Memory and depressive effect on male and female Swiss mice exposed to tannery effluent

Accepted Manuscript Memory and depressive effect on male and female Swiss mice exposed to tannery effluent

Abraão Tiago Batista Guimarães, Raíssa de Oliveira Ferreira, Aline Sueli de Lima Rodrigues, Guilherme Malafaia PII: DOI: Reference:

S0892-0362(16)30160-X doi: 10.1016/j.ntt.2017.03.003 NTT 6691

To appear in:

Neurotoxicology and Teratology

Received date: Revised date: Accepted date:

9 December 2016 5 March 2017 8 March 2017

Please cite this article as: Abraão Tiago Batista Guimarães, Raíssa de Oliveira Ferreira, Aline Sueli de Lima Rodrigues, Guilherme Malafaia , Memory and depressive effect on male and female Swiss mice exposed to tannery effluent. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Ntt(2017), doi: 10.1016/j.ntt.2017.03.003

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ACCEPTED MANUSCRIPT MEMORY AND DEPRESSIVE EFFECT ON MALE AND FEMALE SWISS MICE EXPOSED TO TANNERY EFFLUENT Abraão Tiago Batista Guimarães1,2, Raíssa de Oliveira Ferreira2, Aline Sueli de Lima Rodrigues2,3, Guilherme Malafaia1,2,3,4# 1

Programa de Pós-Graduação em Conservação de Recursos Naturais do Cerrado, Laboratório

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de Pesquisas Biológicas, Instituto Federal Goiano – Campus Urutaí, GO, Brazil. PostGraduation Program in Conservation of Cerrado Natural Resources, Biological Research Laboratório de Pesquisas Biológicas, Instituto Federal Goiano – Campus Urutaí, GO, Brazil.

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Laboratory, Goiano Federal Institute – Urutaí Campus, GO, Brazil.

Biological Research Laboratory, Goiano Federal Institute – Urutaí Campos, GO, Brazil Departamento de Ciências Biológicas, Programa de Pós-Graduação em Conservação de

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Recursos Naturais do Cerrado, Instituto Federal Goiano – Campus Urutaí, GO, Brazil. Biological Sciences Department, Post-Graduation Program in Conservation of Cerrado 4

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Natural Resources, Goiano Federal Institute – Urutaí Campus, GO, Brazil. Programa de Pós-Graduação em Biodiversidade Animal, Universidade Federal de Goiás –

Campus Samambaia, Goiânia, GO, Brazil. Post-Graduation Program in Animal Biodiversity,

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Federal University of Goiás – Samambaia Campus, Goiânia, GO, Brazil. #Corresponding Author: Laboratório de Pesquisas Biológicas, Instituto Federal Goiano – Campus Urutaí, GO, Brazil. Rodovia Geraldo Silva Nascimento, 2,5 km, Zona Rural, Urutaí, Brazil.

CEP:

75790-000.

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GO,

Phone:

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1995.

E-mail:

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[email protected]

Category: Short Communication Number of words: 3862 Figure/Table: 3

ABSTRACT Although tannery industries generate substantial profits to the countries they are located in, they work with one of the most environmentally harmful human activities. Tannery effluents (TE) are highly toxic; thus, their improper release into water bodies may cause severe problems to individuals depending on this water. Therefore, the aim of the current study is to 1

ACCEPTED MANUSCRIPT assess the effects of oral exposure to TE on the anxiety-, memory deficit- and depressionpredictive behaviors in male and female Swiss adult mice. The following experimental groups were set in order to do so, control, positive control (reference drugs) and effluent. The animals in the effluent group were treated with 5% TE diluted in potable water for 15 consecutive days. The neurobehavioral tests started on the 12th experimental day. The results found through the elevated plus-maze test (for anxiety prediction) showed no anxiogenic or anxiolytic effects on animals exposed to TE. On the other hand, animals treated with TE

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showed short- and long-term memory deficit in the object recognition test, as well as depression-predictive behavior in the forced swimming test. These results may concern the

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high concentration of heavy metals and neurotoxic organic compounds in the TE. Therefore,

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the oral exposure to TE, even for a short period-of-time, has effects on the central nervous system (CNS) that lead to neurobehavioral changes. Thus, the current study broadens the

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knowledge on this research field by demonstrating the neurotoxicity of xenobiotics to male and female Swiss mice.

