Are the impacts of carbon nanotubes enhanced in Mytilus galloprovincialis submitted to air exposure ?

Intertidal species are frequently exposed to environmental changes associated with multiple stressors, which they must either avoid or tolerate by developing physiological and biochemical strategies. Some of the natural environmental changes are related with the tidal cycle which forces organisms to tolerate the differences between an aquatic and an aerial environment. Furthermore, in these environments, organisms are also subjected to pollutants from anthropogenic sources. The present study evaluated the impacts in Mytilus galloprovincialis exposed to multi-walled carbon nanotubes (0.01 mg/L MWCNTs) when continuously submersed or exposed to tides (5 h of low tide, 7 h of high tide) for 14 days. Our results demonstrated that mussels were physiologically and biochemically affected by MWCNTs, especially when exposed to tides. In fact, when only exposed to the carbon nanoparticles or only exposed to tides, the stress induced was not enough to activate mussels' antioxidant defenses which resulted in oxidative damage. However, when mussels were exposed to the combination of tides and MWCNTs increased metabolism was observed, associated with a possible higher production of reactive oxygen species (ROS), leading to a significant increase in the activities of antioxidant enzymes (superoxide dismutase, SOD and glutathione peroxide, GPx) and oxidized glutathione content (GSSG), preventing the occurrence of cellular damage, expressed as no lipid peroxidation (LPO) or protein carbonylation (PC). Therefore, organisms seemed to be able to tolerate MWCNTs and air exposure during tidal regime; however, the combination of both stressors induced higher oxidative stress. These findings indicate that the increasing presence of carbon nanoparticles in marine ecosystems can induce higher toxic impacts in intertidal organisms compared to organisms continuously submerged. Also, our results may indicate that air exposure can act as a cofounding factor on the assessment of different stressors in organisms living in coastal systems.


