domain: shs sdv

text:
Alexandre Suire

Melissa Barkat-Defradas

Evolution of human pitch: Preliminary analyses in the French population using INA audiovisual archives of Vox Pops

Introduction

Beyond its linguistic content, the human voice is a formidable tool to convey a wide range of emotions and affective states. Moreover, it also reflects various biological and social information about the emitter such as sex, age, some personality traits and, importantly, his or her socio-cultural background.

Indeed, vocal characteristics are also subjected to the cultural and social expectations of the society the individual belongs to. For instance, van Bezooijen [START_REF] Van Bezooijen | Sociocultural Aspects of Pitch Differences between Japanese and Dutch Women[END_REF] showed that the significant difference in voice pitch observed between Japanese and Dutch women (~230 Hz vs. ~190 Hz respectively) are explained by the fact that they comply to social expectations linked to their gender, which vary considerably between the two cultures. In Japan, traits traditionally associated with femininity include "modesty", "innocence" and "psychological dependence". In order to conform to the preferences of men sharing the same cultural representations, Japanese women vocally express these traits by using a higher pitch, which is known to be perceptually associated with impressions of "docility" and "weakness" [START_REF] Pisanski | The prioritization of voice fundamental frequency or formants in listeners' assessments of speaker size, masculinity, and attractiveness[END_REF]. Conversely, in a culture where the social roles attributed to men and women are relatively more egalitarian like the Netherlands, Dutch men tend to value traits related to female "independence" and "hard-working", which result in a preference for women with lowerpitched voices, which are known to be perceptually associated with "self-confidence" and "leadership" [START_REF] Klofstad | Sounds like a winner: voice pitch influences perception of leadership capacity in both men and women[END_REF]. Thus, Japanese and Dutch men show preferences towards culturally congruent voices related to the values attributed to femininity. More generally, it illustrates the idea that voice pitch reflects how a given culture considers men and women social roles.

Interestingly, some evidence suggests that these gender representations have significantly evolved over time, at least for women from Western's societies. Indeed, several studies have shown that women are increasingly moving away from feminine stereotypes by adopting traits that are generally associated with masculinity, such as competitiveness, independence, leadership, as well as declaring themselves as being more orientated towards higher professional careers [START_REF] Cashdan | Waist-to-Hip Ratio across Cultures: Trade-Offs between Androgenand Estrogen-Dependent Traits[END_REF][START_REF] Donnelly | Masculine and Feminine Traits on the Bem Sex-Role Inventory, 1993-2012: a Cross-Temporal Meta-Analysis[END_REF]. In turn, such changes would shift the use of their voices towards a lower-frequency pitch in order to project those impressions, to which men seem to be more attracted nowadays [START_REF] Pisanski | Voice pitch modulation in human mate choice[END_REF]. However, there is currently a strong lack of information on whether voice pitch has evolved or not across a larger span of time to confirm such results.

In this context, audiovisual archives offer a unique opportunity to tackle this issue and the present project aims at exploring the evolution of both men and women voice pitch in the French population as a first study case. For this purpose, we focused on Vox Pops recorded for television news that are most representative of how individuals vocally behave within a given society at a given time. Therefore, the relevance of this work is twofold: (1) offer an original systematic analysis of how the human vocal pitch has evolved over time and (2) highlight how audiovisual archives can shed light on the evolution of human social behaviors such as speech.

Methods

Voice samples

Voice samples were selected from French speakers interviewed during TV news Vox Pops between 1940 and 2019. These recordings generally took place in public areas, and involved anonymous passers-by who were asked to give their opinion on a particular subject. recordings were provided by the INA (Institut National de l'Audiovisuel, France) as online video clips from which we selected suitable ones for our purpose. The reason for choosing this kind of material is that these individuals' spontaneous speech were felt to be more representative of the general population than, for instance, interviewers, TV presenters, comedians or singers who have generally a better control of their vocal apparatus.

Clips with high background noise or chaotic vocalizations (e.g. screaming, shouting, laughing, crying…) were excluded. Eventually, 96 video clips were selected. Then, we extracted the audio signals associated from each selected video and saved them as .WAV files for further analyses.

Voice analysis

Speech segmentation and acoustic analyses of audio signals were conducted using Praat v. 6.0.43 [START_REF] Boersma | Praat: Doing phonetics by computer[END_REF]. Segmentation consisted in isolating speech samples (i.e. speakers talking) by removing fragments of external noises (i.e. surrounding noises), multi-voicing (i.e. voice overlaps) and non-linguistic vocalizations (e.g. laughter, crying…). For some speakers, multiple speech samples were selected since they could appear several times in the same interview. Then, we used a batch-processing script to measure four parameters of fundamental frequency (F0, Hz): mean (F0mean), range (F0min and F0max) and F0 contour with its standard variation (F0sd). Then, we measured the coefficient of variation F0CV, which corresponds to F0sd / F0mean. This measure represents the logarithmic perception of voice pitch (i.e. controlling for the effect of magnitude on variability, [START_REF] Pisanski | Voice pitch modulation in human mate choice[END_REF][START_REF] Fouquet | Seven and up: individual differences in male voice fundamental frequency emerge before puberty and remain stable throughout adulthood[END_REF]). All F0 parameters were measured using Praat's autocorrelation algorithm with a search range of 100-500 Hz for women speakers and 75-300 Hz for men speakers. We also measured the duration (s) of each voiced segments. For speakers with multiple voice samples, we averaged all five F0 parameters.

Perceptually, F0mean, F0min and F0max refer to voice pitch while F0sd and F0CV refer to global intonation. As such, voices with relatively lower F0mean, F0min and F0max will sound lower-pitched or "deeper" whereas voices exhibiting relatively higher F0mean, F0min and F0max will sound higher-pitched. Voices with relatively lower F0sd and F0CV will sound more monotonous whereas voices exhibiting relatively higher F0sd and F0CV will sound more dynamic and expressive. Note that differences are perceptually noticeable between the two sexes. Women's voices are significantly higher in pitch than men (approximately twice as much) as well as more expressive. These characteristics are consistent across languages. For instance, mean pitch (F0mean) of French speaking women vary between 140 and 240 Hz and between 100 and 150 Hz in men [START_REF] Vaissière | La phonétique[END_REF], while pitch variation F0sd is around 30 Hz in women and 13 Hz in men [START_REF] Suire | Human vocal behavior within competitive and courtship contexts and its relation to mating success[END_REF].

Analyses and results

Except for the period 1940 -1959, women and men's voices were analyzed separately by decade, from 1960 -1969, 1970 -1979, etc. to 2010 -2019 (see Table 1 for details concerning the number of speakers analyzed for each decade). In total, we analyzed 340 women and 432 men unique speakers across 7 decades. Speech samples averaged a duration of 4.7 seconds (± 4.6).

Table 1. Number of analyzed speakers per sex and decade

ANOVAs (Analyses of Variance) were used to examine the evolution of all F0 parameters over time (i.e. per decade) for each sex. Tukey post-hoc comparisons with Bonferroni's correction were then performed to look for specific differences between each decade. Results were considered significant at the α = .05 level.

All results are summarized in Figure 1. Mean pitch (F0mean) was significantly higher in women after 1940 -1959 (all p < .05) but remained constant after that specific period. In men, no significant increase or decrease was observed between each decade and thus mean pitch remained constant over almost ~80 years (all p > .05). Pitch variation (F0sd) did not significantly vary in both women and men across time (all p > .05). Although there was considerable variation, a look at the evolution of CV (F0 coefficient of variation), which corrects for the logarithmic perception of voice pitch [START_REF] Anderson | Preference for Leaders with Masculine Voices Holds in the Case of Feminine Leadership Roles[END_REF], shows that it was only significantly lower between 1940 -1959 and 2000 -2009 (p < .05). Women's F0CV also overlapped the values observed for men between 1970 -1979 and 1990 -1999. In other terms, it appears that women's voices became relatively more monotonous over time. Values of F0CV for men did not significantly change over time (all p > .05). Maximum pitch (F0max) was significantly higher in women after 1940 -1959, with another important increase in 2010 -2019 (all p < .05), while in men it only started to be significantly higher between 2000 In men, a similar pattern was observed (all p < .05). In summary, these results reveal that women's voices have evolved higher pitched voices after 1940 -1959 but remained constant after that. We also observed an increase in vocal range (i.e. simultaneous increase in F0max and decrease in F0min) and a more monotonous voice after 1970 -1979. For men, voice pitch has not significantly changed over time but vocal range also increased after the same period. Our study shows that voice pitch (F0mean) in both men and women has little evolved over time (Figure 1a). Those results seem to contradict the hypothesis of a female vocal masculinization, as initially suggested. In this context, we suggest two possible explanations. First, F0mean may not be the most reliable acoustic parameter to study the evolution of voice over time as it captures relatively little information on how an individual vocally behaves (i.e. static cue). Second, lack of significant changes could also be explained by an important factor we did not control for: individuals could use their voice pitch differently depending on their social positions such as a lower and a higher voice pitch in high-and low-ranking individuals, respectively [START_REF] Klofstad | Sounds like a winner: voice pitch influences perception of leadership capacity in both men and women[END_REF][START_REF] Anderson | Preference for Leaders with Masculine Voices Holds in the Case of Feminine Leadership Roles[END_REF][START_REF] Mayew | Voice pitch and the labor market success of male chief executive officers[END_REF]. Indeed, some studies showed the existence of a link between vocal characteristics and social class [START_REF] Pittam | Voice in social interaction[END_REF][START_REF] Madsen | 10 Stylized voices of ethnicity and social divisions[END_REF][START_REF] Trudgill | The social differentiation of English in Norwich[END_REF]. Here, the relevance of INA archives (and any other audiovisual archives for that matter) should be underlined since a detailed analysis of available visual data could be particularly useful to verify the validity of this hypothesis. For instance, a fine-grained analysis of clothes could allow us to infer the interviewed individual's social background and account for an original method to further investigate this topic. Nonetheless, considering more dynamic acoustic criteria (i.e. F0sd, F0CV, F0min, F0max,) revealed interesting patterns. Figure 1d and 1e (i.e. F0min and F0max) show that both men and women's vocal range increased (simultaneous increase of F0max and decrease of F0min) from the 1970's to nowadays. Considering that F0mean and F0sd did not change significantly, we could interpret these results by suggesting that there are two "vocal profiles" coexisting within both sexes of the French population. All things being equal, this would mean that a part of the population progressively spoke over time in a more lower pitch register and another in a higher pitch register. Again, one possible explanation we suggest is that different social classes are using different ways to express themselves, which became more marked over time. Otherwise, this could also partially confirm that, at least in some parts of the population, there has been a "female vocal masculinization" and, unexpectedly, a "male vocal feminization". Along with results of a respective decrease and increase in F0CV in women and to a lesser extent men (which are considered to be respectively a masculine and feminine vocal trait [START_REF] Leongómez | 2014 Vocal modulation during courtship increases proceptivity even in naive listeners[END_REF]), such results occurring from the 1970's could be interpreted by the fact that some men and women started to depart themselves vocally from the social expectations attributed to their sex and gender at the very beginning of the 1960's because of the relaxation of social taboos and norms [17]. Indeed, the culture of decades prior to the 1960's was described as being unable to contain the demands for greater individual freedom, which saw people breaking free of the social constraints through extreme deviation from the norm. This major socio-historical phenomenon has been studied in the context of several domains, but our study could be, to our knowledge, the first one to investigate its effect on the human voice. Therefore, our work allows us to hypothesize that the evolution of vocal characteristics may be considered as a reliable sign of cultural evolution.