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Keywords: Agro-industrial waste. Experimental models. Tannery industries. Xenobiotics.

1. INTRODUCTION

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The tannery sector in Brazil and in other countries such as India, Pakistan and China is an important socio-economic development source. Tannery industries purchase raw material

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(skin) from slaughterhouses, provide jobs and export fur and leather to furniture, footwear, clothing and automotive industries, besides being one of the main input suppliers in different sectors (Sabumon, 2016). Italy, Hong Kong, China, Brazil, USA, South Korea, Germany,

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India and Argentina are among the greatest leather exporters in the world (Sabumon, 2016). Although tannery has great socio-economic importance, it is also of great concern,

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because of its most environmentally polluting activities (Manivasagam et al., 1987). The pollution potential of tannery industries, mainly of the small ones, is directly linked to the large amount of solid or liquid untreated or inefficiently treated residues they discharge into waterways. The main components in this effluent are sulphide, chromium, volatile organic compounds, solid wastes, suspended solids - such as animal hair - and trimmings, which have negative impacts on ecosystems and on the health of living beings (Shakir et al., 2012). Recently, the research team involved with the present research found that the animal’s sex (mainly mice), as well as exposure time and route (oral or dermal) are factors to be taken into consideration at the time to assess the effects of the herein studied xenobiotics on mammals (Souza al., 2016a). However, these team’s studies did not use 2

ACCEPTED MANUSCRIPT positive pharmacological controls, fact that has limited the researcher’s understanding about the observed behavioral changes. The complexity of tannery effluents (TE) comes from the combination between the different inorganic and organic substances found in them and the metabolic and biotransformation mechanisms developed by mammals. Such combination may have underlain the herein observed results. Thus, the aim of the current study is to assess the effects of the oral exposure to TE on the memory, anxiety and depression behavior of male and female Swiss mice. Given the

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variety of inorganic and organic compounds found in TE, mice treated with these xenobiotics are expected to have neurotoxic effects that could result in neurobehavioral

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disorders; thus, the present article emerges as an incremental step in a series of studies on

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TE.

2.1. Animals and experimental design

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2. MATERIALS AND METHODS

The sample comprised 180 (- 90 males and 90 females) - adult (3 months old) and

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nulliparous Swiss mice who were provided by the Biological Research Laboratory of the Goiano Federal Institute – Urutaí Campus, Brazil. The subjects were kept under standard

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conditions: 12-h light/dark cycle, 22±3ºC and 55-60% humidity. All the adopted procedures were approved by the Ethics Committee on Animal Use of the Goiano Federal Institute,

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GO, Brazil (protocol n. 17/2014).

The animals were divided in experimental groups exposed (or not) to TE. The number of mice per group changed depending on the applied behavioral test. The effluent group was

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composed of animals exposed to 5% wet-blue TE diluted in water, which was made available ad libitum to the mice through water dispensers. The positive control group – which was

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composed of animals treated with intraperitoneal injections of clonazepam (0.5 mg/Kg) and fluoxetine (30 mg/kg), before the behavioral tests - were used to validate the sensitivity of the elevated plus maze and forced swimming tests (Costa et al., 2003). Each experimental group subjected to depression and anxiety-predictive tests was composed of 10 mice (n=10 males and n=10 females). The groups not treated with clonazepam or fluoxetine, received i.p. injections of the drug vehicle (phosphate buffered saline (PBS)). On the other hand, the control and effluent groups subjected to the memory deficit-predictive test were composed of 15 animals (n=15 males and n=15 females). Body mass and water or feed consumption did not differ among the herein analyzed groups (data not shown).