INTRODUCTION
Estuaries are ecologically and economically valuable ecosystems, presenting several essential ecological functions such as high biological productivity, hydrological regulation, biogeochemical cycling of metals and nutrients, as well as habitat and food source for wildlife (Caçador et al., 2007;Mitsch and Gosselink, 2015;Xiao and Li, 2004). However, these ecological functions are strongly influenced by physical and chemical disturbances from natural or anthropogenic sources, typical of transitional coastal ecosystems (Dauvin and Ruellet, 2009;Elliott and Quintino, 2007). Due to their nature, coastal systems, namely estuaries and coastal lagoons, represent one of the hardest environments to endure for the inhabiting organisms.
Among the most stressful conditions to face, species that inhabit these areas are subject to ed tides and a large variation of climatic conditions, such as temperature, salinity, as well as high desiccation risk and marked variation of oxygen availability between aquatic and aerial conditions (Davis, 1985;Freire et al., 2011;Horn et al., 1999, Underwood andKromkamp, 1999).
Furthermore, inputs of chemicals associated with industrial, domestic and agriculture activities from the surrounding areas are another disturbance that these organisms must cope with on a daily basis (Amiard-Triquet andRainbow, 2009, Elliott et al., 2014).
In intertidal areas, as a consequence of air exposure, organisms may face prolonged hypoxia and/or anoxia. Although marine bivalves are among the most hypoxia-tolerant macrofauna (Abele et al., 2009;Gray et al., 2002), the impacts of air exposure on the physiological performance of several bivalves have already been observed (Almeida and Bainy, 2006, Andrade et al, 2018, Chandurvelan et al., 2013, Altieri, 2006Letendre et al. 2008, Yin et al., 2017. It is known that some bivalves, as the mussel Mytilus galloprovincialis, close their valves when exposed to air (Dowd and Somero, 2013;Nicastro et al., 2010). As a consequence, intertidal A C C E P T E D M A N U S C R I P T bivalves may face complete anoxia while closing their shells during low tides to avoid desiccation, although others may prevent anoxia by simply opening the valves for air gaping (Rivera-Ingraham et al., 2013). Different bivalve species have also showed induction of oxidative stress related to air exposure and reoxygenation. Studies demonstrated, for example, an increase on antioxidant defenses in the mussels as a defense mechanism Perna perna and Mytilus galloprovincialis against oxidative stress during re-oxygenation (Almeida andBainy, 2006, Andrade et al., 2018).
A similar response was observed in specimens of the clam daily exposed Ruditapes philippinarum to rhythms of air exposure (Yin et al., 2016). Specimens of the mussel demonstrated M. edulis the generation of over-expression of several proteins involved especially in cytoskeleton, chaperoning, energy metabolism and transcriptional regulation after emergence (Letendre et al., 2011).
Estuaries are dynamic interface zones between water draining from inland river basis and oceans, and for this reason normally receive high concentrations of natural and anthropogenic materials (Amiard-Triquet andRainbow, 2009, Müller et al., 1995;Lopes et al., 2011). Pollutants from different anthropogenic sources are increasing in marine ecosystems which can cause adverse effects (Fu et al., 2003;Maanan, 2008). This is the case of nanoparticles (NPs) which have been increasingly used in numerous applications including medicine, chemistry and electronics (Renn and Roco, 2006) and, thereby, the increased introduction of these materials into the aquatic systems is expected to occur. Among NPs, carbon-based NPs have a diversity of applications (Solarskaciuk et al., 2014;Vlasova et al., 2016;Wu et al., 2013;Muller and Nowack, 2008;Köhler et al., 2008). Among the most important carbon-based NPs are carbon nanotubes (CNTs) (Scown et al., 2010;Eckelman et al., 2012;Sanchez et al., 2012)  and physiological alterations induced in the mussel M. galloprovincialis (Canesi et al., 2008(Canesi et al., , 2010 in the clam , R. philippinarum (De Marchi et al., 2017c, and the polychaetes in
surface to volume ratio and reactivity of CNTs make them highly dynamic in environmental systems and the resulting transformations of these NPs under different environmental conditions (e.g. tidal exposure, with associated salinity and temperature shifts will affect their fate, transport, ) and toxic properties (Velzeboer et al., ). 2013 Among bivalves the mussel species M. galloprovincialis (Lamarck, 1819) is widely distributed across the globe, inhabiting infra littoral areas (FAO, 2016;Vazzana et al., 2016) being present on rocky areas, cliffs, boulders or substrates that are relatively movable and to which it adheres (FAO, 2016;Vazzana et al., 2016). In Portugal, this species exists along the entire coast (Mitchelmore et al., 1998). This species is frequently exposed to tidal changes and, as a sedentary filter feeding organism, has the capacity to accumulate pollutants from the environment and reflect the imposed toxic impacts. Furthermore, bivalves are known to tolerate high concentrations of xenobiotics and provide a specific response to pollutants and, for these reasons, M.
galloprovincialis has been widely used as a bioindicator species (Catsiki and Florou, 2006;Faggio et al., 2016;Kristan et al., 2014;Oliveira et al., 2017;Sureda et al., 2011). These organisms, present in a wave-posure environment associated with rocky intertidal shores, appear to exhibit ex adaptive physiological, behavioral and morphological traits (Dowd et al., 2013, Sherratt andMackenzie, 2016) such as the valve closure to protect from stressful conditions (Gazeau et al., 2013;Ishii et al., 2005;Poulain et al., 2011). However, little is still known about the physiological and biochemical effects of tidal changes in these organisms living under such environmental conditions. Furthermore, in the environment mussels are subjected to tidal changes which may act as a confounding factor when assessing the impacts induced by contaminants, such as CNT exposure. In fact, when mussels are used in environmental monitoring programs and especially I P T  under laboratory conditions, their natural environment and its possible interactions with other stressors, namely pollutants, influencing their toxicity, is not considered. Within this context, the present study aimed to evaluate if physiological and biochemical alterations imposed by the presence of multi-walled CNTs (MWCNTs) were dependent on the submersion/tidal regime, to better understand the possible interactions of both conditions (contamination and exposure to air) in the physiological and biochemical performance of mussels.