Although results are few, the current study offers a more systematic analysis on the evolution of voice pitch in both men and women. Cross-sectional studies are particularly important because they answer questions regarding the evolution of human behavior. As such, the use of audiovisual archives for that purpose is useful and extremely precious. In this context, we suggest two avenues for further research. Firstly, other acoustic parameters should be given attention as the human voice cannot be defined solely on the basis of voice pitch and modulation but also on timbre (vocal resonances), quality (roughness, breathiness…) and rhythm (speech tempo). Furthermore, as all languages can significantly differ across all these aspects, comparing the evolution of voices from different cultures is a particularly interesting aspect. Indeed, this could give us important cues on how gender and social roles have evolved over time across each culture.

Figure 1 .

 1 Figure 1. Evolution of a) Mean pitch (F0mean), b) Pitch variation (F0sd), c) CV (F0CV), e) Maximum pitch (F0max), d) Minimum pitch (F0min). Women's voices are in red and men's voices are in blue.

Acknowledgments

We would like to thank the INA for providing us access to the audiovisual archives. We would also like to thank the International Federation of Television Archives for funding and allowing us to realize this original study.

Access to data

Data can be requested by sending an email to Alexandre Suire: alexandresuire@hotmail.fr


Alessandra Serva

Allan Guerault

Yoshiki Ishii

Emmanuelle Gouillart

Ekaterina Burov

Mathieu Salanne

Structural and dynamic properties of soda-lime-silica in the liquid phase

I. INTRODUCTION

A vast majority of manufactured glass are based on the soda-lime-silica system. It is mainly made of three components: silica (SiO 2 ), which is a network former setting the framework of the glass, 1 while calcium oxide (CaO) and sodium oxide (Na 2 O) are network modifiers [START_REF] Mead | A molecular dynamics study of the atomic structure of (CaO)x(SiO 2 ) 1-x glasses[END_REF][START_REF] Jund | Channel diffusion of sodium in a silicate glass[END_REF] whose concentration strongly impacts many of the materials properties. A large number of additional cations, such as Al 3+ , K + , Mg 2+ , Fe 3+ , Ti 4+ are also present with very low concentrations. Due to the various uses of soda-lime-silica glass, such as windows and bottles to name the most common ones, its properties are very well characterized in the vitreous state.

The liquid state, which forms above ≈ 1300 K, has far less been studied due to the difficulty to conduct experiments at such temperatures. Indeed, the literature on silicate melts at high temperature has focused more on compositions of geological interest [START_REF] Zhang | Silicate melt properties and volcanic eruptions[END_REF][START_REF] Giordano | Viscosity of magmatic liquids: a model[END_REF][START_REF] Zhang | Diffusion data in silicate melts[END_REF] than of industrial interest. A better characterization would be beneficial for many industrial processes. In particular, understanding the impact of the sodium and the calcium ions on the structure is of primary importance, and establishing the link between their structure and their diffusion properties is necessary for a better control of the glass melting and transformation properties. For example, it could provide useful input for controlling the rate of crystal nucleation [START_REF] Fokin | Homogeneous crystal nucleation in silicate glasses: A 40 years perspective[END_REF][START_REF] Yuritsyn | Crystal nucleation in soda-lime-silica glass at temperatures below the glass transition temperature[END_REF] or the interdiffusion with substrates [START_REF] Fonne | Interdiffusion between silica thin films and soda-lime glass substrate during annealing at high temperature[END_REF] during the glass formation. New experimental setups have recently been proposed to study glassy oxides in the liquid state, [START_REF] Ohkubo | Self-diffusion coefficient of lithium in molten x Li 2 O-(1-x)B 2 O 3 system using high-temperature PFG NMR[END_REF][START_REF] Bolore | X-ray imaging of a hightemperature furnace applied to glass melting[END_REF] but so far the only available experimental data for diffusivities in liquid soda-lime-silica were obtained a) Electronic mail: mathieu.salanne@sorbonne-universite.fr through studies of trace elements using electrochemical methods, [START_REF] Rüssel | Self diffusion of polyvalent ions in a soda-lime-silica glass melt[END_REF] or isotopic tracer diffusivity in undercooled melts, close to the glass transition. [START_REF] Njiokep | Tracer diffusion of 22 Na and 45 Ca, ionic conduction and viscosity of two standard sodalime glasses and their undercooled melts[END_REF] In this respect, classical Molecular Dynamics (MD) simulations are a powerful tool to deeply understand both structural and dynamics properties at a molecular level. Many different force fields are dedicated to silica-derived materials. The most generic ones employ fixed partial charges and a Buckingham-type pair potential, [START_REF] Pedone | A new self-consistent empirical interatomic potential model for oxides, silicates, and silica-based glasses[END_REF][START_REF] Pedone | Elastic and dynamical properties of alkali-silicate glasses from computer simulations techniques[END_REF][START_REF] Laurent | Structure and topology of soda-lime silicate glasses: implications for window glass[END_REF][START_REF] Molnár | Microscopic scale simulations of soda-lime-silica using molecular dynamics[END_REF] but they are mostly used to study the structural and mechanical properties in the glassy state. For example, Guillot et al. have developed a series of potentials to study the thermodynamic properties of natural silicate melts (magmatic liquids) at very high temperature. [START_REF] Guillot | A computer simulation study of natural silicate melts. part I: Low pressure properties[END_REF][START_REF] Guillot | A computer simulation study of natural silicate melts. part II: High pressure properties[END_REF][START_REF] Dufils | Properties of magmatic liquids by molecular dynamics simulation: The example of a morb melt[END_REF][START_REF] Dufils | Properties of planetary silicate melts by molecular dynamics simulation[END_REF] The soda-lime-silica structure was studied using such potentials, either from simulations only [START_REF] Cormack | Molecular dynamics simulations of soda-lime-silicate glasses[END_REF] or through combination with diffraction experiments. [START_REF] Cormier | Ca and Na environments in Na 2 O-CaO-Al 2 O 3 -SiO 2 glasses: influence of cation mixing and cationnetwork interactions[END_REF] Nevertheless, when studying the dynamics of such complex oxides, Madden and co-workers [START_REF] Madden | covalent' effects in 'ionic' systems[END_REF][START_REF] Jahn | Transferable interaction model for Al 2 O 3[END_REF][START_REF] Jahn | Modeling Earth materials from crustal to lower mantle conditions: a transferable set of interaction potentials for the CMAS system[END_REF] have shown the importance of accounting for complex interactions, such as polarization effects as well as ionic deformation effects for the repulsion term, leading to the aspherical ion model (AIM). Such potentials are more computationally expensive, but they generally show a high level of agreement with experiments and a high transferability upon composition, temperature and pressure changes. The parameters can be obtained through force and dipole-fitting against firstprinciples calculation, 27 so that no experimental information is used during the development. In recent work, we have validated such a potential for sodium aluminosilicate glasses and melts against a large body of experimental data (bond lengths, neutron and X-ray diffraction, and NMR spectroscopy). [START_REF] Ishii | A DFT-based aspherical ion model for sodium aluminosilicate glasses and melts[END_REF] The potential was then extended to MgO-SiO 2 and CaO-SiO 2 mixtures. [START_REF] Salmon | Pressure induced structural transformations in amorphous MgSiO 3 and CaSiO 3[END_REF] In this study we use this AIM potential to investigate the structure and the dynamics of a model soda-lime-silica system in the liquid state.

II. MODEL AND METHODS

A. Aspherical ion model (AIM)

The AIM potential energy consists in a sum of chargecharge, polarization, repulsion and dispersion terms.

