies (Orger and Polavieja, 2017) about environmental toxicology and ecotoxicology worldwide (Garcia et al., 2016). Models were able to acclimatize to laboratory conditions for 15 days in tanks (85 cm × 40 cm × 40 cm) filled with 70 L of dechlorinated water (naturally, via aerating sprayer), under controlled temperature (27 ± 1 °C) and 14/10 h light/dark photoperiod (respectively), with biological filtration and constant aeration before the experiment had begun. They were fed twice a day with commercial floating pellet (35% protein) at 5% body weight, according to Lawrence et al. (2012). Aquariums were sanitized twice a week during the acclimation and experimental periods. Test animals were distributed into three experimental groups (n = 15 fish/group) to evaluate the behavioral effects of exposure to TE based on their body biomass: control group (C) = unexposed animals; TE0.1, animals exposed to 0.1% crude TE; and TE0.3, animals exposed to 0.3% crude TE - for 30 days (chronic exposure), in all cases. The defined exposure period simulates the time fish are exposed to water courses continuously loaded with TE discharges. 2.2. Untreated tannery effluent and tested dilutions TE previously characterized by the research group responsible for the present study was the same used to evaluate the impact of this pollutant on the reproductive aspects of Swiss mice (Guimarães et al., 2019). It had high concentrations of several metals [i.e. Cr (493 mg/L), Na (11,235 mg/L) and Mg (498.53 mg/L)] and of organic compounds

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(some of known toxicity – e.g. diethyl toluamide and dioctyl phthalate, as well as to not yet used ones). The effluent was kindly provided by a tanning industry located in Inhumas County, Goiás State, (Brazil) - the name of the company was not mentioned for ethical reasons. The climatic periods in tropical regions were taken into consideration in order to make the experimental design as close as possible to real conditions representative of rainy (river flood period) and dry seasons, when natural TE discharges in rivers can reach lower and higher dilution rates, respectively. The work shift in average tanning industries (8 h/day), the amount of skin processed on a daily basis (250 kg/day) and the mean annual flow of a potentially receptive watercourse (333.1 m3/h) were also taken into consideration in the experiment. In addition, the generation of 32 m3 TE per ton of raw hide (Islam et al., 2014), which would correspond to mean daily TE flow of approximately 1 m3/h in the simulated scenario, was also taken into account. The equation in resolution n. 430/2011 of the National Environment Council (Ministry of Environment, Brazil) (http://www2.mma.gov.br/ port/conama/legiabre.cfm?codlegi=646) was applied to calculate the TE dilution rates. This equation should be also used to determine the effluent dilution limits in rivers and streams, because it takes into account the flow of the generated effluent, as well as the reference flow of the receiving water body - dilution rate was expressed in percentage. Accordingly, by applying the above expressed values, 0.1% and 0.3% TE dilutions would correspond to the ones predicted for the rainy and dry seasons, respectively, in the simulated-pollution experiment. The water in the boxes of experimental groups was monitored on a daily basis with the aid of a portable multiparameter and no differences were observed between treatments [(i) pH – C: 7.34 ± 0.115; 0.1% TE: 7.33 ± 0.056; 0.3% TE: 7.35 ± 0.051; F(2,21) = 0.021; p = 0.979; (ii) temperature – C: 25.09 °C ± 0.071 °C; 0.1% TE: 25.20 °C ± 0.103 °C; 0.3% TE: 24.26 °C ± 0.693 °C; F(2,21) = 1.584; p = 0.228; (iii) dissolved oxygen – C: 8.41 mg/L ± 0.050 mg/L; 0.1% TE: 8.47 mg/L ± 0.068 mg/L; 0.3% TE: 8.28 mg/L ± 0.201 mg/L; H = 0.303; p = 0.859]. 2.3. Behavioral tests The neurotoxic potential of TE was evaluated based different behavioral biomarkers used to predict locomotion, anxiety, aggression, social interaction and antipredator defensive responses in zebrafish. All tests were carried out in a room with acoustic insulation and temperature