Sampling and experimental conditions
Mytilus galloprovincialis specimens were collected during low tide in an intertidal area at the Mira Channel (Ria de Aveiro, a coastal lagoon, northwestern Portugal), in September 2017.
After sampling, the collected mussels were placed in aquaria for depuration and acclimation to laboratory conditions for 7 days Artificial seawater (salinity 35 ± 1), made with artificial salt (Tropic . Marin®SEA SALT from Tropic Marine Center) and deionized water, was used During this period .
the organisms were maintained at 18ºC ± 1.0 ºC and pH 8.0 ± 0. the present study to avoid the decrease of carbon content in the water column (due to its dispersion properties). It was already demonstrated that surface areas of CNTs containing carboxyl and hydroxyl groups are widely used as active sites for further functionalization which improves the solubility and biocompatibility of the material (Scheibe et al., 2010). A study conducted by Peng et al. (2009) investigated the precipitation of oxidized CNTs in water by salts.
The results showed that CNT concentration decreases slightly with aging time. CNT concentration after 30 days aging was 85% of the initial CNT concentration, which indicates that only 15% oxidized CNTs settled during 30 days. The stability of oxidized CNTs in water is probably related to the fact that the oxidation process introduces oxyg -containing groups on the CNT surface. en These groups ionize in water and the oxygen-containing groups are negatively charged. In aqueous phase, the electrostatic repulsive forces between negative surface charges of the oxygen-containing groups may lead to stability of oxidized CNTs and the oxidized CNTs can form stable dispersion in water.
During the experimental period (14 days) organisms were fed three times week with per Algamac protein plus (10 7 cells/animal). After 7 days of the beginning of the experiment, seawater was renewed -establishing seawater characteristics and MWCNT concentration. During the re experimental period, water samples were taken immediately before seawater renewal to characterize MWCNTs in the water from aquaria exposed to this NP.
An experimental period of 14 days was chosen considering previous studies in mussels (Andrade et al., 2018;Hu et al., 2015;Huang et al., 2018;Letendre et al., 2011;Verlecar et al., 2007) which observed physiological changes during this period. At the end of the experimental period (14 days), the organisms were immediately frozen at -80 ºC until analysis with the exception of two organisms per aquarium which were immediately used for respiration rate determination.
The concentration of MWCNTs used in this study (0.01 mg/L) was prepared from a stock solution of 50 mg/L concentration. For particle characterization, the average size distribution of MWCNT suspensions in seawater in each exposure condition was analyzed by dynamic light scattering (DLS), using a Delsa TM NanoC Particle Size Analyser (Beckman Coulter).
Measurements were performed on 1 mL of suspension and each analysis was repeated three times.
The hydrodynamic radius and polydispersity index (PDI) of the analysed dispersions were calculated on three replicates of each sample collected after a week of the experimental period by using the cumulant method. Undetected colloidal material at the end of each measurement was indicated as Invalid data (I.d.).

Respiration Rate
The respiration rate (RR) was measured at the end of the experimental period. Six M. galloprovincialis per specimens condition (2 individuals aquarium/replicate, 6 individuals per per treatment) were used to determine the respiration rate of organisms. Measurements were performed by simple static respirometry, filling the respirometric chambers with the same artificial seawater used during the experimental period and two organisms of the same aquarium as per chamber. Organisms were in the dark and oxygen concentrations were measured at each 15 put min for 2 hours with an oxygen meter (model 782, with an oxygen electrode model 1302, Strathkelvin Instruments, Glasgow). Organisms were afterwards dried weighed. The oxygen and consumption rate was determined by calculating the differences between the oxygen content in the water before (Tinitial = 0h) and after (Tfinal = 2h) the process. Respiration rate was expressed in g dry tissue (DW) and an hour. Two blank controls (chambers with no per organisms) were employed to correct the ambient oxygen depletion due to other factors than the respiration of organisms.

Biological responses biochemical parameters :
After the 14 days of the experimental period, shells of the frozen organisms individuals (4 per replicate, 12 individuals treatment were removed and the frozen whole soft tissue was per ) pulverized with liquid nitrogen using a mortar and pestle. The homogenized tissue of each organism was then distributed in 0.5 g aliquots.