φ tot = φ charge-charge + φ pol + φ rep + φ disp (1)
All of them are provided (in atomic units) in the following. Firstly,

φ charge-charge = i j>i q i q j r ij (2)
where q i is the charge of each ion i, and r ij the distance between i and j. Contrarily to many rigid ion models, [START_REF] Pedone | A new self-consistent empirical interatomic potential model for oxides, silicates, and silica-based glasses[END_REF][START_REF] Guillot | A computer simulation study of natural silicate melts. part I: Low pressure properties[END_REF] formal charges are used for all the ions (-2 for O, +4 for Si, +3 for Al, +2 for Ca and +1 for Na in the present work). The polarization energy term φ pol includes charge-dipole and dipole-dipole interactions,

φ pol = i j>i q i r ij • µ µ µ j r 3 ij f ij D (r ij ) - µ µ µ i • r ij q j r 3 ij f ji D (r ij ) (3) + i j>i µ µ µ i • µ µ µ j r 3 ij - 3(r ij • µ µ µ i )(r ij • µ µ µ j ) r 5 ij + i |µ µ µ i | 2 2α i
where µ µ µ i is the induced dipole moment of particle i. The f ij D are functions [START_REF] Tang | An improved simple model for the van der Waals potential based on universal damping functions for the dispersion coefficients[END_REF] that correct the charge-dipole interactions at short range, [START_REF] Wilson | Quadrupole polarization in simulations of ionic systems: application to AgCl[END_REF][START_REF] Wilson | Molecular dynamics simulations of compressible ions[END_REF]

f ij D (r ij ) = 1 -c ij D e -b ij D rij 4 k=0 (b ij D r ij ) k k! (4)
in which b ij D and c ij D are two parameters giving the range and the strength of the correction. Note that the range of damping of the charge of i on the dipole of j is the same as the one of the charge of j on the dipole of i (b ij D = b ji D ), but this reciprocity is not true for the correction strength (c ij D = c ji D ). The effect of the latter on anions is generally to reduce the dipole from its asymptotic value [START_REF] Madden | covalent' effects in 'ionic' systems[END_REF][START_REF] Jemmer | Dipole and quadrupole polarization in ionic systems: ab initio studies[END_REF] while the situation is more complex for cations dipoles. [START_REF] Domene | Short-range contributions to the polarization of cations[END_REF] The induced dipoles are calculated by solving self-consistently the set of equations

µ µ µ i = α i E i {q j } j =i , {µ µ µ j } j =i , (5)
where E i is the electric field generated at r i by the whole set of charges and induced dipoles from the ions j = i, and α i is the polarizability of ion i. In practice, the instantaneous dipole moments are determined at each time step by minimization of the total energy (at fixed positions and charges -the latter being constant parameters throughout the whole simulation) using the conjugate gradient method. [START_REF] Jahn | Structure and dynamics in liquid alumina: simulations with an ab initio interaction potential[END_REF] The charge-charge, charge-dipole and dipole-dipole contributions to the potential energy and forces of each ion are evaluated under the periodic boundary condition by using the Ewald summation technique. [START_REF] Aguado | Ewald summation of electrostatic multipole interactions up to the quadrupolar level[END_REF] The repulsive term is given by: 26,37

φ rep = i∈cation j∈O A ij 1 exp(-b ij 1 ρ ij ) + A ij 2 exp(-b ij 2 ρ ij ) (6)
+ i∈cation j∈O

A ij 3 exp(-b ij 3 r ij ) + i∈O j∈O,i<j A ij 1 exp(-b ij 1 r ij ) + i∈O [D {exp(βδσ i ) + exp(-βδσ i )} + exp(ζ 2 |ν i | 2 ) -1
where ρ ij plays the role of an "effective" distance between ions i and j. It is calculated at each time step using

ρ ij = r ij -δσ i -δσ j - 1 r ij r r r ij • (ν ν ν i -ν ν ν j ), (7)
where δσ i represents the deviation of the ionic radius, and ν ν ν i expresses the distortion of the dipolar shape. These variables therefore account for the anisotropic deformation of the ionic shape. They are treated as additional degrees of freedom of the simulation. As the induced dipoles, they are computed at each timestep by minimizing the total energy at fixed positions through a conjugate gradient procedure; the fourth summation term of eq 6 consists of the self-energy terms to account for the energy cost of these deformation. The repulsive interaction thus includes many-body effects. Finally, the dispersion term is given by an usual asymptotic expansion

φ disp = - i j>i C ij 6 r 6 ij f ij 6 (r ij ) + C ij 8 r 8 ij f ij 8 (r ij ) (8)
where similar damping functions are used as for the charge-dipole term to screen the interactions at short range , except that they involve only a single range parameter in each case:

f ij 6 (r ij ) = 1 -e -b ij 6 rij 6 k=0 (b ij 6 r ij ) k k! (9)
f ij 8 (r ij ) = 1 -e -b ij 8 rij 8 k=0 (b ij 8 r ij ) k k! (10)
All the parameters for such an AIM are fitted from first-principles calculations , except from the dispersion coefficients (C ij 6 and C ij 8 ) which were adjusted in order to reproduce experimental glass densities under ambient condition (because the functional used for the firstprinciples calculations notoriously underestimates dispersion effects [START_REF] Corradini | The effect of dispersion interactions on the properties of lif in condensed phases[END_REF] ). This parameterization was already performed for Si, Al, Na and O in Ref. 28 4). All the parameters are provided in atomic units. The system was studied at six different temperatures (1400, 1800, 2000, 2200, 2400 and 3000 K). The simulation cells were first equilibrated by performing a simulation in the NPT ensemble, using the method of Martyna et al. [START_REF] Martyna | Constant pressure molecular dynamics algorithms[END_REF] with relaxation times of 0.5 ps for both the ther-TABLE III. Pair parameters for repulsive and dispersion terms in soda-lime silicates. All the parameters are provided in atomic units. The cations interact between themselves only through electrostatics (the corresponding repulsion terms are null). mostat and the barostat, for at least 1 ns, with a time step of 0.5 fs. Production simulations were carried out in the NVT ensemble, using again a timestep of 0.5 fs and saving a configuration every 100 steps. The Nosé-Hoover chain thermostat, [START_REF] Martyna | Nosé-Hoover chains: the canonical ensemble via continuous dynamics[END_REF] with a relaxation constant of 0.5 ps, was used to control the system temperature. The total simulation time was 10 ns for the 1400 K, 1800 K, 2000 K and 2200 K temperatures and 6 ns for the 2400 K and 3000 K temperatures. The short-ranged repulsion interactions were calculated with a cut-off distance fixed to half the simulation cell (typically 10 Å) while the Ewald summation method was used for dispersion 41 and electrostatics [START_REF] Aguado | Ewald summation of electrostatic multipole interactions up to the quadrupolar level[END_REF] interactions. It employed the same cut-off distance as for repulsion for the short-range sum, and an accuracy of 10 -7 .

i-j O-O Si-O Al-O Ca-O Na-O b ij D 2.
i-j O-O Si-O Al-O Ca-O Na-O A ij

III. RESULTS AND DISCUSSIONS

A. Short-range structure

We start the structural analysis by characterizing the coordination shell of the various cationic species. The radial distribution functions (RDFs) between the network formers Si and Al and the oxygen atoms (Si-O and Al-O) show very intense first peaks followed by a first minimum close to 0 (see Figure 1, left panels). This is typical of a well-defined first coordination shell. The position of the first peak gives Si-O and Al-O distances 1.62 and 1.75 Å, respectively. These distances do not vary with temperature, but there is a decrease in intensity and a broadening of the peak with increasing temperature. The corresponding average coordination number, calculated by integration of the RDF up to the first minimum, is 4 for both of them for all the investigated temperatures. These numbers are consistent with our previous simulation works on aluminosilicate systems, [START_REF] Ishii | A DFT-based aspherical ion model for sodium aluminosilicate glasses and melts[END_REF] and experimental values of Ref. 42.

Figure 1 also shows the RDFs between the network modifiers, Na and Ca, and the oxygen atoms. We can see that the Na-O and Ca-O most likely distances are very close since the corresponding RDFs first peaks are respectively centered at 2.22 and 2.20 Å, with an average coordination number of about 5 for both cations. These numbers are in agreement with previous theoretical studies of soda-lime-silicate system carried out at 300 K, [START_REF] Laurent | Structure and topology of soda-lime silicate glasses: implications for window glass[END_REF][START_REF] Cormack | Molecular dynamics simulations of soda-lime-silicate glasses[END_REF] while a coordination number of about 7 for Ca and 6 for Na has been found in another XAS/MD study. [START_REF] Cormier | Ca and Na environments in Na 2 O-CaO-Al 2 O 3 -SiO 2 glasses: influence of cation mixing and cationnetwork interactions[END_REF] However, as discussed below, the distribution of coordination number is much wider than for Si and Al, which can explain such discrepancies. The position of this first peak also does not change with increasing temperature. In particular, the Ca-O RDF's first peak is somewhat narrower as compared to the Na-O one, denoting a more rigid coordination shell for Ca 2+ ions. Anyway, in both cases the RDF's first minimum does not go to zero, meaning that the first coordination shell is more flexible in comparison to the network formers. This feature is reflected in the instantaneous coordination number analysis (see Figure 2), which shows that the Si and Al cations are almost only 4-fold coordinated by oxygen atoms at all investigated temperatures, while Ca and Na cations can adopt a very large number of different coordinations. In particular, the Ca cations can take all values from 3 to 8, with 5 as favored configuration and 4/6 as second favored configurations. For Na cations the distribution of coordination numbers is even broader, ranging from 2 to 9, with preferential values of 4, 5 and 6. Moreover, the Na-O instantaneous coordination number probability seems not to be affected by the temperature, while a small trend can be seen for the Ca cations. For the latter the 5-and 6-fold coordinated species probabilities decrease with increasing temperature, while the 3-and 4-fold ones increase, with a consequent slight decrease of the average coordination number (from 5.2 at 1400 K to 5.0 at 3000 K).

If we then look at long-ranged interactions, such as the ones occurring between Na-Na and Ca-Ca, the RDFs show a first contribution around 3.5 Å. An example for the simulation at 1400 K is reported in Figure 3. According to the literature, [START_REF] Cormack | Molecular dynamics simulations of soda-lime-silicate glasses[END_REF] this distance is smaller than the one required for a homogeneous distribution and it is generally interpreted using the modified random network model with preferential regions concentrated with modifiers and non-bridging oxygens. Also the Na-Ca RDF shows a peak between 3 and 4 Å, suggesting a mixing between Na and Ca atoms. In particular, the average coordination numbers are 2.50 for Na-Na, 1.48 for Na-Ca, 1.05 for Ca-Ca and 3.90 for Ca-Na. According to these numbers, 63% (2.50/3.98*100) of the 3.98 network mod-ifiers found on average around a Na atom are Na atoms while 37% (1.48/3.98*100) are Ca atoms. In the same way, 21% of the network modifiers around a Ca atom are Ca atoms while 79% are Na atoms. If we assume a random distribution of Na and Ca in the glass and considering that at the investigated glass composition 72% of the network modifiers are Na (N N a /(N N a +N Ca )*100) and 28% are Ca, we would expect to find around any network modifier 72% of Na atoms and 28% of Ca atoms. However, our numbers clearly show that around Na atoms there are less Na atoms (63%) than expected from a random distribution (72%), and around Ca atoms there are less Ca atoms (21%) than expected from a random distribution (28%). Our result thus indicates a preference for Na and Ca to mix, in agreement with a previous experimental NMR study, [START_REF] Lee | Nature of cation mixing and ordering in Na-Ca silicate glasses and melts[END_REF] where it has been shown a significant non random distribution of the Na and Ca modifying cations and this nonrandomness appears to be primarily governed by charge differences among cations.