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control, equipped with cameras (infrared) to record and file the videos. All tests were performed between 08:00 am and 04:00 pm, with 24 h intervals between test sessions (Fig. 1). Intervals were established to reduce stress caused by animal handling and the effects tests can have on each other. All tests were carried out under conditions similar to the ones set for exposure tests, i.e., in tests with water containing the same TE dilution used throughout the experimental period. Water was replaced between tests and between model exchange in order to avoid interferences caused by chemical communication between animals and to ensure good levels of dissolved oxygen. Apparatus position in the test room changed between sessions to avoid space-preference effects. Test tanks were coated with yellow paper - this color is not aversive to D. rerio - to avoid the influence of external factors (Avdesh et al., 2012), except for the front side, which was kept translucent to enable the video recording. 2.3.1. Anxiety-like behavior and aggressiveness Animals were subjected to novel tank diving test to evaluate the possible anxiogenic effect of the herein assessed treatments after 28-days of exposure to TE (Blaser and Rosemberg, 2012; Huang et al., 2019), as well as to evaluate assumed locomotor deficit in the models. Zebrafish aggressiveness due to TE effects was evaluated through mirror test (Zabegalov et al., 2019) conducted in the same tank used for the novel tank diving test - trapezoidal tanks (15 cm height × 28 cm top × 23 cm base × 10 cm width) filled with 1.5 L of dechlorinated water with, or without, TE were used for such purpose. Fish models were individually transferred to the aquarium test and video recorded for 5 min, based on Oliveira et al. (2011). The tank was virtually divided into two equal horizontal segments for the anxiogenic-like behavior analysis; the following parameters were subsequently evaluated: top-up latency (s) (top half), top-off frequency, time (s) display and freezing frequency. Models' locomotor ability was assessed by recording the number of crossings through the virtually divided quadrants (15 quadrants ≈ 23 cm2), because this parameter helped estimating the travelled distance (in cm) and zebrafish swimming speed (cm/s) during the test. The freezing behavior was depicted by the absence of movements, except for the eyes and gill arches. Animals were left in the tank for 5 min after the novel tank diving test; subsequently, the wall paper was removed from one of the sides

Fig. 1. (A) Schematic drawing of the experimental design and behavioral tests carried out with adult Danio rerio representatives in the control and tanning effluent groups. (B) Sympatric predator of D. rerio (Indian leaf fish, Nandus nandus) and (B) Orechromis niloticus used as representative predator in the antipredator defensive response test. Colors are merely illustrative.

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of the aquarium and a mirror was positioned at right angle on this uncoated wall (Fig. 2A), which enabled the animal's reflection as it swam back and forth. Models were monitored for 5 min, based on Moretz et al. (2007); the following parameters were recorded: number of aggressive tail beats against the mirror, permanence time and travel frequency in zones 1 (20 cm), 2 (2.5 cm) and 3 (0.5 cm), the so-called “mirror aversion”, “approach” and “attack” zone, respectively, which were virtually drawn in the aquarium. 2.3.2. Visual social preference The visual social preference test was carried out at the 29th experimental day, based on Engeszer et al. (2004a, 2004b), Barba-Escobedo and Gould (2012) and Crittenden et al. (2015). Three tanks (20 cm length × 20 cm height and 15 cm width) were interconnected and individually filled with 5 L of dechlorinated water with, or without, TE. Compartments were separated by transparent walls in the tanks themselves, and this procedure allowed animals to visualize the interconnected compartments (Fig. 2B). The test consisted in subjecting each animal to three different sessions (4 min per session). Animals were carefully introduced into the central aquarium and were free to explore it for 4 min for acclimation purposes in the first session - opaque acrylic plates prevented animals from viewing the sides of the aquarium. Plaques were removed in the second session to allow visual contact with the extremities of the aquariums; one of these aquariums hosted six adult D. rerio representatives (three males and three females) and the other ones did not host any model. Test fish were video recorded for 4 min in this session. The aquarium only filled with water was replaced by another one containing six adult Poecilia sphenops representatives (three males and three females) (38,4 ± 5 mm length; 2.1 ± 0.5 mg body mass) in the third session (4-min duration). The test animals received visual stimuli from one side of the aquarium (stimulus A), whereas heterospecific individuals entered the aquarium from the other side (stimulus B). The spatial preference of unexposed zebrafish was evaluated during the acclimation period for test validation; therefore, animals' residence time (s) in virtually drawn zones A and B (Fig. 2B), which would be the closest zones to the co- and heterospecific stimuli, respectively, were recorded. The aim this evaluation was to assess whether zebrafish behavior would be influenced by spatial preferences or by the stimuli coming from the sides of the aquariums. The “co-specific vs. empty” and “cospecific vs. heterospecific” side biases changed from individual to