Metabolic capacity
The ETS activity was measured based on the method of Packard (1971 and modifications ) by Coen and Janssen (1997 The GSSG content was determined following the method described in Rahman et al. (2007), using GSSG as standard (0 results were per g FW. The quantification of PC levels followed the DNPH alkaline method described by Mesquita et al., (2014) Absorbance was measured . 0.022 mM -1 cm -1 ) and the results were expressed in nmol of protein carbonyl groups formed g of FW. per

Antioxidant enzymes
The activity of SOD was determined following the method of Beauchamp and Fridovich (1971). The standard curve was generated with SOD standards (0.25-60 U/mL). Absorbance was measured at 560 nm after 20 min of incubation at room temperature. The SOD activity was g of FW where one unit (U) of enzyme activity corresponds to a reduction of per 50% of nitroblue tetrazolium (NBT).
The activity CAT was quantified based on the method of Johansson and Borg (1988). The standard curve was determined using formaldehyde standards (0-150 Absorbance was measured at 540 nm. CAT activity was also expressed in U g of FW. In this case, one unit (U) per is defined as the formation of 1 nmol formaldehyde min. per The activity of GPx was quantified following Paglia and Valentine (1967). Absorbance was 0.00622 µM -1 cm -1 ) in 10 s intervals during 5 min. Results were expressed in U g FW where one unit (U) represents the quantity of enzyme which catalyzes the per conversion of 1 µmol nicotinamide adenine dinucleotide phosphate (NADPH) min. per

Data analysis
Due to a lack of homogeneity of variance, RR, ETS, GLY, LPO, PC, GSSG, SOD, CAT and GPx were separately submitted to a non-parametric permutational analysis of variance (PERMANOVA Add-on in Primer v7) with a two-factor design: submersion condition (submersed (sub) or exposed to tide (tide)) as factor 1 and contamination condition (non-contaminated (ncont) or contaminated (MWCNTs)) as factor 2. PERMANOVA main test was performed to test the effect of submersion condition, contamination condition and the interaction between these two factors on each biomarker. PERMANOVA main tests were considered significant when p < 0.05 and followed by PERMANOVA pair-wise tests. Pair-wise tests were used to test the effect of contamination condition (ncont MWCNTs) within each submersion condition and the effect of and submersion condition (sub and tide) within each contamination condition. PERMANOVA pair-wise tests results are represented in figures with lower case letters and in the main text by p-values.

MWCNT characterization
The mean size (nm) and the polydispersity index (PDI) of simulated seawater samples collected from aquaria containing mussels (Mytilus galloprovincialis) were measured by dynamic light scattering (DLS). Results obtained by DLS analysis did not evidence the presence of dispersed materials in seawater samples collected from aquaria where organisms were subjected to tidal simulation, while samples kept submersed in water throughout the course of the experiments were found to be contaminated by micro-sized suspensions (Table 1).

Mortality
After 14 days of exposure, no mortality was observed in any tested treatment.

Respiration rate
Concerning respiration rate (RR), no significant differences were observed between contaminated and non-contaminated organisms either under submersion or tide exposure conditions ( Figure 1) Comparing submersion and tide exposure conditions, -contaminated . non and contaminated organisms submersed during the entire experiment tended to show lower RR values than organisms exposed to tides but the difference was not statistically significant ( Figure  , 1 No significant effect of the interaction between tide exposure and the presence of MWCNTs ).
on the RR was observed (p=0.5709).

Metabolic capacity
Concerning ETS activity, no significant differences were observed between -non contaminated contaminated organisms either submersion or tide exposure conditions and under ( Figure 2A) Significantly lower ETS values were observed mussels submersed for the entire . in experiment in comparison to mussels exposed to tides, both for non-contaminated and contaminated organisms (Figure 2A The interaction between tides and MWCNTS showed no ).
significant effects on the ETS activity (p=0.6504).

