Emna Djebbi
email: amnadjebbi@gmail.com

Delphine Bonnet

Olivier Pringault

Khawla Tlili

Mohamed Néjib

Daly Yahia

Effects of nickel oxide nanoparticles on survival, reproduction, and oxidative stress biomarkers in the marine calanoid copepod Centropages ponticus under short-term exposure

Keywords: Metal oxide nanoparticle, Copepods, Acute toxicity test, egg production, egg hatching success, antioxidant enzymes

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Nanoparticles (NPs) represent a serious environmental threat due to their toxicity and persistence [START_REF] Geissen | Emerging pollutants in the environment: A challenge for water resource management[END_REF]. According to the British Standards Institution, these compounds are defined as discrete pieces of material with all three external dimensions in the nanoscale (size <100 nm). Size, shape, chemical composition and structure of particle are main factors which determine the nanoparticle properties (optical, electrical, and magnetic properties) [START_REF] Khan | Nanoparticles: properties, applications and toxicities[END_REF]. Based on their chemical composition, NPs can be divided into various groups: e.g., metallic, polymeric metallic oxide, semiconductor, and metallic oxide nanoparticles. Among the metallic oxide group, nickel oxide nanoparticles (NiO NPs) have widespread use in many industrial and commercial products (e.g., solar cells, lithium-ion batteries, resistive random-access memory, and biosensors), due to their magnetic properties [START_REF] Tadik | Magnetic properties of NiO (nickel oxide) nanoparticles: Blocking temperature and neel temperature[END_REF]. There are also multiple medical and biotechnological applications of NiO NPs, such as remediation of heavy metal contaminated water and enhancement of biogas production from macrophytes of wastewater treatment plants [START_REF] Mahmoud | Adsorption of heavy metal ion from aqueous solution by nickel oxide nano catalyst prepared by different methods[END_REF][START_REF] Salama | Nickel oxide nanoparticles application for enhancing biogas production using certain wastewater bacteria and aquatic macrophytes biomass[END_REF]. A recent study has reported an annual production of NiO NPs of 20 tons only in the United states [START_REF] Gomes | High-throughput transcriptomics: Insights into the pathways involved in (nano) nickel toxicity in a key invertebrate test species[END_REF]) which increases progressively the levels of NiO NPs exposure as stated by [START_REF] Avila-Arias | Impacts of molybdenum, nickel, and lithium oxide nanomaterials on soil activity and microbial community structure[END_REF]. The excessive use of NiO NPs leads to their possible widespread distribution in terrestrial, estuarine, freshwater, and marine ecosystems through industrial and domestic wastewaters and aerial deposition [START_REF] Wiesner | Assessing the Risks of Manufactured Nanomaterials[END_REF]). Despite the number of studies on NiO NPs, the environmental concentrations and distribution in field environmental matrices (water, sediment, and biota) have not yet been explored.

Research investigating environmental nanoparticle concentrations in aquatic ecosystems has shown that these compounds are detectable in aquatic matrices at concentrations up to µg/L [START_REF] Zhang | Detection of engineered nanoparticles in aquatic environment: 2 state-of-art and challenges in enrichment, separation and analysis[END_REF]. Overall, the behavior of nanoparticles varies with the environmental matrix (water, sediment, soil, etc.). For NiO NPs, aggregation behavior in seawater has been observed due to their low solubility and high stability [START_REF] Gong | Acute and chronic toxicity of nickel oxide nanoparticles to Daphnia magna: The influence of algal enrichment[END_REF][START_REF] Oukarroum | Toxicity of nickel oxide nanoparticles on a freshwater green algal strain of Chlorella vulgaris[END_REF].