individual. Animals' residence time (s) at the end of the test in all experimental groups were recorded in different test session in virtually designed zones close to A and B stimuli (Fig. 2B). 2.3.3. Anti-predator defensive response The effect of exposure to TE was assessed at the last experimental day to evaluate whether it would influence antipredator defensive responses in zebrafish. A joined system composed of two tanks at the same dimensions of tanks in the previous test was used in the experiment. The test started when the model was placed in one of the tanks and video recorded for 5 min for environmental acclimation, but the model did not have visual contact with the sides of the aquarium because a yellow opaque acrylic plate was used to isolate the lateral walls. The plaque was removed after the acclimation period to allow the test animal to visualize (for 5 min) the sides of the aquarium hosting a Oreochromis niloticus (Nile tilapia) representative, who was used as predatory stimulus given its similar external morphology (Fig. 2C) to the sympatric predator of D. rerio, (i.e., Indian leaf fish, Nandus nandus) (Fig. 1) (Jayaram, 1981; Datta Munshi and Srivastava, 1988; Bass and Gerlai, 2008). Three O. niloticus specimens (total weight: 14.35 ± 1.07 g; total length: 98.69 ± 7.71 mm) were used in the alternating system test sessions so that the predatory stimulus between experimental groups was similar. The aquarium was divided into two equal-sized horizontal zones and into vertical zones called “aversive” (closest zone to the predator), “neutral” and “safety” stimulus (the farthest zone from the predator) in order to analyze the animals' behavioral tests (Fig. 2C). The following parameters were evaluated in sessions with, or without, predators: latency (s) to top, time (s) spent in the upper half of the tank, and residence time in the “aversive”, “neutral” and “safety” zones. 2.3.3.1. Predator behavior. Parameters related to O. niloticus, namely: total crossings between the upper and lower zones of the aquarium where animals were kept during each test session; frequency of predator's turn to the prey (i.e., frontal position), and dwell time (s) with the predator in front of the prey in the proximal area of the partition separating the tanks, were evaluated to assess whether the predator's behavior can influence defensive responses in preys. These evaluations were carried out to assess whether the predator's behavior could have any influence on the behavior of zebrafish in the control and TE-exposed groups.

Fig. 2. Schematic drawings of the apparatus used in the behavioral tests conducted with adult Danio rerio representatives from the control group and after their exposure to tanning effluent. (A) Front view of the mirror test, (B) social preference test (C) predator response test. Stimulus A: D. rerio (co-specific), Stimulus B: Poecilia sphenops (heterospecific); SZ: safety zone; NZ: neutral zone; AZ: aversive zone; SAZ: A zone stimulus; SBZ: B zone stimulus; UH: upper half; LH: lower half.

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for 4 h. The watch glasses placed on the beakers were removed after acid digestion and the heating process continued at 80 °C until the sample was completely dry. Next, 5% HNO3 was added to dilute sample residues and to increase the final volume to 5 mL. The solution was analyzed through flame atomic absorption spectrometry according to procedures described by Dutra et al. (2006), by using calibration standards for each chemical element. Results were expressed in mg/g of body biomass recorded at the end of the experiment. 2.5. Statistical analyses Residual data normality was assessed through Shapiro-Wilk test. Parametric data of the novel tank diving and mirror tests, as well as predators' general activity were subjected to one-way ANOVA, and

Fig. 3. (A) Latency to top, time in the top and time at freezing behavior in the novel tank diving test conducted with Danio rerio representatives from the control group and after their exposure to different tannery effluent dilutions (TE). (B–D) Frequency of aggressive tail beats against the mirror and times in virtually drawn zones 1 and 2 in the tanks during the mirror test. Bars indicate the mean + standard deviation. Parametric data were subjected to one-way ANOVA and non-parametric data were subjected to Kruskal-Wallis test; at 5% probability level. C: control; TE0.1% and TE0.3%: groups composed of animals kept in dechlorinated water containing 0.1 and 0.3% TE (n = 15/ group), respectively. Statistical analysis summaries are shown at the top of the figures.