Energy reserves .
Concerning GLY content no significant differences were observed between contaminated , and non-contaminated organisms either under submersion or tide exposure conditions ( Figure   2B) Also, no significant differences were observed between submersion and tide exposure .
conditions, either for -contaminated contaminated organisms (Figure The interaction non or 2B).
of both stressors (tides and MWCNTs) showed no significant effect on the GLY content (p=0.8487).

Oxidative damage .
With regard to LPO levels, under submersion conditions, non-contaminated organisms presented significantly lower LPO values than contaminated organisms. When exposed to tides, significantly lower LPO levels were observed in contaminated organisms compared to noncontaminated mussels ( Figure 3A) Comparing submersion and tide exposure conditions, -. non contaminated mussels submersed during the entire experiment showed significantly lower LPO values than organisms exposed to tides. In the presence of MWCNTs, mussels submersed during the entire experiment showed significantly higher LPO values than organisms exposed to tides (Figure The interaction between tides and MWCNTs showed a significant effect on LPO 3A). levels (p=0.0234).
Concerning GSSG, no significant differences were observed between contaminated and non-contaminated organisms maintained under submersion conditions during the entire experiment. When exposed to tides significantly higher GSSG values were observed in the presence of MWCNTs ( Figure 3B). Comparing submersion and tide exposure conditions, significant differences were only observed for contaminated mussels, with higher values in mussels exposed to tides in the presence of MWCNTs ( Figure 3B Nevertheless, the interaction ).
between tides and MWCNTs showed no significant effect on GSSG content (p=0.1524).
Concerning no significant differences were observed between contaminated and non-PC, contaminated organisms always submersed or exposed to tides ( Figure 3C). Comparing submersion and tide exposure conditions, no significant differences were observed either for -non contaminated contaminated organisms (Figure No significant effects (p=0.4293) were or 3C).
observed on the PC resulting from the interaction between tides and MWCNTs.

Antioxidant enzymes
Concerning SOD activity under submersion conditions, no significant differences were , observed between non-contaminated and contaminated organisms. When exposed to tides significantly higher SOD values were observed in contaminated compared to non-contaminated mussels ( Figure 4A) Comparing submersion and tide exposure conditions, mussels in the .
absence of MWCNTs and submersed during the entire experiment showed significantly higher SOD values than organisms exposed to tides, while contaminated mussels exposed to tides showed significantly higher SOD values in comparison to contaminated organisms submersed the entire experimental period (Figure The interaction between tides and MWCNTs showed 4A). a significant effect (p=0.0003) on the SOD activity.
Regarding CAT activity, no significant differences were observed between contaminated and non-contaminated organisms always submersed or exposed to tides. Comparing submersion and tide exposure conditions, no significant differences were observed between -non contaminated and contaminated mussels ( Figure  ). No significant effects (p=0.6662) were 4B observed due to the interaction between tides and MWCNTs.
Concerning GPx activity, no significant differences were observed between contaminated and non-contaminated organisms always submersed, while when exposed to tides significantly higher GPx values were observed in contaminated mussels compared to non-contaminated organisms ( Figure 4C). Comparing submersion and tide exposure conditions, mussels submersed in the absence and presence of MWCNTs showed significantly lower GPx values than organisms exposed to tides ( Figure 4C). The interaction between tides and MWCNTs showed no significant effects (p=0.7146) on the GPx activity.

DISCUSSION
The present study evaluated the physiological and biochemical performance of M. galloprovincialis when exposed to MWCNTs both under continuous submersion and exposed to tidal regime aiming to understand if air exposure during simulated tidal regime would influence , the toxic impacts induced by the NPs. This topic is of upmost importance as very little information is available on the impacts of air exposure in mussels, and especially when organisms are exposed to contaminants.