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ACCEPTED MANUSCRIPT 2.2. Tannery effluent The TE used in the current study was provided by a tannery industry located in Goiás State, Brazil. The analysis applied to the organic compounds found in the TE was performed in a desorption electrospray ionization device coupled to a high-resolution mass spectrometer (HRMS), as described by Guimarães et al. (2016). The physicochemical and chemical analyses applied to the raw TE, to the potable water and to the water added with 5% TE were carried out according to recommendations by the American Public Health

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Association (APHA, 1997). All the analyzed contaminants found in the tannery effluent are



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identified and shown in Table 1.

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The rate of TE diluted in water bodies depends on hydrological factors such as the large variation in tannery effluent production by the industry, or on local climatic factors,

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which are often estimated. The effluent dilution rate (5%) in the current study is probably higher than the one typically faced by animals or humans. Accordingly, the aim was to simulate illegal effluent discharge scenarios and dilutions found throughout long drought

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periods or during months of increased bovine skin processing production.

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2.3. Neurobehavioral tests 2.3.1. Elevated plus maze (EPM) test

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The EPM test was performed on the 12th experimental day. The EPM device consisted of two opposing open arms (30 x 5 x 25 cm) and two opposing closed arms (30 x 5 x 25 cm) extending from a common central platform (5 x 5 cm). The adopted apparatus

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was made of wood and elevated 45 cm from ground level. The edges (0.25 cm) of the open arms were tested to prevent the mice from falling. The behavior rehearsal room was

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soundproof and the light intensity was kept at 100 lx. All experimental groups were kept in the rehearsal room for 30 minutes for acclimatization purposes. Subsequently, each animal was individually placed in the center of the EPM device with its face turned to one of the open arms. The animal was allowed to freely explore the apparatus for 5 min. All mice were tested once. The EPM device was cleaned with 10% ethanol before each test. The anxiety index was calculated as follows: Anxiety index = 1 - [([time the animal stayed in the open arms, in seconds/test duration in seconds (300 s)] + [input frequency in the open arms/total number of entries])/2]. The total number of entries was defined as the sum of the number of times the animal entered the open and closed arms. An input was accounted each time the animals’ four paws overtook the initial limit of the arm. The locomotor activity of the 4

ACCEPTED MANUSCRIPT animals in this test was accounted as the total number of entries (line crossing) in the arms. In addition, the frequency of open arm entries and the exploration time spent in the open arms were assessed.

2.3.2. Object recognition test The object recognition test was performed on the 13th and 14th experimental days using a (30 x 20 x 13 cm3) box. The test was divided in three sessions, the training session was

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followed by two test sessions (1h after training and another 24h after training). The animals were exposed to two identical objects (in size, form, and color) defined as familiar objects, F1

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and F2 (squared Lego toys), for five minutes during the training session. A familiar object

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was replaced by a new object (N) during the test sessions, so that the animals could explore a familiar object and a new one for three minutes. A triangular Lego toy was used 1 hour after

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the training test; and a circular Lego toy, 24h after it.

At the beginning of each trial, the animals were placed in front of the objects with their faces turned to the wall. The time each animal spent exploring each object was

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recorded. A crossed-over design was used in all test sessions, so that the positions of the new and familiar objects was alternated in order to exclude the potential preference of the animals

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for a certain spatial location of the objects in the box. Exploration behaviors consisted of having the animals smelling and touching the objects with their nose or forepaws, and

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standing 2 cm or closer to the objects. The recognition index of each animal was calculated according to Rabelo et al. (2016), and expressed by the ratio: TOX/(TF + TN) , wherein TOX = time spent exploring the familiar (F) or new (N) object; TF = time spent exploring the

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familiar object; TN = time spent exploring the new object. The boxes used in the tests were

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cleaned with 10% alcohol after each session.