2.4. Bioaccumulation Models were euthanized to quantify Cr, Na and Mg accumulation at the end of the exposure period in order to correlate possible behavioral changes in D. rerio exposed to TE, based on Souza et al. (2018), with modifications. These chemical elements were chosen because they appeared at high concentrations in the used TE, based on the chemical characterization previously reported by Guimarães et al. (2019). The test animals (whole body) were macerated and digested in beakers filled with 2 mL of digestion solution (75% nitric acid (HNO3): 70% perchloric acid (HClO4) = 1: 1 v/v) and placed on hot plate at 80 °C

Fig. 4. A) Total crossings through the virtually drawn quadrants in the tanks, (B) estimated travelled distance (C) swimming speed of Danio rerio representatives in the control group and after their exposure to different tannery effluent – TE dilutions subjected to the novel tank diving and mirror tests. Bars indicate the mean + standard deviation. Data were subjected to one-way ANOVA, at 5% probability level. C: control; TE0.1 and TE0.3: groups composed of animals kept in dechlorinated water containing 0.1% and 0.3% of TE (n = 15/group), respectively. Statistical analysis summaries are presented at the top of the figures.

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Tukey post-test, at 5% probability level. The Kruskal-Wallis test was used to compare non-parametric data through the Dunn's post-test, at 5% probability level. Data about social visual preference tests and antipredator defensive responses were analyzed through paired test, because the same animals were used in the first and second sessions. Student's t-test was used to compare parametric data and the MannWhitney test was applied to the non-parametric ones. Different groups in the first and second sessions were compared through one-way ANOVA (parametric data) or through Kruskal-Wallis test (non-parametric data), at 5% probability level. Correlation analyses were performed based on the Spearman's method. All videos recorded during behavioral tests were analyzed in the PlusMZ software v1.1. 4.3.2. Statistical analyses and graphics were generated in the GraphPad Prism software (version 7.0). 3. Results and discussion Models were initially subjected to the novel tank diving test, which is one of the most used test to evaluate anxiety and locomotion in D. rerio, because it explores zebrafish's natural trend to dive into the new environment and to gradually increase their vertical locomotor activity throughout the test (Blaser and Rosemberg, 2012; Stewart et al., 2012). Based on the herein recorded results, there were not differences in latency to climb to the top of the aquarium, in the time staying in the top of the aquarium and in the time at freezing behavior (Fig. 3A). Changes in these variables are commonly predictive of anxiety disorder in D. rerio (Egan et al., 2009; Stewart et al., 2012); therefore, it is possible inferring that TE did not induce anxiogenic or anxiolytic effects on zebrafish after their chronic exposure to it (30 days). There was no evidence of changes in the boldness and sociability of D. rerio exposed to TE. The mirror test did not show differences in the number of aggressive tail beats against the mirror between groups (Fig. 3B), as well as in the frequency and time staying in zones 1 and 2 of the apparatus (Fig. 3C–D). Animals in all groups presented behaviors typical of the herein assessed species: biting, touching or remaining close to their reflection in the mirror, without signs of locomotor deficits. The total number of crossings through aquarium quadrants, the distance travelled and swimming speed did not differ between unexposed animals and TE-exposed ones in the novel tank diving and mirror tests (Fig. 4A, B and C, respectively). Neurotoxic effects on fish due to exposure to TE at environmentally relevant dilutions were not evaluated; however, these data diverge from results in previous studies, which have assessed the neurotoxicity of TE in rodents. Guimarães et al. (2016a, 2016b) and Souza et al. (2017a, 2017b) reported the anxiogenic effect of TE on Swiss and C57Bl/6J mice, respectively. According to Almeida et al. (2016), female Swiss mice exposed to the pollutant showed behaviors suggestive of anxiolytic effect. On the other hand, data in the present study are similar to those collected by Guimarães et al. (2017), who did not find anxiolytic or anxiogenic effect when male and female Swiss mice were exposed to TE, at 5% dilution, for 15 days. Such differences result from complex factors including the biological features of the herein assessed model systems and the experimental designs adopted in each study, as well as the TE chemical composition, dilution rates and exposure times. Nevertheless, data in the current study allow discarding the emotionality Fig. 5. (AB) Frequency and time (s) in the “empty” zone and in the zone “with Danio rerio” (CD), frequency and time in the zone with “Danio rerio” or with “Poecilia sphenops” of D. rerio from control group and after their exposure to different tannery effluent dilutions – TE (subjected to the visual social preference test). Bars indicate the mean + standard deviation. Data collected in the sessions with, and without, stimuli or in those with stimulus A (D. rerio) and B (P. sphenops), were analyzed through paired test. Student's t-test was used to compare parametric data and the Mann-Whitney test was applied to the non-parametric ones. Statistical analysis summaries are shown at the top of the figures - - asterisks indicate significant differences at 5% probability level. C: control; TE0.1 and TE0.3: groups composed of animals kept in dechlorinated water containing 0.1% and 0.3% of TE (n = 15/group), respectively.