Physiological responses Respiratory capacity
Respiration rate has been used in different intertidal organisms to assess the alterations induced by different stressors (De Marchi et al., 2017b;Gestoso et al., 2016;Freitas et al., 2017;Wang et al., 2015). Our results demonstrated that the exposure to MWCNTs did not change M.
galloprovincialis respiratory capacity when compared to non-contaminated mussels neither when , individuals were exposed to tides n when they were continuously submersed. Nevertheless, t or he increase of RR due to contaminant exposure has been demonstrated as a physiological adaptation-response (Relexans et al., 1988 In fact, this increase has been observed in different ). neopolitana H. diversicolor and exposed to 0.01 mg/L of MWCNTs did not show any changes in RR, but at higher concentration (1.00 mg/L of MWCNTs) an increase of RR in H. diversicolor was observed Therefore, the fact that in the presen study the RR did not increase with contamination . t may result from the low concentration of MWCNTs tested that was not enough to induce any changes on the respiratory capacity of mussels.
Our study also showed that RR slightly creased when the mussels were under the tidal in regime, but the absence of significant changes compared to submersed mussels is probably due to the short experimental period tested. Yin et al. (2017) showed a significant increase oxygen on consumption in the clam with the daily rhythms of air exposure (3h, 6h Ruditapes philippinarum and 9h) followed by immersion, explaining that it was caused by the need to compensate for the oxygen debt resulting from the hypoxia caused by air exposure, when the clams where reimmersed.

Biochemical responses Metabolic capacity and energy reserves
An indication of the metabolic status of organisms can be assessed by the determination of the electron transport system (ETS) activity, which allows an estimation of the energy consumption at the mitochondrial level (Coen and Janssen 1997). In the present study, mussels exposed to MWCNTs were able to maintain their metabolic capacity compared to noncontaminated organisms under both continuous submersion and tidal regime The lack of .
significant differences in the ETS activity between contaminated and non-contaminated mussels can be associated to the similar respiratory capacity of mussels independently on the presence or absence of MWCNTs These findings indicate that the concentration tested was not stressful .
enough to increase the metabolic activity of mussels to activate defense mechanisms or to inhibit metabolism, for example by valve closure to avoid contamination Similarly, De Marchi et al. .
(2017c, 2018) observed no significant differences of ETS activity in R. philippinarum exposed for 28 days to the same nanoparticles and at the same concentration (0.01 mg/L).
Nevertheless, our results further revealed that mussels tended to increase the ETS activity when exposed to tides, in comparison to submersed mussels, which may result from re-immersion periods to which the mussels are subjected Andrade et al. (2018) also demonstrated an increase .
of ETS activity in under daily air exposure conditions (during 3h or 6h) followed M. galloprovincialis by immersion. It is known that metabolic depression may occur under oxygen limitation (Guppy et al., 1994). However, little is still known about the physiological responses of mussels exposed to tidal regimes. The present findings point out that re-oxygenation after air exposure (during low tide simulation) induced high metabolic capacity in mussels, necessary to -establish their re physiological and biochemical performance after oxygen absence.
Also, the general condition of an organism can be assessed by the determination of the differences between the energy consumption and available energy reserves (Coen and Janssen 2003). The availability of energy reserves, such as GLY content, can be affected not just by chemical stressors, but also by general physiological stressors (Scott-Fordsmand and Weeks, 2000). Our results demonstrated no differences in GLY content between contaminated and noncontaminated mussels, indicating that either GLY was not the reserve used as a resource of energy to fuel the defense mechanisms of mussels when preservation of GLY content observed in the present study could thus result from the low MWCNT concentration used and/or short exposure period that did not result in the increased expenditure of this reserve.
The present results further demonstrated that exposure to tides did not alter GLY content in comparison to submersed mussels. Similarly, Ivanina et al. (2011) did not observe differences in the GLY content in the oyster Crassostrea virginica exposed to hypoxic conditions for 2 weeks. re-immersion periods. In the present study, although under tidal regime mussels showed increased metabolism, this activation was not reflected in the use of GLY content which, once again, may indicate that GLY was not used to fuel the defense mechanisms of mussels to fight against the stress caused by air exposure or this condition was not stressful enough to cause the net expenditure of energy reserves. Therefore, we may hypothesize that under tidal regimes mussels preferentially use lipids to fuel up their defense mechanisms.