B. Structure factors

In Figure 4 (left panel), the calculated neutron weighted structure factors, S(Q), are plotted as a function of the temperature. They are obtained through the following equation:

S(k) = 1+ 1 | α c α w α (k) | 2 α β c α c β w α (k)w * β (k)[S αβ (k)-1] ( 11
)
where k is the scattering vector magnitude, c α is the atomic fraction (of chemical species α ), w α is the coherent neutron scattering length. S αβ are the partial structure factors, obtained from the partial RDFs (noted g αβ (r)) by performing a Fourier transform:

S αβ (k) = 1 + 4πρ k ∞ 0 r[g αβ (r) -1] sin(kr)dr ( 12
)
where ρ is the atomic number density of the system. The total structure factor at 1400 K compares well with previous experiments carried out at 1273 K for a very similar system (75SiO 2 -15Na 2 O-10CaO). [START_REF] Cormier | Structural changes between soda-lime silicate glass and melt[END_REF] Moreover, we can see that the high-Q range, accounting for short-range interactions, is quite similar for all temperatures, while the low-Q region is characterized by a peak at around 1.7 Å-1 , which becomes broadened and less intense when

C. Network structure

In silicate glasses, the network structure is generally analyzed by splitting the oxygen population depending on their bonding. A bridging oxygen (BO) is defined as an oxygen atom connected to two network formers (Si/Al) in a sphere with radius corresponding to the first minimum of the Si-O/Al-O RDFs, while a non-bridging oxygen (NBO) is connected to only one Si/Al. Finally triple bonding oxygen (TBO) atoms are connected to 3 or more Si/Al atoms. The results of the analysis are summarized in Table IV and show that the vast majority of oxygens are BO, with a ratio BO/NBO of about 2.6, independently of the temperature (the TBO percentage is almost negligible).

T(K) BO NBO TBO Q 0 Q 1 Q 2 Q 3 Q 4 1400
Once BO, NBO and TBO have been identified, it is also possible to analyze the structure in terms of Q n distribution. Q is defined as a SiO 4 tetrahedra and n is the number of oxygen atoms belonging to this tetrahedra that are BO. At the calculated BO/NBO ratio, one should expect that on average a Si tetrahedron has 1 NBO and 3BO, i.e. is a Q 3 . Based on this, we should find in the Q n analysis a majority of Q 3 , followed by a minority of Q 4 and Q 2 , the latters having almost similar percentages. This is clearly not the case (see Table IV), suggesting that the NBOs are not homogeneously distributed, but preferentially localized close to the network modifiers. For both BO/NBO and Q n distribution analysis, an overall agreement is obtained with the values previously determined for various modelling and simulation studies on systems with a similar composition. [START_REF] Cormier | Structural changes between soda-lime silicate glass and melt[END_REF]

D. Structure around the bridging/non-bridging oxygen atoms

The decomposition of the Ca-O and Na-O RDFs, shown in Figure 5 (left panels), clearly indicates that the first coordination shell of Ca is largely formed by NBOs, with just a very small contribution from BOs. A different situation is depicted for Na cations, where there is a more balanced contribution of NBOs and BOs to the first coordination shell. Note that for both cations, the Ca/Na-NBO RDF's first peak is found at shorter dis- tances than the Ca/Na-BO one. In particular, at 1400 K the Ca ions are on average coordinated by 4.4 NBOs and 0.8 BOs, while there are 2.5 NBOs and 2.5 BOs around Na cations (see Figure 6). The greater affinity of NBOs for Ca than Na cations is preserved in the investigated temperature range, with a very slight decrease in the number of NBOs from 1400 K to 3000 K accompanied by a small increase in the number of BOs, in order to mantain the total average coordination number almost constant. These observations agree with the numerical study of Tilocca and de Leeuw, [START_REF] Tilocca | Structural and electronic properties of modified sodium and soda-lime silicate glasses by carparrinello molecular dynamics[END_REF] which also found a greater affinity of NBOs for calcium than sodium ions, and with experimental NMR [START_REF] Jones | MAS NMR study of soda-lime-silicate glasses with variable degree of polymerisation[END_REF] or Raman 46 results showing preferential arrangement of Ca in the vicinity of Q 2 rather Q 3 species.

E. Diffusion coefficients

The self-diffusion coefficients have been calculated from the slope of the mean-squared displacements (MSD) versus time using the Einstein relation:

D i = lim t→∞ 1 6t |δr a (t) | 2 (13)
where δr a is the displacement of a given ion of species i in time t. They are shown as an Arrhenius plot on the lowest investigated temperatures, the motion of the network modifiers starts to decouple from the one of the network formers, and the Na and Ca cations diffuse into a silica matrix that is basically frozen along all the simulation time. Finally, we can see that Na ions always diffuse faster than the Ca ones, and both diffusion coefficients follow a linear Arrhenius behaviour in all the investigated range of temperature, i.e D ∝ e -Ea/RT , with an activation energy, E a , of 1.32 eV (127 kJ mol -1 ) and 0.62 eV (59.9 kJ mol -1 ) for Ca and Na, respectively. The activation energy of Na is consistent with values found in the literature for similar compositions. [START_REF] Pedone | Elastic and dynamical properties of alkali-silicate glasses from computer simulations techniques[END_REF] However, activation energies are significantly smaller than the experimental values found closer to the glass transition. [START_REF] Njiokep | Tracer diffusion of 22 Na and 45 Ca, ionic conduction and viscosity of two standard sodalime glasses and their undercooled melts[END_REF] Diffusion values of Si, Al and O are close to each other, consistently with the literature observations that diffusivities of network formers and oxygen are governed by the Eyring law relating diffusivity and viscosity.

The different diffusivities of Na and Ca can be linked to the different structural organization adopted by these two cations. Indeed, the RDF and coordination number analysis at 1400 K (Figures 5 and6) have shown that the difference in the first coordination shell of the two cations relies on the number of NBOs that they coordinate (4.4 for Ca vs 2.5 for Na). From the RDFs it is also possible to calculate the potential of mean force (PMF) through the following expression:

P M F = -K B T ln(g(r)) (14)
where K B is Boltzmann's constant and T is the temperature of the system. Note that a maximum in RDF corresponds to a minimum in the PMF and vice-versa.

The difference in energy between the first minimum and the first maximum in the PMF provides an estimate of the activation energy (E a ) needed to break one Ca/Na-O bond (see Figure 5, right panels). The energy that a Na or Ca cation needs to escape from its first coordination shell can hence be estimated as:

E tot = E a • N ( 15
)
where N is the average coordination number. When considering the total Ca-O and Na-O RDFs, we obtain E O tot = 1.30 eV for Ca and E O tot = 0.56 eV for Na. Thus, the Ca cation needs ∼ 2.5 times more energy to escape its coordination shell than Na. However, the difference in E O tot may not arise from the total average coordination number, that is very similar between the two cations, but rather from their different NBO/BO ratio. This becomes evident when calculating the PMFs from the Ca/Na-BO and Ca/Na-NBO RDFs instead. As shown in Figure 5, right panels, the PMF for Ca/Na-NBO exhibits a clear first minimum, which is deeper for Ca than Na, while the Ca/Na-BO PMF does not have any first minimum for both cations. In particular, E N BO tot has been found equal to 1.92 eV and 0.66 eV for Ca and Na, respectively. This means that the energy cost to break the first solvation shell observed from both Na-O and Ca-O PMF solely comes from the Ca/Na-NBO bonds. It is therefore higher for Ca than Na because Ca is on average coordinated to more NBOs (4.4 vs 2.5 for Na) and with stronger bond (E Ca-N BO a = 0.44 eV vs E N a-N BO a = 0.26 eV). Interestingly, E O tot obtained from PMF compares well with the activation energy previously determined from the diffusion coefficients, suggesting that escaping from the first oxygen coordination shell is the limiting step for the diffusion of Na and Ca cations. In the light of all these findings, the diffusion of the network modifiers is clearly ruled by the non-bridging oxygens coordinated to them, and thus Na diffuses faster than Ca because it is bonded to less NBOs and less strongly than Ca. This results also suggests that the diffusion mechanism is probably more interstitial-based (i.e an ion escapes its first coordination shell to an empty space in the network) than pair-based (i.e two cations exchange positions), the two mechanism being previously identified for the diffusion of network modifiers in silicate melts. [START_REF] Jund | Channel diffusion of sodium in a silicate glass[END_REF][START_REF] Pedone | Elastic and dynamical properties of alkali-silicate glasses from computer simulations techniques[END_REF][START_REF] Tilocca | Sodium migration pathways in multicomponent silicate glasses: Car-Parrinello molecular dynamics simulations[END_REF]

IV. CONCLUSION

In this work a soda-lime-silica system in the liquid state (from 1400 to 3000 K) has been modelled by means of classical Molecular Dynamics simulations, using an aspherical ion model that accounts for atomic polarization and deformation effects. Overall, no structural important modifications occur going from 1400 to 3000 K and the calculated neutron structure factor compares well with previous experiments. In particular, we evaluated the structure in terms of bridging (BO) and non-bridging oxygens (NBO), showing the greater affinity of Ca 2+ ions for NBOs with respect to Na + , which is preserved in all the investigated temperature range. The different structural organization adopted by the two cations has been further linked to their different diffusivities. By computing the potential of mean force from the radial distribution functions, we find evidence that the limiting step for the diffusion of Ca 2+ and Na + ions is escaping from their first oxygen coordination shell. This step requires more energy for Ca 2+ than Na + , since Ca 2+ is on average coordinated to more NBOs and more strongly, thus the lower diffusivity of Ca 2+ .