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factor (e.g., related to anxiety and aggressiveness), which has been influential to the behavior shown by the animals in the later tests. The assessed TE dilutions were not able to induce hyper- or hypoactivity in the models, and this outcome could have negative influence on animals' behavior in subsequent parameters. The innate trend of D. rerio was used to develop fish visual choice tests to assess the species' preference for social interaction and for novelty in order to evaluate possible TE impacts on its sociability parameters. The residence time of control animals in different areas of the aquarium (Fig. 2) was assessed during the acclimation period in order evaluate whether their behavior was the consequence of stimuli provided or driven by previous spatial preferences. Zones 1 and 2 were explored in the acclimation session by animals in the control group (Zone 1: 13.27 s ± 2.90 s, zone 2: 16.33 s ± 2.96 s; t = 0.739; p = 0.465) and frequency was higher and time spent in the zone near the co-specific zones was longer than in the zone next to the empty aquarium or to the heterospecific ones (Fig. 5). These data confirm previous studies that have shown that D. rerio are visually drawn to co-specifics and that adult representatives belonging to this species instinctively aggregate into shoals, fact that validates the herein suggested paradigm (Miller and Gerlai, 2011; Barba-Escobedo and Gould, 2012). The frequency in exploration of the “empty” zones and in zones “with D. rerio” did not differ in the TE-exposed groups (Fig. 5A–B). Fish in the water with 0.3% TE had no preference for the zone close to their co-specific ones in comparison to the empty zone (parameter: residence time in the zones - Fig. 5B) or to the zone holding their heterospecific (parameters: frequency and residence time in the zones Fig. 5C–D, respectively). Based on these data, D. rerio exposed to TE (mainly at higher dilution - 0.3%) did not recognize the shoal of their co-specific species; thus, they expressed behavior contrary to the natural tendency of their group and schools. According to Buske and Gerlai (2012), shoaling provides multiple benefits to fish, including access to mate, efficient foraging and defense against predators. Therefore, the effects of TE on the natural populations of shoaling fish inhabiting areas that receive these pollutants can be drastic; however, the biological mechanisms triggering this change were not the object of our study, although they could be as complex as the chemical constitution of the assessed ET. Assumingly, TE had negative influence in biological pathways involved in the synthesis of neuropeptides such as vasotocin and isotocin, which are known to regulate social behavior in D. rerio (Braida et al., 2012). These molecules are synthesized by neurons located in the parvocellular and magnocellular nuclei of the preoptic area of Teleostei; this area protrudes into the neurohypophysis, releases the synthesized neuropeptides into the bloodstream and into multiple regions of the brain (Holmqvist and Ekstrom, 1995; Saito et al., 2004). The preoptic area includes different nuclei that are functionally related to the ventricular and tubal regions of the hypothalamus and that form a functional and structural unit (Meek and Nieuwenhuys, 1998). Therefore, changes in this structural unit can be related to social behavior differences observed in unexposed D. rerio and in the ones exposed to TE. Giusi et al. (2005) associated several neurodegenerative events in the preoptic areas of the hypothalamus with behavioral changes shown by Thalassoma pavo exposed to endosulfan and Cd, which is a toxic metal also identified in the herein tested TE sample [see details in Guimarães et al., 2019]. This finding reinforces the hypothesis that TE Fig. 6. Frequency and time (s) in the aversive (A–B) and safety zones (C–D) recorded for Danio rerio representatives in the control group and after their exposure to different tannery effluent – TE dilutions (antipredator defensive response test). Bars indicate the mean + standard deviation. Data collected in sessions with, and without, predatory stimulus were analyzed through paired test. Student's t-test was used to compare parametric data and the Mann-Whitney test was applied to the non-parametric ones. Statistical analysis summaries are shown at the top of the figures - -asterisks indicate significant differences at 5% probability level. C: control; TE0.1 and TE0.3: groups composed of animals kept in dechlorinated water containing 0.1% and 0.3% of TE (n = 15/group), respectively.