Oxidative damage
With the abiotic changes in the environment, marine bivalves may be exposed to stressful A C C E P T E D M A N U S C R I P T levels increased in MWCNT-contaminated mussels submersed during the entire experiment, while when exposed to tides, contaminated mussels decreased their LPO in comparison to -non contaminated mussels These results were accompanied by a significant increase on the GSSG . content in contaminated mussels exposed to tides, evidencing that under this condition the organisms were experiencing oxidative stress. In fact, GSSG results from the oxidation of GSH, which participates in the antioxidant defense system as the most abundant cytosolic scavenger and neutralizing ROS directly, but also acts as a co-factor of antioxidant enzymes such as GPx, the activity of which, in fact, significantly increased in contaminated organisms exposed to tides.
Thus, our findings showed that increased LPO levels in contaminated mussels under submersion conditions was not associated with increased GSSG content, which shows that ROS production was not high enough to either increase GSSG production activate antioxidant enzymes activity or in C. grayanus exposed to 12-14nm diameter MWCNTs (100mg/L) for 48h. De fact that MWCNTs were not detected in water from aquaria submitted to tides could explain the lower LPO levels observed at this condition, indicating that organisms were exposed to low contamination levels.
However, higher GSSG content indicates that ROS production was extremely high under this condition inducing oxidation of GSH and thus the decrease of LPO resulted from the activation of antioxidant enzymes that also contributed to the elimination of ROS and, whereby the formation A C C E P T E D M A N U S C R I P T of L was avoided. Therefore, our results highlight that although no MWCNTs were identified in PO water from Tides+MWCNT condition, mussels were exposed to these NPs, which caused cellular damages in organisms.
The present study further demonstrated that -contaminated mussels increased their non LPO levels when exposed to tides compared to submersion conditions but an opposite response was observed for contaminated mussels with higher LPO levels in mussels submersed during the entire experiment. These results demonstrated that in the absence of MWCNTs the exposure to air leads to cellular damages, because of the lack of activation of antioxidant defenses, which, in fact, shows that submersion was the least stressful condition to mussels. Similarly, Andrade et al.
(2018) showed an increase of LPO in mussels exposed to daily cycles of 6h of air exposure.
Likewise, in clams exposed to 3h, 6h and 9h of daily air exposure, Yin et al. (2017) observed a non-significant increase of LPO levels. For mussel exposed to anoxic conditions, M. edulis,

Rivera-Ingraham et al. (2013) demonstrated an increase of ROS after reoxygenation and
induction of LPO in the mantle. These authors suggested that the shell closure strategy during emersion as a typical behavior of intertidal bivalves to avoid oxidative stress during frequent anoxia-hyperoxia conditions. Furthermore, under anoxic conditions, ATP degrades in AMP, which is further converted to hypoxanthine. Hypoxanthine and xanthine are then oxidized upon reoxygenation, generating ROS (Jones 1986), and thus explaining the increase of LPO levels in mussels exposed to tides. On the other hand, when mussels were contaminated the decrease of LPO levels under tidal regime may indicate that the limited presence of MWCNTs, only during high tide periods, caused a less stressful condition or the combination of air exposure and MWCNT contamination induced higher oxidative stress compared to organisms submersed the entire experiment in the presence of these which was surpassed by the activation of NPs antioxidant enzymes that eliminated ROS and prevented the occurrence of LPO.
A C C E P T E D M A N U S C R I P T ROS can alter cellular functions through a reversible or irreversible post-translational modification (PTM) which may inactivate critical proteins (Sultan et al., 2018). In fact, ROS may promote the oxidation of proteins, a process known as protein carbonylation (PC) which constitutes the most common type of PTM triggered by oxidative stress (Cattaruzza and Hecker, 2008;Suzuki et al., 2010 In general, the present results showed no alteration on PC levels in ). mussels, with similar PC levels in non-contaminated and contaminated mussels exposed to submersion and tide conditions Similarly, Marisa et al. (2016) showed no significant differences .
in either protein carbonyl content or LPO levels in exposed to zinc oxide R. philippinarum 2) for 7 days, putting in hypothesis that antioxidant defenses were enough to cope with the increase of oxidative damage and this way protect the cells.
Our results further demonstrated similar PC levels between submersed and tides exposure conditions both for non-contaminated and contaminated mussels. Rivera-Ingraham et al. (2013) observed that the mussel maintained its protein carbonyl content after M. edulis reoxygenation, although a burst of LPO was observed for the same condition. Thus, air exposure during low tide may not induce protein oxidation.