2.3.3. Forced swimming test The forced swimming test was performed on the 15th experimental day. It consisted of individually placing the mice in a cylindrical tank (height 39.0 cm, diameter 20.0 cm) containing water at 25°C (20.0 cm depth) for 6 minutes. Subsequently, the animals were removed from the water and left to dry under light heating. Next, they were taken back to their crates. All test sessions were video recorded using a camera located 30 cm above the tank. The videos were used to assess the time the animals were motionless. Such time is often used as depression predictor in the forced swimming test (Petit-Demouliere et al., 2005). Immobility was herein defined as the absence of movement in the whole body - the mouse 5

ACCEPTED MANUSCRIPT stops fighting and keeps motionless, floating in water, or only does the necessary movements to keep its head above the water. The time spent swimming was used to measure the locomotor activity in the forced swimming test, as described by Costa et al. (2013). The understanding of swimming concerns the large and horizontal movements of the forepaws leading the body displacement around the cylinder. The swimming parameter was recorded for the first 2 minutes of the test, as described by Costa et al. (2013). Three trained observers (blind to the treatments) reviewed

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the videos, each video was analyzed twice, thus totaling inter-observer consistency > 85%.

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2.4. Statistical analysis

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Initially, the residual normality was checked by means of the Shapiro-Wilk test. The Bartlett test was used to check the residual homoscedasticity. Next, data from the EPM and

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forced swimming tests were subjected to analysis of variance (ANOVA) using the factors “sex” - male and female mice - (factor 1), and “treatment” - control = water; positive control = water added with the reference drug; and effluent - (factor 2). The data resulting from the

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ANOVA and the new object recognition indices were subjected to analysis of variance (ANOVA) using the factors “sex” - male and female mice - (factor 1), “treatment” - control

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and effluent - (factor 2), and “time” - 1 and 24 hours - (factor 3). The Fisher LSD test was applied at 5% probability in case of significant (p < 0.05) F value. All graphs were generated

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and the statistical analyses were performed in the GraphPad Prism software, version 7.02.

3. RESULTS

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The animals exposed (or not) to the TE, as well as to clonazepam (reference drug used as positive control), in the EPM test showed no differences in the locomotor activity. The

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"sex" (Factor 1 – F(1,54) = 1.376; p = 0.261 – % of total variation = 0.98), "treatment" (Factor 2 – F(2,54) = 0.598; p = 0.442 – % of total variation = 5.77) (Figure 1A), as well as the interaction between “sex x treatment” (Factor 1 vs. Factor 2 – F(2,54) = 1.759; p = 0.182 – % of total variation = 4.52) have shown no effect. On the other hand, the treatment had effect on all other behavioral parameters assessed in the EPM test, namely: anxiety index, F(2,54) = 72.89 and p < 0.0001 (% of total variation = 71.89) (Figure 1B); mean time exploring the open arms, F(2,54) = 53.061 and p < 0.0001 (% of total variation = 65.34) (Figure 1C); and mean entries into the open arms, F(2,54) = 78.600 and p < 0.0001 (% of total variation = 72.80) (Figure 1D). According to the pairwise comparison, just as expected, clonazepam had

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ACCEPTED MANUSCRIPT anxiolytic effects on the animals, whether they were male or female (Figure 1). However, effluent-exposed animals did not differ from the control in any EPM measure. The new object recognition index showed the effect of factor “treatment” - Factor 2, F(2.54) = 44.136; p < 0.001 (% of total variation = 58.02).. The new object recognition indices of animals in the control group, 1h and 24h after the training session, were higher than the



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same indices of animals exposed to 5% TE, fact that evidences memory deficit (Figure 2A).