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chemical constituents can act in key areas modulating the social behavior of the assessed fish. Besides, one can assume that TE chemical constituents have changed the models' visual system, fact that would explain D. rerio inability to distinguish their co-specific from their heterospecific ones. Based on Engeszer et al. (2004a, 2004b), Rosenthal and Ryan (2005) and Hutter et al. (2011), D. rerio demonstrates clear visual preference for shoals presenting similar size and color. LeFauve and Connaughton (2017) reinforce this hypothesis by showing that the short exposure of D. rerio embryos to low Cd and Ni concentrations (both metal were also identified in the herein tested TE) was able to change the optomotor responses of exposed larvae in all tested ages. However, it is possible advocating that the retinal histological analysis, the expression of

Fig. 8. Chromium, sodium and magnesium concentrations in Danio rerio representatives in the control group and after their exposure to different tannery effluent dilutions. Bars indicate the mean + standard deviation (mg/g dry weight). Parametric data were subjected to one-way ANOVA (with Tukey's post-test) and the non-parametric ones were subjected to the Kruskal-Wallis test (with Dunn's post-test); at 5% probability level. Statistical analysis summaries are shown at the top of the figures. C: control; TE0.1 and TE0.3: groups composed of animals kept in dechlorinated water containing 0.1% and 0.3% of TE (n = 15/group), respectively.

Fig. 7. (A) Frequency of crossings between upper and lower zones, (B) time predators stayed in a frontal position towards Danio rerio and (C) time (s) spent by the predator in frontal position towards the prey in the zone proximal to the partition of O. niloticus, which was used as potential predators of D. rerio in the control group and after their exposure to different tannery effluent dilutions. Bars indicate the mean + standard deviation. Parametric data were subjected to one-way ANOVA and non-parametric data were subjected to the Krukal-Wallis test; h at 5% probability level. Statistical analysis summaries are shown at the top of the figures. C: control; TE0.1 and TE0.3: groups composed of animals kept in dechlorinated water containing 0.1% and 0.3% of TE (n = 15/group), respectively.

mRNA encoding photoreceptor opsin genes and genes related to apoptosis are interesting biomarkers to be addressed in future studies involving fish exposure to TE, as used by Liu et al. (2018) in D. rerio exposed to bisphenol S [BPS, (4,4′-sulfonyldiphenol)]. The present investigation also assessed whether exposure to TE would change the anti-predatory defensive responses of zebrafish; results showed that, unlike the control group, zebrafish exposed to TE did not recognize Nile tilapia as a potential threat. The frequency and residence time of this fish species in the aversive zone, i.e., in the closest zone to the partition separating the models from the predator, were statistically the same in sessions with, and without, tilapia (Fig. 6A–B). These animals recorded lower frequency and remained shorter in the safety zone in the presence of O. niloticus (Fig. 6C–D). On the other hand, the used potential predators showed similar behavior in test sessions imposed to all experimental groups; this outcome ensures the same stimuli and stimulus intensity to the experimental groups. The total number of crossings recorded for the tilapias in the upper and lower zones of the aquarium (Fig. 7A), the number of times predators turned towards D. rerio (Fig. 7B) and the time the predator spent in front of the prey in the zone proximal to the partition (Fig. 7C) did not differ between predators (Fig. 7C). The collected data confirmed the hypothesis that exposure to TE induces behavioral changes suggestive of antipredator response deficit. These data are similar to findings observed in recent studies about how the exposure to aquatic pollutants (even at small concentrations or dilutions) can compromise animals' perception of risks stimulating their natural predators. This is the case of Gallus gallus domesticus chicks exposed to ZnO nanoparticles (Mesak et al., 2018), Japanese quail (Coturnix coturnix japonica) exposed to the pesticide abamectin (Faria et al., 2018), Lithobates catesbeianus tadpoles exposed to abamectin (Amaral et al., 2018b) and to a mixture of different pharmaceutical drugs (Amaral et al., 2019); as well as the case of mice exposed to haloxyfop-β-methyl ester (Mendes et al., 2018), among others. If deficit in response to predatory stimuli is a common feature among the aforementioned studies, action mechanisms against the pollutants would explain why the results recorded in their study remain unknown, similar to results in the current study. TE complexity makes the understanding about such mechanisms even more difficult. D. rerio is known to have diurnal habits and good visual acuity (Connaughton and Nelson, 2010), and these features were essential for animals in the control group to recognize O. niloticus as a predatory threat, since acoustic, olfactory and vibratory stimuli were blocked in the test apparatus. Therefore, the hypothesis that changes in the