Antioxidant enzymes
Marine organisms can increase antioxidant defenses, including the activities of SOD and CAT enzymes, as a way to eliminate the excess of ROS produced under stressful conditions and to prevent the occurrence of LPO (Freitas et al. 2016b;Regoli and Giuliani, 2014;Velez et al., 2016). In addition to SOD and CAT, there is a third mechanism involving the antioxidant enzyme GPx, which reduces lipid hydroperoxides, by oxidizing GSH to oxide GSSG, the animals neutralize ROS directly (Regoli and Giuliani, 2014 Our results showed that under submersion ). conditions contaminated mussels d similar antioxidant enzyme activities compared to non-ha contaminated ones, which may explain increased LPO levels in mussels exposed to MWCNTs. conditions, being only significant increased at higher MWCNT concentrations (0.10 mg/L and ly 1.00 mg/L). However, under tidal regime condition, contaminated organisms showed increased antioxidant enzyme activities compared to non-contaminated mussels which may result from higher ROS production, also associated with higher GSSG content at this condition that is related to higher GPx activity, leading to lower LPO levels at this condition. Therefore, it seems that higher stressful condition was generated by the combination of both stressors (air exposure and the presence of MWCNTs) which in turn leads to the highest activation of defense mechanisms that were able to prevent LPO and PC. Nevertheless, Letendre et al. (2011) showed no significant differences in CAT and Cu/Zn SOD activities in the mussel M. edulis submitted to an artificial tidal cycle for 14 days with and without PAHs.
Our results also revealed that exposure to tides resulted, in general, in higher antioxidant enzyme activities. These results demonstrated that mussels increase antioxidant defenses as an adaptation to high levels of ROS resulting from -oxygenation typical of tidal cycles, which was re especially noticed when mussels were contaminated Andrade et al. (2018) observed an increase .
of SOD activity in non-contaminated mussels submitted to 3h and 6h of air exposure followed by submitted to an artificial tidal cycle for 14 days. Therefore increased antioxidant enzyme activity , in mussels exposed to tides and especially under the combined effect of tides and MWCNTs indicate higher stress under this condition which was not accompanied by higher cellular damage due to the effective response of these defense mechanisms.
The present study demonstrated that although , response for submersed organisms, contamination by MWCNTs may change physiological and biochemical performance of M. galloprovincialis by inducing alterations on the oxidative status of organisms, which was especially noticed when organisms were exposed to tides The present .
findings revealed that mussels seemed to be able to tolerate oxidative stress caused by the high production of ROS induced by the contaminant and by exposure to air, being able to increase their metabolism to activate their defense mechanisms and, therefore, preventing cellular damages. Nevertheless, although being able to avoid cellular damages, the physiological and biochemical alterations induced in mussels exposed to tides and MWCNTs may have negative impacts on the physiological performance of organisms, including reproductive success and growth. Therefore, longer exposure periods should be tested in the future, as cellular damage and/or higher negative physiological injuries may possibly occur.    Total Environ. 541, 1106-1114. . http://doi.org/10.1016/j.scitotenv.2015.09.149 Fu, J., Mai, B., Sheng, G., Zhang, G., Wang, X., Peng, P., Xiao, X., Ran, R., Cheng, F., Peng,X., Wang, Z., Tang, U.W., 2003 Gomes, T., Pereira, C.G., Cardoso, C., Bebianno, M.J., 2013. Differential protein expression in mussels exposed to nano and ionic Ag. Aquat.