The treatment showed effects through the forced swimming test. Animals in the

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positive control group (fluoxetine) showed shorter motionless time than animals in the control

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group, as expected. In turn, the mice exposed to 5% TE showed longer motionless time than the control ones: F(2,54) = 62.807; p < 0.001 (% of total variation = 67.20) (Figure 2B). Such

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result differs from that by Siqueira et al. (2011) and Moysés et al. (2014), who found no effect of Cr-rich TE on the depressive behavior of male Swiss mice and male Wistar rats, respectively. The herein tested animals showed no change in their locomotor activity, Factor

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1: F(1,54) = 0.034; p = 0.853 (% of total variation = 0.12); Factor 2: F(2,54) = 2.206; p = 0.139 (% of total variation = 5.05); and Factor 1 vs. Factor 2: – F(2,54) = 1.638; p = 0.215 (% of total

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variation = 2.58) (Figure 2C).

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4. DISCUSSION

The literature about the exposure of experimental models (mammals) to TE is scarce, fact that makes it difficult comparing results. Nevertheless, the present data (Figure 1) does

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not meet the results found in previous studies, which have shown controversial effects on the anxiety-predictive behavior of mice exposed to such xenobiotic. Siqueira et al. (2011) and

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Souza et al. (2016b) found that the exposure to TE increased anxiety-like behavior in adult male Swiss and C57Bl/6J mice. On the other hand, Almeida et al. (2016) noticed that female Swiss mice exposed to water containing 5% and 10% TE showed anxiolytic-like behavior. In addition, the exposure of male Wistar rats to TE [photoelectrooxidation (POE)-treated or nonPEO] did not change any of their EPM parameters (Moysés et al., 2014). It is known that TE has very diverse and complex chemical composition; moreover, this composition may change from one tannery industry to another. Thus, the different results found in the studies conducted so far may concern the different experimental designs or the differences in the time and route of exposure, as well as the age of the animals, the TE type, the sex of the animals, and the assessed species and rodent strains. 7

ACCEPTED MANUSCRIPT Untreated TE, rich in heavy metals such as Cr (chromium), Pb (lead), Cd (cadmium), and a variety of compounds were used in the current study to analyze the organic compounds known as neurotoxic-effect producers (Caito & Aschner, 2015). The effects of the plasticizer contaminants identified in the TE on the anxiety-related behavior of mice were shown in previous studies (Park et al., 2015; Yan et al., 2016). However, no anxiolytic or anxiogenic effect was observed in the animals. Possibly, the time of exposure adopted in the current study (15 days) was insufficient and the longer exposure to these xenobiotics may cause

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harmful effects, such as anxiety-related behaviors on mice.

The present data collected through the object recognition test (Figure 2A) are

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consistent with Rabelo et al. (2016). These authors exposed male and female Swiss mice to

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TE oral treatment and found that the new object recognition indices, calculated for male and female animals exposed to the effluent, were significantly lower than the same index

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calculated for the control group. Rabelo et al. (2016) exposed the animals to xenobiotics through gavage, whereas in the current study they were treated (ad libitum) with water containing TE. Regardless of the exposure form, the results found by Rabelo et al. (2016) and

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those obtained through the current study have shown that the short exposure to wet-blue TE xenobiotic may lead to memory impairments in Swiss mice.

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As it was previously mentioned, the effluent used in the current study contained high concentrations of heavy metals, as well as of organic compounds of acknowledged

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neurotoxicity, such as Cd. Experimental studies on the central nervous system of experimental mammalian models exposed to high Cd dosage have shown extensive hemorrhages in the cerebral and cerebellar cortices of these animals, as well as several pyramidal cells with

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pyknotic nuclei, neuroglial cells with cytolisis and altered Purkinje cells (Méndez-Armenta & Ríos, 2007). Another important element found in TE is Ni (nickel), whose exposure is linked