Establishing at a molecular level the link between the structure and diffusion properties of Ca 2+ and Na + ions is the first step for a better understanding of glass melting and transformation processes. Some works remain still to be done to further investigate if there are also multicomponent diffusion effects [START_REF] Liang | Multicomponent diffusion in molten silicates: theory, experiments, and geological applications[END_REF][START_REF] Claireaux | Atomic mobility in calcium and sodium aluminosilicate melts at 1200 C[END_REF] that take place in such systems.

FIG. 1 .

 1 FIG. 1. X-O RDFs calculated for the various temperatures (X = Si, Ca, Al, O or Na). The insets for the Si-O, Al-O and O-O pairs show a magnified view of the RDF first peaks.

FIG. 2 .FIG. 3 .

 23 FIG.2. Instantaneous coordination number (n) probability distributions, expressed in percentage, for the various temperatures.

FIG. 4 .

 4 FIG. 4. Left panel: calculated neutron weighted total structure factor as a function of temperature, and experimental structure factor at 1273 K from Ref. 42. Right panel: neutron weighted partial structure factors calculated 1400 K.

FIG. 5 .

 5 FIG.5. Left panels: Ca/Na-O, Ca/Na-BO and Ca/Na-NBO RDFs calculated at 1400 K. Right panels: corresponding potential of mean force (PMF, eV) extracted from the radial distribution functions.

Figure 7 .FIG. 6 .

 76 FIG.6. Average coordination number of oxygen, as well as its decomposition between BOs and NBOs, around Ca (top) and Na (bottom) as a function of the temperature. The cutoffs used are 3.09 and 3.23 Å for Ca and Na, respectively.

FIG. 7 .

 7 FIG. 7. Diffusion coefficients plotted as a function of the inverse of temperature.

  and for Ca in Ref.29. The parameters are listed in Tables I, II and III.

TABLE I .

 I Individual parameters of the AIM force field. The Si and Al atoms are not polarizable in the AIM model (because their polarizabilities are negligible), and only the O atoms are deformable. All the parameters are provided in atomic units.

	i	O	Si Al Ca	Na
	qi	-2 +4 +3 +2 +1
	αi 10.74 --3.183 0.991
	D 0.5287 --	-	-
	ζ 1.6838 --	-	-
	β 1.5723 --	-	-

TABLE II .

 II Pair parameters for charge-charge and polarization terms in soda-lime silicates (dipole damping functions provided in Equation

  SiO 2 system with composition in mol% 10.71 CaO, 14.53 Na 2 O, 0.3 Al 2 O 3 and 74.46 SiO 2 were carried out in a range of temperature 1400 K -3000 K. The corresponding numbers of ions in the simulation box are 293 O 2-, 125 Si 4+ , 1 Al 3+ , 47 Na + and 18 Ca 2+ .

	513 1.939 1.908 1.769 1.964
	c ij D 2.227 1.446 1.627 1.881 3.493
	c ji D 2.227	0.144 0.066

B. Molecular dynamics simulations

Classical MD simulations of CaO-Na 2 O-Al 2 O 3 -

TABLE IV .

 IV Distribution (expressed in percentage) of the number of bridging (BO), non-bridging (NBO) and triple bonding oxygens and distribution of the Q n as a function of the temperature. See the text for more details.

ACKNOWLEDGMENTS

This work was supported by the French National Research Agency (project MAGI, Grant No. ANR-17-CE08-0019-04).

DATA AVAILABILITY STATEMENT

The data that support the findings of this study (input files for the simulations, raw data used for the various figures) are available on a GitLab repository (https://gitlab.com/magi4) as well as on Zenodo (http://dx.doi.org/10.5281/zenodo.4266009).


Diana A Monteiro
email: dianamonteiro@ufscar.br

Edwin W Taylor

David J Mckenzie

Francisco T Rantin

Ana L Kalinin

Interactive effects of mercury exposure and hypoxia on ECG patterns in two Neotropical freshwater fish species: Matrinxã, Brycon amazonicus and traíra, Hoplias malabaricus

Keywords: cardiac electrical excitability, heavy metal, ECG, bradycardia, action potential, Brycon amazonicus, Hoplias malabaricus

Hypoxia and mercury contamination often co-occur in tropical freshwater ecosystems, but the interactive effects of these two stressors on fish populations are poorly known. The effects of mercury (Hg) on recorded changes in the detailed form of the electrocardiogram (ECG) during exposure to progressive hypoxia were investigated in two Neotropical freshwater fish species, matrinxã, Brycon amazonicus and traíra, Hoplias malabaricus. Matrinxã were exposed to a sublethal concentration of 0.1 mg L-1 of HgCl2 in water for 96 h. Traíra were exposed to dietary doses of Hg by being fed over a period of 30 days with juvenile matrinxãs previously exposed to HgCl2, resulting in a dose of 0.45 mg of total Hg per fish, each 96 h.

Both species showed a bradycardia in progressive hypoxia but Hg exposure impaired cardiac electrical excitability in both fish species, leading to first-degree atrioventricular block, profound extension of the ventricular action potential (AP) plateau, and the development of cardiac arrhythmias and anomalies (atrial fibrillation, extra systoles, negative QRS complex, T wave inversion), especially in hypoxia below O2 partial pressures (PO2) of 40 mmHg.

Moreover, the chronic dietary Hg exposure induced intense bradycardia in normoxia in traira, lengthening of ventricular AP duration coupled with prolonged QRS intervals. This indicates slower ventricular AP conduction during ventricular depolarization. Overall, the data indicate that acute waterborne and chronic trophic inorganic mercury exposures, at sub lethal doses, cause damages in electrical stability and rhythm of the heart beat, leading to amyocardial dysfunction which is further intensified during hypoxia. These changes could lead to impaired cardiac output, with consequences for swimming ability, foraging capacity, and hence growth and/or reproductive performance.

Introduction

The freshwater aquatic environments inhabited by fishes, particularly in the tropics, are often highly variable in terms physical and chemical properties [START_REF] Carpenter | State of the world's freshwater ecosystems: physical, chemical, and biological changes[END_REF].

Hypoxia is a phenomenon that occurs over large areas in aquatic systems worldwide and may affect resident animals, leading to population decline and changes in community composition due to elimination of sensitive species [START_REF] Shang | Aquatic hypoxia is a teratogen and affects fish embryonic development[END_REF]. Hypoxia can be common in tropical freshwater habitats and fishes possess a variety of regulatory mechanisms to respond to this environmental stressor. These include increased gill ventilation and an increase in functional gas exchange surface in the gills [START_REF] Glass | Cardiorespiratory synchronization and myocardial function in hypoxic carp, Cyprinus carpio L[END_REF]. In most species , hypoxia also provokes braycardia, due to increased vagal tone on the heart [START_REF] Taylor | The role of the cardiac vagus in the response of the dogfish, Scyliorhinus canicula to hypoxia[END_REF][START_REF] Taylor | Nervous control of the heart and cardiorespiratory interactions[END_REF][START_REF] Rantin | Effects of environmental oxygen changes on cardio-respiratory function in fish[END_REF]. Relatively little is known about how responses to hypoxia might be influenced by other concurrent changes in water quality, such as increasing pollutant concentrations.

Of all contaminant metals mercury (Hg) is by far the most toxic, due to its persistence in water and sediments and its ability to bioaccumulate and biomagnify through aquatic food webs [START_REF] Lacerda | Mercury contamination in aquatic ecosystems: an analysis of the critical areas[END_REF]. Hg transfer and biomagnification have been demonstrated in both freshwater and marine food webs and can lead to hazardous concentrations in fishes and fish-eating wildlife [START_REF] Lavoie | Biomagnification of mercury in aquatic food webs: a worldwide meta-analysis[END_REF]. Although Hg contamination in aquatic ecosystems can derive both from processes, the main sources today are anthropogenic activities, such as agricultural materials, mining and smelting operations, chloralkali, pulp, and paper industries [START_REF] Wang | Sources and remediation for mercury contamination in aquatic systems -a literature review[END_REF], among others. The environmental impact of Hg can be intensified by ecological disasters, a case in point being the collapse of a mining dams in the Brazilian state of Minas Gerais (Mariana on November, 05 th 2015 and Brumadinho on January 28 th 2019).

The aim of this study was to analyze the effects of sub-lethal doses of inorganic mercury (HgCl2) on cardiac electrical activity (electrocardiogram, ECG) during hypoxia in two ecologically distinct Neotropical fish species, matrinxã Brycon amazonicus (Characidae) and traíra Hoplias malabaricus (Erythrinidae). These species were exposed, respectively, to acute water borne HgCl2 for 96 h or chronic dietary HgCl2 for 30 days. The matrinxã is an omnivorous and a rheophilic species native to the Amazon basin that inhabits clear and welloxygenated waters, frequently contaminated by Hg. The traíra is a widespread sedentary piscivorous species, which commonly inhabits tropical lakes.

According to [START_REF] Pollock | The effects of hypoxia on fishes: from ecological relevance to physiological effects[END_REF], low O2 rarely occurs in isolation in aquatic systems and it is of utmost importance to investigate interactions between hypoxia and contaminants in fishes. A previous study from our group demonstrated that waterborne and dietary Hg exposure decreased the plasticity of cardiorespiratory responses to hypoxia in these two fish species, reducing their ability to increase gill ventilation and respiratory frequency and, therefore, to maintain O2 extraction and regulate their metabolic rate [START_REF] Monteiro | Cardiorespiratory responses to graded hypoxia in the Neotropical fish matrinxã (Brycon amazonicus) and traíra (Hoplias malabaricus) after waterborne or trophic exposure to inorganic mercury[END_REF] .