There are numerous concerns about the risks posed by nanoparticle pollution to the environment and human health [START_REF] Ates | Toxicity of engineered nickel oxide and cobalt oxide nanoparticles to Artemia salina in seawater[END_REF][START_REF] Oukarroum | Toxicity of nickel oxide nanoparticles on a freshwater green algal strain of Chlorella vulgaris[END_REF]. Previous laboratory studies have shown that NiO NPs have the potential to cross biological membranes and to accumulate in target tissues, causing harmful toxic effects [START_REF] Ates | Toxicity of engineered nickel oxide and cobalt oxide nanoparticles to Artemia salina in seawater[END_REF][START_REF] Oukarroum | Toxicity of nickel oxide nanoparticles on a freshwater green algal strain of Chlorella vulgaris[END_REF]). In addition, [START_REF] Ates | Toxicity of engineered nickel oxide and cobalt oxide nanoparticles to Artemia salina in seawater[END_REF] showed that NiO NP accumulation increases with exposure concentration and duration. Acute and chronic exposures to NiO NPs have been associated with mortality effects on aquatic organisms, depending on species sensitivity [START_REF] Kovrižnych | Long-term (30 days) toxicity of NiO nanoparticles for adult zebrafish Danio rerio[END_REF][START_REF] Oukarroum | Toxicity of nickel oxide nanoparticles on a freshwater green algal strain of Chlorella vulgaris[END_REF]. NiO NPs can limit cell division, as observed for the green algae Chlorella vulgaris, and can also limit reproduction in zebrafish (Danio rerio) [START_REF] Oukarroum | Toxicity of nickel oxide nanoparticles on a freshwater green algal strain of Chlorella vulgaris[END_REF]. Moreover, NiO NPs have been shown to be associated with cytotoxic and genotoxic effects even under acute exposure in algae, rats, and human cells [START_REF] Ada | Cytotoxicity and apoptotic effects of nickel oxide nanoparticles in cultured HeLa cells[END_REF][START_REF] Oukarroum | Toxicity of nickel oxide nanoparticles on a freshwater green algal strain of Chlorella vulgaris[END_REF]. The toxicity of nanoparticles is influenced by their intrinsic characteristics (size, surface area, chemical composition, crystal structure, shape, etc.) and their behavior (aggregation, dissolution) [START_REF] Sukhanova | Dependence of nanoparticle toxicity on their physical and chemical properties[END_REF].

Studies examining the toxicity mechanism of NiO NPs have found that they cause toxicity in aquatic organisms by induction of oxidative stress through generation of high levels of reactive oxygen species (i.e., hydrogen peroxide: H 2 O 2 , the superoxide anion: O 2-, and the hydroxyl radical: OH) and disruption of antioxidant enzymes such as glutathione (GSH), as observed in algal cells [START_REF] Manke | Mechanisms of nanoparticle-induced oxidative stress and toxicity[END_REF][START_REF] Oukarroum | Toxicity of nickel oxide nanoparticles on a freshwater green algal strain of Chlorella vulgaris[END_REF]. Disruption of the antioxidant system can be associated with major risks, such as neurotoxicity, lipid peroxidation, and apoptosis [START_REF] Ada | Cytotoxicity and apoptotic effects of nickel oxide nanoparticles in cultured HeLa cells[END_REF][START_REF] Ates | Toxicity of engineered nickel oxide and cobalt oxide nanoparticles to Artemia salina in seawater[END_REF].

Despite the known toxicity of NiO NPs, marine species have received little attention, as noted by [START_REF] Gong | Stability of nickel oxide nanoparticles and its influence on toxicity to marine algae Chlorella vulgaris[END_REF], in comparison with freshwater species [START_REF] Oukarroum | Toxicity of nickel oxide nanoparticles on a freshwater green algal strain of Chlorella vulgaris[END_REF]Sousa et al. 2018a, b). Thus, ecotoxicological studies of the effects of NiO NPs on marine organisms are needed. Copepods may be interesting marine biological models for such studies, considering their essential role in marine ecosystems as main secondary producers in food webs and as vital links between phytoplankton and higher trophic levels, to which they transfer carbon and energy [START_REF] Jayalakshmi | A microcosm study on the impact of acidification on feeding, survival, nauplii production rate, post-embryonic development and nutritional composition of marine copepod[END_REF]. In addition, copepods can represent a pathway for contaminant entry for higher trophic links [START_REF] Lauer | Chronic copper toxicity in the estuarine copepod Acartia tonsa at different salinities[END_REF][START_REF] Cailleaud | Uptake and elimination, and effect of estrogen-like contaminants in estuarine copepods: An experimental study[END_REF].