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to a variety of adverse nervous system effects including significant decrease in the rat’s the total brain antioxidant status and significant increase in the acetylcholinesterase activity (Liapi et al., 2011), among others. In addition, the effects of the plasticizing contaminants identified in the TE on the cognitive function of rodents and humans have been shown in previous studies (Miodovnik et al., 2014). Such cognitive deficits may be related to diffuse neuronal cell death and to significant neuronal cytoskeletal abnormalities in the motor cerebral cortex, in the hippocampal formation and in the cerebellum, as well as related to apoptosis of hippocampal cells and to morphologic changes in mitochondria mybe (Miodovnik et al., 2014). Thus, such data reinforce the hypothesis that the TE constituents may have been responsible for the 8

ACCEPTED MANUSCRIPT object recognition deficit found in animals exposed to it. It is tempting speculating that the memory impairment in male and female mice exposed to TE can be linked to hippocampus damages caused by one or more effluent constituents, because the hippocampus is an important brain region for memory and object recognition. Finally, although the mechanisms - through which the treatments have led to a depression-predictive behavior (Figure 2C) in the herein assessed animals - were not specifically investigated,

the TE constituents may have changed the functioning of the

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hypothalamic-pituitary-adrenal axis (HPA axis) or the serotonergic neurotransmission, which

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are functions linked to depression.

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4. CONCLUSION

The present data suggest that the exposure to TE (5%) causes behavioral disruptions in

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male and female Swiss mice. The results partially support previous findings, but contradict others. Thus, the current data, in association with previous findings, suggest that anxiety, depression and memory may all be affected by long and strong exposure to TE; however, the

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mechanism through which TE causes behavioral changes remains unknown.

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ASL, Malagaia G (2016) Behavioral changes in female Swiss mice exposed to tannery effluents. Ambiente & Água, 11(3): 519-534, 2016. American Public Health Association (APHA) (1997) Standard methods for the examination

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10.1016/B978-0-444-62627-1.00011-1. Costa AP, Vieira C, Bohner LO, Silva CF, Santos EC, De Lima TC, et al (2013). A proposal for refining the forced swim test in Swiss mice. Prog Neuropsychopharmacol Biol Psychiatry, 45: 150-155. Guimarães AT, Ferreira RO, Rabelo LM, E Silva BC, de Souza JM, da Silva WA, de Menezes IP, Rodrigues AS, Vaz BG, de Oliveira Costa DR, Pereira I, da Silva AR, Malafaia G. The C57BL/6J mice offspring originated from a parental generation exposed to tannery effluents shows object recognition deficits. Chemosphere, 164: 593-602, 2016. Liapi C, Zarros A, Theocharis S, Voumvourakis K, Anifantaki F, Gkrouzman E, et al. (2011) Short-term exposure to nickel alters the adult rat brain antioxidant status and 9

ACCEPTED MANUSCRIPT the activities of crucial membrane-bound enzymes: neuroprotection by L-cysteine. Biological Trace Element Research, 143: 1673-1681. Manivasagam, N (1987) Industrial effluents origin, characteristic effects, analysis and treatment Kovaipudur, India. Shakti Publication, 42. Miodovnik A, Edwards A, Bellinger DC, Hauser R (2014) Developmental neurotoxicity of ortho-phthalate diesters: review of human and experimental evidence. Neurotoxicology 41: 112-122.

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trait anxiety in boys or anxiety-like behavior in male mice. J Child Neurol, 30(1):48-52. Petit-Demouliere B, Chenu F, Bourin M (2005). Forced swimmnig test in mice; a review of antidepressant activity. Phychophamacology 177, 245-255.