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models' visual acuity caused by ET, were responsible for changes in their social behavior, can also explain the observed antipredator deficit. Previous studies have shown the importance of D. rerio's visual system to drive anti-predatory defensive behavior under stimuli from live, robotic or computerized image predators, and it reinforces the plausibility of the hypothesis advocated in the present research (Bass and Gerlai, 2008; Gerlai et al., 2009; Gerlai, 2010; Ahmed et al., 2012; Ladu et al., 2015; Oliveira et al., 2017). On the other hand, evidences indicate that predator-associated cue processing and predator-associated fear response depend on a specific hypothalamic subsystem. Accordingly, one can assume that TE may have caused site-specific hypothalamic changes. Canteras et al. (1997) showed that bilateral cytotoxic lesions restricted to the most responsive site to predatory threats and to dorsal premammillary nucleus significantly reduced defensive responses to a predator. Therefore, the absence of TE-induced anxiogenic state that could be associated with anti-predatory defense deficit can be explained by the independent functioning of neuronal circuits that modulate models' response to potential predators (Pereira and Moita, 2016). However, future studies will help better understanding the adjacent mechanisms related to the biological impact of TE on fish behavioral responses. Bioaccumulation results revealed higher concentrations of Cr, Na and Mg elements in the body of zebrafish exposed to TE in comparison to the control group (Fig. 8). Although not all inorganic and organic TE components were quantified, the collected data evidence TE absorption by the exposed animals (by not yet investigated pathways), as well as the consequent sublethal effect. The present research has certain limitations, but they can be the starting point for future studies. Effects observed are unlikely to be extrapolated to native or endemic species of aquatic TE receptor systems. Would the effects of this pollutant be more comprehensive and harmful on these species? What are the TE constituents responsible for the observed effects? Identifying the main chemical components responsible for the observed effects is essential to plan a specific remediation strategy against pollution caused by TE. A physiologically more comprehensive investigative approach can elucidate many action mechanisms of different TE xenobiotics, and it would be an important basis for assessing the ecotoxicological risk posed by these pollutants to aquatic biota. To the best of our knowledge, the present study is pioneer in reporting behavioral effects on fish species exposed to environmentally relevant TE dilutions. The experimental design was the closest possible to realistic pollution conditions. From an experimental viewpoint, the behavioral responses of D. rerio were sensitive to low TE dilutions (0.1 and 0.3%); this outcome certainly opens room for better exploring the approached model system in ecotoxicological studies with mixed pollutants and/or aquatic contaminants. 4. Conclusions The current study confirms the hypothesis that even highly diluted TE (0.1 and 0.3%), induces behavioral changes in adult zebrafish, fact that suggests neurotoxic effect. Thus, the present study can be included in the list of scientific evidences about the ecotoxicological potential of these effluents, with special attention to behavioral effects that are not always taken into consideration in assessments focused on biological damages caused by the disposal of these pollutants in aquatic systems. Given the pioneering nature of the study, further investigations must better explore the subject, not only to elucidate mechanisms adjacent to the observed changes, but also to investigate sublethal effects undetectable through conventional toxicological evaluations. Acknowledgment The authors are grateful to the Brazilian National Research Council (CNPq, Brazil) for the financial support (Proc. No. 426531/2018-3) and

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for granting scholarships to the students involved in this project. In addition, they would like to thank Goiano Federal Institute for the financial support [Resolution 64/2014 and Ordinance 24/2016 (Proc. No. 23219.000553/2019-21)]. Malafaia G. receives researcher productivity grants by CNPq (Proc. N. 307743/2018-7). Compliance with ethical standards The authors declare that they have no conflict of interest. Meticulous efforts were made to assure that the animals suffered the least possible and to reduce external sources of stress, pain and discomfort. The current study did not exceed the number of animals necessary to produce trustworthy scientific data. This article does not contain any studies with human participants performed by any of the authors. References Acosta, D.D.S., Danielle, N.M., Altenhofen, S., Luzardo, M.D., Costa, P.G., Bianchini, A., Bonan, C.D., da Silva, R.S., Dafre, A.L., 2016 Jul-Aug. 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