In the present study, we used electrocardiograms (ECGs) to evaluate the effects of hypoxia and Hg exposures on electrical activity of the heart. ECG recordings provide information about cardiac rhythm, the duration of the ventricular action potential (AP), and the rate of impulse propagation through different chambers of the heart. The rate, rhythm and orderly spread of contraction depend on the electrical excitation of cardiomyocytes [START_REF] Badr | Effects of seasonal acclimatization on temperature dependence of cardiac excitability in the roach, Rutilus rutilus[END_REF]. The ECG can, therefore, reveal disturbances in cardiac function, in particular of impulse conduction [START_REF] Vornanen | Electrical excitability of the fish heart and its autonomic regulation[END_REF]. As a result, the utility of ECG monitoring in toxicological studies has gained traction because it is noninvasive means of obtaining a mechanistic, cause and effect, understanding of how animals might be affected by pollutants.

Material and Methods

Animals

Matrinxã (Wt = 154.5 ± 7.9 g), were obtained at the Águas Claras fish farm, Mococa,São Paulo State,Brazil. Traíra (Wt = 246.3 ± 7.5 g), were obtained at the Parque do Lago fish farm, Dourado, São Paulo State, Brazil. Fishes were acclimated for 60 days prior to experimentation in 500 or 1000 L holding tanks provided with a continuous supply of wellaerated and dechlorinated water at a constant temperature (24 ± 1 ºC). During this period, matrinxã were fed ad libitum with commercial fish pellets (40% of protein). Traíra will only accept live prey, therefore they were fed at a ratio of 2 % of biomass day -1 [START_REF] Rios | Erythrocyte senescence and haematological changes induced by starvation in the notropical fish traíra, Hoplias malabaricus (Characiformes, Erythrinidae)[END_REF] with small matrinxã (5-8 g).

Mercury exposures

For waterborne Hg exposure, B. amazonicus were divided into two experimental groups: control (Ctrl; n = 12) and Hg-exposed (wHg; n = 12), given ~20% of their LC50-96 h as previously established by [START_REF] Monteiro | Inorganic mercury exposure: toxicological effects, oxidative stress biomarkers and bioaccumulation in the tropical freshwater fish matrinxã, Brycon amazonicus (Spix and Agassiz, 1829)[END_REF], namely 0.1 mg L -1 of HgCl2 (> 99.5 % purity, Sigma-Aldrich) for 96 h in static holding tanks. Concentrations of total mercury ranging from 1 to 230 μg L -1 are usually detected in water bodies near industrialized areas [START_REF] Hypolito | Comportamento de espécies de mercúrio no sistema sedimento-água do mangue no município de Cubatão, São Paulo[END_REF][START_REF] Alinnor | Assessment of elemental contaminants in water and fish samples from Aba River[END_REF][START_REF] Bollen | Mercury speciation analyses in HgCl2-contaminated soils and groundwater -implications for risks assessment and remediation strategies[END_REF].

For trophic Hg exposure, H. malabaricus were divided into two treatment groups: control (Ct, n = 12) and Hg-exposed (tHg, n = 12), via contaminated food. The food supply consisted of four live juvenile B. amazonicus (5-8 g) each 96 h. The Ct received animals from clean water whereas tHg received animals exposed to 0.1 mg L -1 of HgCl2 for 96 h, as described above. Feeding rate was 2% of the total body weight per day, over 30 consecutive days. The total Hg concentration in the whole body of each living prey was 8.1 ± 0.9 mg kg -1 , analyzed by a Cold Vapor Atomic Absorption Spectrometry (USP, 2000. Considering the average weight of the prey, each traíra received about 0.45 mg of total Hg every 96 h. This dose was calculated to be similar to fish collected in Hg-impacted ecosystems [START_REF] Alho | Fish and wildlife resources in the Pantanal wetlands of Brazil and potential disturbances from the release of environmental contaminants[END_REF][START_REF] Hylander | Fish mercury concentration in the Alto Pantanal, Brazil: influence of season and water parameters[END_REF].

The water and trophic contamination refers mainly to inorganic Hg, since no methyl mercury level was detected in the experimental water or in the matrinxã exposed to HgCl2 and used as prey for the traíra. Methyl mercury was analyzed by gas chromatography -mass spectrometry (GC-MS) according to the method of [START_REF] Aoac | Mercury (methyl) in fish and shellfish: rapid gas chromatographic method[END_REF] and the quantification limits were 20 ng L -1 (water) and 1 µg kg -1 (tissues).

All treatments and experimental designs are described in detail in previous studies from our laboratory [START_REF] Monteiro | Inorganic mercury exposure: toxicological effects, oxidative stress biomarkers and bioaccumulation in the tropical freshwater fish matrinxã, Brycon amazonicus (Spix and Agassiz, 1829)[END_REF]2013).

In vivo experimentation: surgery and ECG recording

After waterborne and trophic exposures, fishes were anaesthetized by immersion in a solution of benzocaine (0.1 g L -1 ) then placed on a surgical tray with the gills irrigated by a flow of water containing the anaesthetic (0.05 g L -1 ). Two ECG electrodes were placed in a ventral position, the first (positive) between the gills and the heart, and the second (negative) in a ventral position close to the pelvic fins. After surgery, fishes were placed in the experimental chamber and left undisturbed to recover for 12 h in normoxic water (140 mmHg) at 25 o C (temperature for all experiments). A reference electrode was placed in the water of the experimental chamber. This preparation allowed electrocardiographic (ECG) recordings of the first diagonal branch (D1) lead. The ECG was obtained by coupling the electrode set to an amplifier (ML 136 Animal Bio Amp, AD Instruments) which was connected to a data acquisition system PowerLab (Powerlab 8/30, AD Instruments, Castle Hill, NSW, Australia). A 3 Hz-40 Hz band pass filter was used to remove muscle movement and power line artifacts. This computer-based ECG system permits detailed analysis of wave durations (P, T, and QRS complex), intervals (RR, QT, and PR), and segment (ST). The PR and QT intervals are equivalent, respectively, to the interval from onset of atrium depolarization and ventricle depolarization to total repolarization (i.e., the atrial AP duration -AAPD; and ventricular AP duration -VAPD). The ST segment represents the plateau of the ventricular AP.

After recovery from surgery, normoxic (PO2 = 140 mmHg or 7.3 mg L -1 of dissolved O2) control values were recorded and, subsequently, the PO2 was gradually reduced to 120, 100, 80, 60, 40, and 20 mmHg. Each tension was maintained for 45 min with recordings collected over the last 15 min . Normoxia was maintained by bubbling the water with compressed atmospheric air while hypoxic levels were created by dispersing pure N2 into the water of the experimental tank. The O2 tension of water was controlled by O2 electrodes (FAC001-O2) connected to a FAC-204A O2 Analyzer (FAC, São Carlos, SP, Brazil). All experiments were conducted at 25 °C.

Statistical analysis

Results are presented as means ± S.E.M. Differences between means at a 5% (P < 0.05) level were considered significant. Data from control and Hg exposed groups were tested for statistically significant differences using the Student t-test. The Kolmogorov-Smirnov method was applied to evaluate normality of the samples and the F-test was applied for homogeneity of variances (GraphPad Instat, GraphPad Software, USA). Analysis of variance one-way Anova complemented by Dunnett's multiple comparison test (GraphPad Instat version 3.00, GraphPad Software, USA) was used to check the possible existence of significant variations between the values obtained at different O2 tensions for the same experimental group. Two-way analysis of variance Anova complemented by Tukey's multiple comparison test (SigmaStat version 2.0 Jandel SigmaStat Statistical Software, USA) was used to determine interactions between water/diet Hg concentrations and water O2 levels on ECG responses.

Results

Effects of acute exposure to waterborne HgCl2 in matrinxã

Figure 1 shows the effects of graded hypoxia on ECG parameters of matrinxã in both experimental groups (wCt and wHg).

Fig 1

The Ct group maintained a constant P wave duration (38.6 ± 0.6 ms) at all O2 tensions, from normoxia to deep hypoxia. For the wHg group, the P wave duration was kept constant (38.2 ± 0.4 ms) from normoxia down to a PO2 of 80 mmHg, below which it was higher (nearly 30 %) than the Ct at O2 tensions from 60 to 20 mmHg. No significant differences were observed in the QRS complex duration, either for gradual hypoxia or between Ct and wHg groups. Conversely, the durations of T wave were significantly lower (26 to 38 %) than the Ct ones in the PO2 range between 140 and 20 mmHg. Furthermore, the T wave duration of the Ct group was maintained constant until 40 mmHg (169.1 ± 6.3 ms) and increased significantly in the O2 tension of 20 mmHg (247.1 ± 1.8 ms). The same tendency was observed for the wHg group, but T wave duration values increased significantly below 60 mmHg, reaching maximum values at 20 mmHg (175.5 ± 7.9 ms).

The RR intervals of both experimental groups remained nearly constant (1125.3 ± 80.1 ms) until a PO2 of 60 mmHg. Below this O2 tension, RR intervals increased significantly and progressively in both groups, reaching maximum values at 20 mmHg (Ct: 2759.3 ± 349.5 ms, and wHg: 5996.7 ± 715.0 ms). This indicates the typical reflex bradycardia in response to hypoxia for teleost fishes. The P wave durations of the wHg group were significantly higher than those of tthe Ct group at PO2's between 40 and 20 mmHg (48 and 117 %, respectively).

The Ct and wHg groups maintained a constant PR interval from normoxia to deep hypoxic conditions (74.2 ± 2.2 ms and 89.1 ± 3.1 ms, respectively). However, PR durations of the wHg group were significantly higher (~ 20 %) than the Ct ones at all O2 tensions.

The ST segment was maintained constant in the Ct group at all O2 tensions, whereas a significant increase in ST segment occurred in the wHg group at 20 mmHg. However, the values of ST segment of the wHg group were significantly higher (25 to 54 %) than those of the Ct group at O2 tensions from 140 to 20 mmHg.

Table 1 shows the effects of graded hypoxia on the QT interval or ventricular AP durations of B. amazonicus. For the wHg group, VAPD values increased significantly during deep hypoxia, while this tendency was not observed in the Ct group. QT interval was significantly higher (~20%) in the wHg group than in the Ct group, but only at a PO2 of 20 mmHg.