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tannery efluente. Neurotoxicology and Teratology, 55(3):45-49. Sabumon PC (2016) Perspectives on biological treatment of tannery effluent. Advances in

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Recycling &Waste Management: Open Acess, 1: 3-10. Siqueira IR, Vanzella C, Bianchetti P, Rodrigues MAS, Stülp S (2011) Anxiety-like behaviour in mice exposed to tannery wastewater: the effect of photoelectrooxidation

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treatment. Neurotoxicology and Teratology, 33: 481-484. Shakir L, Ejaz S, Ashraf M, Qureshi NA, Anjum AA, Iltaf I, Javeed A. Ecotoxicological risks

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ACCEPTED MANUSCRIPT Yan B, Guo J, Liu X, Li J, Yang X, Ma P, Wu Y (2016) Oxidative stress mediates dibutyl phthalate induced anxiety-like behavior in Kunming mice. Environ Toxicol Pharmacol, 45:45-51. Méndez-Armenta M, Ríos C. Cadmium neurotoxicity. Environmental Toxicology and

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Pharmacology, 23: 350-358, 2007.

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Figure 1. (A) Total number of crossings; (B) anxiety index; (C) rate of time spent in the open arms; and (D) rate of frequency of entries that the (male and female) Swiss mice exposed (or not) to the tannery effluent took into the open arms of the Elevated Plus-Maze. The bars indicate the

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or not to tannery effluent calculated 1 and 24 hours after the training session of the object recognition test. (B) Spent in time swimming (in 120 s), and (C) swimming time (in 360 s) in the forced swimming test applied to (male and female) Swiss mice exposed

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(or not) to the tannery effluent. The bars indicate the mean + standard deviation (SD).

PT E

The data were subjected to analysis of variance with factors “sex” (male and female) (factor 1) and “treatment” (control, fluoxetine and effluent) (factor 2). In case of significant (p < 0.05) F value, Fisher LSD test was applied at 5% probability. (n=10, for

CE

each experimental group). C: control; E: effluent. In “C”, the mean values + SD with

probability.

AC

the same lower-case letter in the figures do not differ in the Fisher LSD test, at 5%

14

ACCEPTED MANUSCRIPT Table 1. Physico-chemical and chemical features of the water, 100% tannery effluents and 5% tannery effluents and general information from the analysis of organic compounds of the 100% tannery effluent used in the present study.

Attributes

pH at 25ºC (UpH) Total dissolved solids (mg.L-1) Zn (mg.L-1) Na (mg.L-1) Ca (mg.L-1) Mg (mg.L-1) Pb (mg.L-1) As (mg.L-1) Cr (mg.L-1) Cd (mg.L-1) Ni (mg.L-1)

Physico-chemical and chemical features WHO Guidelines for Potable water Quality1 Normally Tannery found in Potable effluent fresh water Health-based guideline by the WHO (5%) water/surface water/ground water

T P

Tannery effluent (100%)

I R

4.05

7.19

4.87

37,380.00

80.00

0.30 9,690.00 601.20 364.80 0.32 <0.01 859.00 0.95 5.50

0.03 5.01 4.00 2.43 <0.01 <0.01 <0.05 <0.001 <0.01

1,945.00 No guideline

ID

m/z

1 2 3

197.11803 292.13458 308.12958

M

N A

6.5 – 8.5 No guideline

0.04 No guideline 3.00 mg.L-1 489.26 <20 mg.L-1 200 mg.L-1 33.86 No guideline No guideline 20.55 No guideline No guideline 0.02 No guideline 0.01 mg.L-1 <0.01 No guideline 0.01 mg.L-1 -1 42.95 <0.002 mg.L 0.05 mg.L-1 0.05 <0.002 mg.L-1 0.003 mg.L-1 0.28 <0.02 mg.L-1 0.02 mg.L-1 Organic compounds of the 100% tannery effluent Molecular RDBa Name Formula 3.5 C11H17O3 5-Cyclohexyl-5-oxopentanoic acid 11.5 C19H18NO2 1-benzoyl-2,2,4-trimethyl-1,2-dihydro-6-quinolinol 11.5 C19H18NO3 (S)-3-Hydroxy-pyrrolidine-1-carboxylic acid 9H-fluoren-9-ylmethyl