Moreover, copepods are known as good bio-indicators of ecosystem pollution, because of their sensitivity to several environmental disturbances [START_REF] Bianchi | Can plankton communities be considered as bio-indicators of water quality in the Lagoon of Venice?[END_REF][START_REF] Hussain | A novel index based on planktonic copepod reproductive traits as a tool for marine ecotoxicology studies[END_REF]. These organisms can develop adaptive responses to oxidative stress using a sophisticated biochemical defense mechanism [START_REF] Kim | Heavy metals induce oxidative stress and trigger oxidative stress-mediated heat shock protein (hsp) modulation in the intertidal copepod Tigriopus japonicus[END_REF]. The marine calanoid copepod Centropages ponticus is a cosmopolite species and has been used previously as an experimental model for ecotoxicological tests due to its short life cycle, its small size, and its high sensitivity to contaminants such as metals [START_REF] Ensibi | Effects of cadmium exposure on reproduction and survival of the planktonic copepod Centropages ponticus[END_REF][START_REF] Ensibi | Toxicity assessment of cadmium chloride on planktonic copepods Centropages ponticus using biochemical markers[END_REF]. In this study, we conducted acute toxicity tests to investigate the lethal and sublethal effects of NiO NPs on C. ponticus under short-term exposure. Indeed, acute tests mimic the effects of NPs under accidental and short-term release into the environment. In addition, due to their lifestyle and ability to swim, planktonic organisms, like copepods, should experience shorter time exposure than benthic organisms for example. Moreover, due to their rapid transformation behavior, NPs, such as NiO NPs, aggregate in marine waters and deposit in the sediment which reduces their bioavailability for aquatic pelagic organisms and raises the risk for benthic organisms [START_REF] Miglietta | Characterization of nanoparticles in seawater for toxicity assessment towards aquatic organisms[END_REF]. Finally, results from acute tests allow selecting a range of NiO NPs concentrations to be used in sublethal tests. Copepod reproductive traits and molecular responses (e.g., egg production rate, hatching success, nauplius survival rate, biomarkers, and genomic profiles) are reliable tools to evaluate the impacts of different types of environmental contaminants [START_REF] Zhou | De Novo Transcriptome Assembly and Differential Gene Expression Analysis of the Calanoid Copepod Acartia Tonsa Exposed to Nickel Nanoparticles[END_REF][START_REF] Hussain | A novel index based on planktonic copepod reproductive traits as a tool for marine ecotoxicology studies[END_REF]. Egg production rate (EPR) and hatching success (HS) were studied here as sub-lethal endpoints under different NiO NPs concentrations. The response of the antioxidant defense system to NiO NPs was investigated by determining the levels of some antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), and glutathione S-transferase (GST), and the level of acetylcholinesterase (AChE) as a neurotoxicity indicator of oxidative damage.

This study is the first to focus on marine planktonic copepods, with the aim of providing new knowledge to clarify the mechanisms behind NiO NPs toxicity in this species.

Materials and methods

Copepod sampling and algal cultures

Copepods were captured in the Bizerte channel, north of Tunisia (37°16'1"N; 9°52'50"E), using a WP2 plankton net (200 µm mesh size) drawn by vertical tows.

Sampling was carried out during summer and autumn 2017. Copepods were placed in cold boxes and immediately transported to the laboratory. Identification of C. ponticus was performed based on their morphological and swimming characteristics, using a Leica MZ125 stereomicroscope [START_REF] Soler | Morphological and taxonomical revision of Centropages ponticus Karavaev, 1895 (Copepoda, Calanoida)[END_REF]. Prior to exposure to nanoparticles, adult females of C. ponticus were acclimated in seawater at 23 °C and 37 psu for 24 h, and fed the marine microalga Isochrysis galbana. Culture of this algal species was performed in filtered seawater (0.2 µm) with f/2 enriched media at 20 °C and on a 12-:12-h light: dark cycle. All glassware was soaked in 10% nitric acid (NHO 3 ) for 24 h and autoclaved at 121 °C for 15 min before use.

Nickel nanopowder preparation

Nickel oxide nanoparticles (product reference 637130) were purchased from Aldrich Sigma Chemicals. A test solution was freshly prepared before the beginning of the experiment and then diluted in filtered seawater. The NiO NPs were dispersed in ultra-pure water to obtain a concentration of 100 mg/L and the suspension was then sonicated for 20 min at 4 °C (40 KHz, 100W). The particle size of the NiO NPs was ≤50 nm with purity >99.8% and the particle morphology was cubic, according to the supplier (https://www.sigmaaldrich.com/catalog/product/aldrich/637130?lang=en&region=TN). The size of these nanoparticles (before its dispersion in ultrapure water or biological test media (powder form) was confirmed (< 50 nm) by Sousa et al. (2018a) using transmission electron microscopy. Right after dispersion in deionized water, Sousa et al. (2018a) reported a main hydrodynamic size of 342 nm which exceeds 1000 nm after 24 h and displays a negative zeta potential value.