The two-way Anova analysis indicated a significant interaction effect between HgCl2 exposure and water O2 tensions just for RR interval, indicating that the Hg exposure changed the predictable pattern of response of heart rate (fH) of the matrinxã in hypoxia, making the reflex bradycardia more profound. No interactive effects were found for the other parameters evaluated.

The electrocardiograms representing the characteristic waveforms during normoxia and hypoxia for B. amazonicus are presented in Figure 2. These reveal a prolongation of the PR interval and ST segment with concomitant reduction of T wave duration/amplitude after acute waterborne Hg exposure. Moreover, in severe hypoxia, about 30% of fish from Ct group showed some ventricular extrasystoles (approximately 2 per min), whereas 80% of fish from wHg group exhibited these (3-4 per min). Arrhythmias, such as atrial fibrillation, were also observed predominantly in wHg group during gradual hypoxia, suggesting myocardial ischemia.

Fig 2

Effects of chronic exposure to trophic HgCl2 in traíra

The effects of graded hypoxia on the ECG of the tCt and tHg groups are shown in Figure 3.

Fig 3

The two groups exhibited no statistically significant differences in P duration (Ct: 95.5 ± 1.8 ms, and tHg: 104.8 ± 1.5 ms), QRS complex (Ct: 82.9 ± 0.8 ms, and tHg: 98.2 ± 2.7 ms), T wave duration (Ct: 283.2 ± 5.9 ms, and tHg: 208.9 ± 10.3 ms), or ST segment (Ct: 308.9 ± 5.8 ms, and tHg: 365.1 ± 2.5 ms) during hypoxia. However, at all PO2, trophic Hg exposure induced significant increases in the duration of P wave (14 -27 %), QRS complex (34 -75%), and ST segment (33 -52%), with concomitant reduction of T wave duration (36 -49%), in comparison to the controls.

The Ct group maintained a constant RR interval from normoxia down to 40 mmHg (1341.2 ± 22.9 ms), below which these values increased markedly, reaching maximum values at 20mmHg (2052.1 ± 197.1 ms). On the other hand, RR intervals of the tHg group (1797.8 ± 43.5 ms) increased progressively and significantly above a PO2 of 80 mmHg, reaching maximum values at 20 mmHg (2796.8 ± 230.1 ms). Furthermore, the duration of RR intervals for the tHg group were significantly higher (36 -74%) than the tCt at all PO2.

The PR interval was maintained constant until 20 mmHg in the Ct group (161.4 ± 6.4 ms) and tHg group (189.1 ± 6.6 ms). PR duration for the tHg group was significantly higher (12 to 23%) than the Ct group at PO2 between 140 and 20 mmHg.

The effects PO2 changes on the QT (VAPD) of H. malabaricus are presented in Table 2.

Discussion

Bradycardia has been characterized for many fish species when exposed to acute hypoxia, including in matrinxã and traíra [START_REF] Monteiro | Cardiorespiratory responses to graded hypoxia in the Neotropical fish matrinxã (Brycon amazonicus) and traíra (Hoplias malabaricus) after waterborne or trophic exposure to inorganic mercury[END_REF][START_REF] Kalinin | Dependence on body size of respiratory function in Hoplias malabaricus (Teleostei, Erythrinidae) during graded hypoxia[END_REF]. In most species the cardiac frequency remains constant with moderate decreases of PO2, whereas bradycardia (increase in RR intervals) develops with severe hypoxia [START_REF] Glass | Cardiorespiratory synchronization and myocardial function in hypoxic carp, Cyprinus carpio L[END_REF][START_REF] Taylor | Nervous control of the heart and cardiorespiratory interactions[END_REF]. Generally, the development of bradycardia in fish coincides with ECG waveform changes, such as alterations in T wave duration and amplitude, suggesting a relationship between cardiac dysfunction and hypoxic bradycardia (Glass et al., 1992;[START_REF] Rantin | Electrocardiographic characterization of myocardial function in normoxic and hypoxic teleosts[END_REF].

In our previous study, we demonstrated an effect of Hg on electrocardiographic recordings and ex vivo myocardial contractility under normoxic conditions [START_REF] Monteiro | Impact of waterborne and trophic mercury exposures on cardiac function of two ecologically distinct Neotropical freshwater fish Brycon amazonicus and Hoplias malabaricus[END_REF]. As a novel aspect, this study reports Hg-induced changes in ECG waveforms from two tropical fish species facing a hypoxic challenge. The present data indicate that Hg exposures, via water or food, intensified hypoxia-induced myocardial anomalies, causing severe cardiac dysfunction and reduction in fH. Similar data on impairment of cardiac function is available following exposure of fish to other toxic metals. Trout apparently died from heart failure following exposure to lethal levels of copper in freshwater [START_REF] Wilson | The physiological responses of freshwater rainbow trout (Oncorhynchus mykiss) during acutely lethal copper exposure[END_REF] and exposure to sublethal copper reduced swimming speed due to the indirect effects of ammonia accumulation on depolarization of muscle that was accompanied by changes in fH [START_REF] Beaumont | Exposure of brown trout, Salmo trutta, to a sub-lethal concentration of copper in soft acidic water: effects upon muscle metabolism and membrane potential[END_REF][START_REF] Taylor | Lethal and sublethal effects of copper upon fish: a role for ammonia toxicity?[END_REF].

In a fish ECG, the P wave represents the atrium depolarization, QRS complex indicates the ventricular depolarization, and the T wave corresponds to the repolarization of the ventricle [START_REF] Rantin | Electrocardiographic characterization of myocardial function in normoxic and hypoxic teleosts[END_REF][START_REF] Liu | Improvement of surface ECG recording in adult zebrafish reveals that the value of this model exceeds our expectation[END_REF]. Both PR interval and the ST segment were increased at all O2 levels, after waterborne and trophic Hg exposures. In the heart of B. amazonicus, acute Hg exposure also intensified the duration of the RR interval, P wave, and ST segment during deep hypoxia. On the other hand, these changes in the heart of H. malabaricus were observed at all O2 tensions, from normoxia to severe hypoxia. Separating direct and indirect effects of toxic metals on cardiac function in intact fish is difficult. This was achieved in the present study by trophic exposure to Hg, thus bypassing any direct impacts of hg on the gills. Trophic exposure to Hg caused an overall dysfunctional effect on the heart. The main effects of Hg were on the time-dependent processes such as the QRS complex, T wave and AP durations. The RR, PR, and QT intervals and ST segment were increased at all O2 levels as were the duration of P and QRS waves, though the duration of the T wave was reduced at all O2 levels. Subsequent hypoxia caused a significant increase in RR interval, marking a hypoxic bradycardia that was intensified in the presence of Hg.

The PR interval is equivalent to the interval from onset of atrium depolarization to total repolarization (i.e., the atrial AP duration AAPD). PR elongation, also known as firstdegree atrioventricular block (AV-block), happens when conduction through the AV node is slowed. Thus, Hg induced an AV-block, impairing transmission of the the AP from atrium to the ventricle at all O2 tensions. Cardiac contractile rhythmicity in the fish heart is determined by specialized pacemaker cells located in the sinoatrial (SA) node [START_REF] Hassinen | Small functional If current in sinoatrial pacemaker cells of the brown trout (Salmo trutta fario) heart despite strong expression of HCN channel transcripts[END_REF].

These Hg-induced abnormalities may be due to the ability of the metal to inhibit the inward Na + [START_REF] Visentin | Sodium current block caused by group IIb cations in calf Purkinje fibers and in guinea-pig ventricular myocytes[END_REF], Na + /K + -ATPase activity [START_REF] Anner | Mercury blocks the Na-K-ATPase by a ligand-dependent and reversible mechanism[END_REF], and sarcoplasmic reticulum (SR) function [START_REF] Hechtenberg | Inhibition of sarcoplasmic reticulum Ca (2+) -ATPase activity by cadmium, lead and mercury[END_REF][START_REF] Monteiro | Impact of waterborne and trophic mercury exposures on cardiac function of two ecologically distinct Neotropical freshwater fish Brycon amazonicus and Hoplias malabaricus[END_REF].

After Hg exposures, there was a remarkable prolongation of the plateau (ST segment) of ventricular AP, with concomitant reduction of ventricular repolarization time (T wave duration), in all O2 tensions. The AP plateau represents the cardiac systole duration, which changes with fH to allow both adequate time for ventricular filling and a force of contraction that sustains a steady cardiac output [START_REF] Tikkanen | Effects of prolonged anoxia on electrical activity of the heart in crucian carp (Carassius carassius)[END_REF]. A prolongation of the ventricular AP duration can be important to maintain the cardiac tissue perfusion, mainly during bradycardia that occurs when hypoxia is prolonged and/or after dietary exposure to Hg.

According to [START_REF] Driedzic | Energy metabolism and contractility in ectothermic vertebrate hearts: hypoxia, acidosis, and low temperature[END_REF], a prolonged AP probably indicates a prolonged time with open Ca 2+ channels and should also favor a possible Ca 2+ influx via the Na + -Ca 2+ exchange at positive membrane potentials. Patch-clamp experiments demonstrated that inorganic Hg amplified the voltage-dependent Ca 2+ current through L-type channels [START_REF] Rossi | Modification of Ca 2+ signaling by inorganic mercury in PC12 cells[END_REF]. Reactive oxygen species (ROS) can also regulate L-type Ca 2+ channel function by direct redox modification of cysteines on the channel protein. Exposing guinea-pig ventricular myocyte to ROS enhanced Ca 2+ influx through the L-type Ca 2+ channel (Hool, 2007). Our previous studies found that the same exposures to the same doses of Hg increased ROS generation and antioxidant enzyme activities in the heart of these fish species, causing oxidative stress, evidenced by significant increases in lipid and protein oxidation [START_REF] Monteiro | Inorganic mercury exposure: toxicological effects, oxidative stress biomarkers and bioaccumulation in the tropical freshwater fish matrinxã, Brycon amazonicus (Spix and Agassiz, 1829)[END_REF]2013).