D E

T P E

C C

A

No guideline

C S U

Error (ppm) -1.461 0.950 1.178

15

ACCEPTED MANUSCRIPT

4

320.12949

0.853

12.5

C20H18NO3

5

336.12433

0.590

12.5

C20H18NO4

6 7

344.10432 423.32705

0.741 0.463

15.5 8.5

C20H14N3O3 C29H43O2

8

485.28424

-0.200

9.5

C28H41N2O3S

9

617.26770

0.226

8.5

C26H41N4O13

10

666.19287

0.989

31.5

C44H28NO6

11

794.42456

1.545

15.5

C43H60N3O11

12

214.12059

1.657

4.5

13

251.10241

-0.890

a

E C

PT

D E

6.5

ester 2-(4-Butoxy-phenyl)-quinoline-4-carboxylic acid (2S)-1-(9H-fluoren-9-ylmethoxycarbonyl)pyrrolidine-2-carboxylic acid (5,6-Diphenyl-furo[2,3-d]pyrimidin-4-ylamino)-acetic acid 4,4'-Methylenebis[2,6-bis(1,1-dimethylethyl)phenol] 1,2-Naphthalenediol, 5,6,7,8-tetrahydro-6-[[6-[[2-[(2methoxyphenyl)thio]ethyl]amino]hexyl]propylamino] 3-Amino-3-désoxy-β-D-glucopyranoside de (1S,2R,3S,4S,6R)-4,6diamino-3-[(6-{[(benzyloxy)carbonyl]amino}-6-désoxy-β-Dglucopyranosyl)oxy]-2-hydroxycyclohexyle N-[3'-Hydroxy-6'-(1-naphthylmethoxy)-3-oxo-3H-spiro[2benzofuran-1,9'-xanthen]-5-yl]-4-biphenylcarboxamid 2-propenoic acid, 3-(4-hydroxyphenyl)-, (3aR,4R,6S,7aR)-6-[[[4[[(1,1-dimethylethoxy)carbonyl]amino]-1-[[[4(octyloxy)phenyl]amino]carbonyl]butyl]amino]carbonyl]hexahydro6-hydroxy-2,2-dimethyl-1,3-benzodioxol-4-yl ester, (2E)Diethyl toluamide Ethyl 6-(2-furyl)-2-hydroxy-4-methyl-1,6-dihydropyrimidine-5carboxylate

T P

I R

C S U

N A

M

C12H17NONa C12H15N2O4

RDB – ring/double bond equivalent. The ions were detected as deprotonated [M-H]- protonated [H+H]+ and aduccts [M+Na]+ and [M-S]-. 1The WHO's guidelines for potable-water quality, set up in Geneva, 1993, are the international reference point for standard setting and for potable-water safety (http://www.lenntech.nl/toepassingen/drinkwater/normen/who-s-drinking-water-standards.htm). The physicochemical and chemical analyses were carried out according to the recommendations of the American Public Health Association (APHA, 1997).

C A

16

ACCEPTED MANUSCRIPT

RI SC

Depression-predictive behavior in the forced swimming test

NU

Intake of water contaminated with tannery effluent

PT

Water contaminated with tannery effluent

Natural watercourse

MA

15 days

Short- and long-term memory deficit in the object recognition test

PT E

D

Adult Swiss mice

AC

CE

Graphical abstract

17

ACCEPTED MANUSCRIPT Highlights

Title: MEMORY AND DEPRESSIVE EFFECT ON MALE AND FEMALE SWISS MICE EXPOSED TO TANNERY EFFLUENT

Oral exposure to tannery effluent has effects on the central nervous system.



Exposure to tannery effluent causes behavioral disruptions in male and female mice



Memory deficit was observed in male and female mice exposed to tannery effluent



Depression-predictive behavior was observed in Swiss mice exposed to tannery

RI

PT



AC

CE

PT E

D

MA

NU

SC

effluent

18