On the other hand, Hg exposures reduced ventricular repolarization time (T wave duration) probably due its effects on activation of the membrane K + conductance. According to [START_REF] Jungwirth | Activation of cell membrane potassium conductance by mercury in cultured renal epitheloid (MDCK) cells[END_REF] Hg 2+ increasescell membrane K + selectivity, and decreases cell membrane resistance, enhancing the K + conductance of the cell membrane in cultured kidney cells. Abnormalities in ventricular repolarization were also revealed by a flattening and/or position inversion of T wave, predominantly in Hg exposed groups during severe hypoxia.

Alterations of the T wave (e.g., increase in amplitude and area, inversion, and biphasic aspect) indicate acute ischemia with insufficient O2 supply to the myocardium, leading to drastic changes in the direction of ventricular repolarization [START_REF] Rantin | Electrocardiographic characterization of myocardial function in normoxic and hypoxic teleosts[END_REF]. [START_REF] Mittal | Hypoxia induces Kv channel current inhibition by increased NADPH oxidase-derived reactive oxygen species[END_REF] demonstrated that sustained hypoxia induced K + channel current inhibition by increased ROS production in pulmonary artery smooth muscle cells. ROS are generated by mitochondria during hypoxia in cardiomyocytes [START_REF] Duranteau | Intracellular signaling by reactive oxygen species during hypoxia in cardiomyocytes[END_REF].

The prolonged P wave duration (atrial depolarization) in matrinxa below 60 mmHg and and in traíra at all O2 tensions indicates a slowing of conduction velocity in the atrium by conduction delay or blockage. Conduction delay happens when the propagating impulse fails to conduct, and gap junction coupling plays a pivotal role in sustaining the impulse propagation [START_REF] Gaztañaga | Mechanisms of cardiac arrhythmias[END_REF]. Connexins are ubiquitous channel forming gap junction channels that directly connect cardiomyocytes and conductive tissues to synchronize the atrial and ventricular contraction/relaxation cycles [START_REF] Leybaert | Connexins in cardiovascular and neurovascular health and disease: pharmacological implications[END_REF]. It is known that Hg reduce functional electric coupling mediated by connexin 43 gap junction channels [START_REF] Schirrmacher | Effects of lead, mercury, and methyl mercury on gap junctions and [Ca 2+ ]i in bone cells[END_REF].

Hypoxia evokes a reflex bradycardia mediated by the cholinergic fibers of the cardiac vagus, which allows the blood to stay in the gill lamellae longer to promote gas exchange when PO2 of the water falls [START_REF] Satchell | Physiology and Form of Fish Circulation[END_REF]. [START_REF] Tikkanen | Effects of prolonged anoxia on electrical activity of the heart in crucian carp (Carassius carassius)[END_REF] studied the effects of sustained anoxia on cardiac electrical excitability in crucian carp (Carassius carassius) and demonstrated that the oxygen shortage induced a strong bradycardic reflex and remarkable lengthening of ventricular AP duration (VAPD), which is equivalent to the interval from onset of ventricle depolarization to total repolarization (QT interval). In H. malabaricus, Hg exposure also induced bradycardia in normoxia and anticipated the reflex bradycardia in response to hypoxia, besides VAPD prolongation. These results suggest that Hg exposure deteriorated cardiac electrical behavior that would challenge the survivalof this species under hypoxic conditions. These findings are aggravated by the prolonged QRS intervals, which indicate slower ventricular conduction of AP during depolarization. QRS interval widening has been associated with low gap junction conductance and/or reduced sodium channel activity [START_REF] Posnack | Bisphenol A exposure and cardiac electrical conduction in excised rat hearts[END_REF]. In mammals, exposure to inorganic Hg affects the cardiovascular systems, triggering significant prolongations of the PR, QRS and QT intervals, which are associated with increased risk of arrhythmias and cardiac failure [START_REF] Wildemann | Combined exposure to lead, inorganic mercury and methylmercury shows deviation from additivity for cardiovascular toxicity in rats[END_REF][START_REF] Peregrina-Chávez | Full atrioventricular block secondary to acute poisoning mercury: a case report[END_REF].

Abnormalities in the generation or conduction of electrical impulses has been associated with arrhythmias. Several types of cardiac arrhythmias and anomalies (extra systoles, atrial fibrillation, negative QRS complex) appeared in the current study after Hg exposure, in both fish species in deep hypoxia. That is, mercury exposures disturbed the coordinated electrical activation of the heart, which is crucial for the maintenance of a regular fH and contractility, which might cause myocardial dysfunction.

Negative effects of Hg on ex vivo cardiac contractility have been reported in these two species of fish, in a study measuring myocardial isometric twitch force development, contraction/relaxation dynamics and cardiac pumping capacity of ventricular muscle strips [START_REF] Monteiro | Impact of waterborne and trophic mercury exposures on cardiac function of two ecologically distinct Neotropical freshwater fish Brycon amazonicus and Hoplias malabaricus[END_REF]. According to [START_REF] Vornanen | Electrical excitability of the fish heart and its autonomic regulation[END_REF], the pumping function of fish heart depends on the electrical excitability of cardiac myocytes, to maintain the excitability at a level that ensures steady cardiac contractility under various physiological and environmental stressors. Consequently, electrical excitability is crucial for the adjustment of cardiac performance to attend physiological demands, especially concerning physiological adaptations to metal contamination.

Oveall, the results highlighted the negative impacts of environmental factors, like Hg contamination vand hypoxia, on rhythm of the cardiac pacemaker, rate of impulse propagation, duration of ventricular AP, and genesis of cardiac arrhythmias. These alterations in electrical cardiac conduction could lead to pathophysiological outcomes, most noticeably an increase in hypoxia sensitivity. They could also cause sub-lethal impairments of cardiac output, swimming performance, capacity to forage and capture prey, with consequences for growth and/or reproductive rates.

Environmental stressors, like metal contamination, might elevate maintenance costs, leading to energy deficiency [START_REF] Sokolova | Interactive effects of metal pollution and temperature on metabolism in aquatic ectotherms: implications of global climate change[END_REF]. Furthermore, understanding the interactive effects of low levels of dissolved O2 and sublethal exposures to contaminants are crucial for understanding how hypoxia may affect communities of organisms [START_REF] Pollock | The effects of hypoxia on fishes: from ecological relevance to physiological effects[END_REF]. Hg-induced changes in cardiac electrophysiology could have severe implications for fishes living in hypoxic tropical waters. The present results together with the earlier data lead to the conclusion that acute waterborne and chronic trophic inorganic Hg exposures can both cause impairments in electrical stability and rhythm of the heart beat,leading to amyocardial dysfunction that is further intensified in hypoxia. Knowledge of interactions among multiple potential stressors is crucial to understanding of how populations are impacted and predicting the consequences of simultaneous exposure to environmental stressors. Moreover, cardiac electrical activity might be a useful ecotoxicological endpoint biomarkers for biological monitoring.

For

  Fig 4

Figure 1 .

 1 Figure 1. ECG parameters of matrinxã, B. amazonicus. Effects of decreasing O2 tensions

Figure 2 .

 2 Figure 2. Electrocardiograms of B. amazonicus. A and C: control (Hg-free water), B and D:

Figure 3 .

 3 Figure 3. ECG parameters of traíra, H. malabaricus. Effects of decreasing O2 tensions of

Figure 4 .

 4 Figure 4. Electrocardiograms of H. malabaricus. A and C: control (Hg-free diet), B and D:

  Fig 1

  Fig 2

  Fig 4

Table 1 .

 1 Effects of graded hypoxia on the QT intervals or ventricular action potential durations (VAPD) for B. amazonicus from groups Ct and wHg (n = 12). Values are mean ± SEM. Bold values indicate a significant difference in relation to the normoxic values, while asterisks indicate significant difference in relation to the control group (p < 0.05).

	PO2 (mmHg)	Ct	QT interval (VAPD -ms)	wHg
	140	373.9 ± 9.4		373.5 ± 14.9
	120	361.7 ± 10.4	373.8 ± 21.6
	100	358.3 ± 11.8	371.3 ± 22.7
	80	364.9 ± 15.3	384.9 ± 23.3
	60	379.9 ± 16.5	394.2 ± 25.4
	40	402.1 ± 12.3		431.8 ± 9.0
	20	422.4 ± 23.7	506.3 ± 17.3*

Table 2 .

 2 Effects of graded hypoxia on the QT intervals or ventricular action potential

	durations (VAPD) for H. malabaricus from groups Ct and tHg (n = 12). Values are mean ±
	SEM. Bold values indicate a significant difference in relation to the normoxic values (140
	mmHg), while asterisks indicate significant difference in relation to the control group (p <
	0.05).
	PO2 (mmHg)	Ct	QT interval (VAPD -ms)	tHg
	140	587.7 ± 28.0	704.1 ± 38.6 *
	120	579.3 ± 28.0	707.9 ± 34.5 *
	100	578.8 ± 28.6	690.3 ± 37.4 *
	80	570.1 ± 19.7	709.9 ± 32.8 *
	60	563.2 ± 23.5	724.4 ± 33.4 *
	40	572.4 ± 20.3	698.7 ± 29.1 *
	20	584.8 ± 24.9	706.4 ± 34.1 *

Acknowledgments

The authors are thankful to Águas Claras (Mococa-SP) and Parque do Lago (Dourado-SP) fish farms, which provided the fishes.

Atrial and ventricular depolarization and repolarization are represented on each ECG recordings as a series of waves: the P wave followed by the QRS complex and the T wave. In (D), arrowheads represent atrial fibrillation. Note altered ventricular repolarization displayed as flattened, inverted, or biphasic T wave as well an altered direction of the ventricular depolarization as indicated by negative QRS complex.

Funding Information

This study was supported by São Paulo State Research Foundation (FAPESP, Proc. #06/50772-6).

Ethics in Animal Experimentation

This study was performed with the approval of the Committee of Ethics in Animal Experimentation (CEUA -approval 04/2007) and Committee of Environmental Ethics (CEAapproval 003/2006) of the Federal University of São Carlos.

Conflict of interest

The authors declare that they have no conflict of interest.

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