Previous physical characterizations of other manufactured NiO NPs, different from the one we used, in seawater showed that these NPs display negative zeta potential value, non-uniform morphology and a size > 100 nm revealing their tendency to aggregate in seawater [START_REF] Gong | Biotoxicity of nickel oxide nanoparticles and bio-remediation by microalgae Chlorella vulgaris[END_REF][START_REF] Ates | Toxicity of engineered nickel oxide and cobalt oxide nanoparticles to Artemia salina in seawater[END_REF][START_REF] Gong | Stability of nickel oxide nanoparticles and its influence on toxicity to marine algae Chlorella vulgaris[END_REF].

Toxicity test procedures

Acute lethal toxicity test

To determine the lethal toxicity (median lethal concentration, LC 50 ) of NiO NPs, 48-h acute toxicity tests with adult females of C. ponticus were performed according to ISO 14669:1999[START_REF]Water quality determination of acute lethal toxicity to marine copepod (Copepoda, Crustacea). International Organization for Standardisation[END_REF][START_REF] Zhou | Toxicity of nickel in the marine calanoid copepod Acartia tonsa: nickel chloride versus nanoparticles[END_REF], with some modifications. All glass beakers were washed with 10% NHO 3 and rinsed three times with deionized water. For each NiO NP concentration tested, triplicate groups of 10 adult females were placed in 150 mL glass beakers containing 100 mL of the test solution. Five NiO NP concentrations were tested: 0, 0.5, 1, 5, 10, and 50 mg/L. Photoperiod was 12-:12-h light: dark cycle, salinity was 37 psu, and temperature was fixed at 23 °C. During the experiment, the copepods were not fed. Mortality was recorded after 24 h and 48 h of exposure. A copepod was considered dead if no swimming or appendage movements were observed for 10 seconds.

Acute sub-lethal toxicity tests

Once the LC 50 (48 h) for NiO NPs had been determined, acute sub-lethal toxicity tests were performed using NiO NP concentrations below the LC 50 value. Adult females of C. ponticus were exposed to four sub-lethal concentrations: 50%, 5%, 0.5%, and 0.05% of the 48 h LC 50 to study NiO NPs effect on reproduction and oxidative stress.

Reproduction

Reproductive toxicity tests were conducted on adult females according to [START_REF] Zhou | Toxicity of nickel in the marine calanoid copepod Acartia tonsa: nickel chloride versus nanoparticles[END_REF] with few modifications, and then EPR and HS were monitored over 48 h of NiO NPs exposure. Three replicates were prepared for each concentration. Each replicate comprised 3-4 ovigerous females of C. ponticus incubated in a 150 mL glass beaker with 100 mL of filtered seawater. To prevent cannibalism, the females were separated from their eggs using a Perspex® chamber with a 200 µm mesh false bottom. The experiments were run at 37 psu and 23 °C in an incubator under a fixed 12-:12-h light:dark cycle. After 24 h, the females from each replicate were gently transferred to a new glass beaker containing fresh test solution, to measure EPR after 48 h. The number of eggs spawned was counted every 24 h under a Leica binocular microscope for each condition, and EPR was expressed as number of eggs spawned per female per day (eggs/female/day). To monitor the effect of exposure to NiO NPs on hatching success, the eggs spawned after 24 h and 48 h were then incubated for 48 h in the same solution of NiO NPs. The numbers of nauplii and unhatched eggs in each treatment were determined at the end of the incubation period.

Oxidative stress

Prior to biomarker analysis, C. ponticus adult females were exposed for 48 h to the same range of NiO NP concentrations as used in the reproduction tests. The experiments were conducted in triplicate for each treatment and control, following the protocol of [START_REF] Fossi | Preliminary results of biomarker responses in zooplankton of brackish environments[END_REF]. Each replicate received 150 individuals, which were placed in a 1 L glass beaker filled with filtered seawater. The temperature and salinity were 23 °C and 37 psu, respectively. A light regime of 12-:12-h light: dark cycle was maintained. The experiment was run for 48 h and the organisms were not fed. A sample of 75 copepods was taken every 24 h from each replicate per treatment and immediately frozen at -80 °C until measurement of biomarkers.

Biomarker analysis

Total protein

In order to determine the protein content in copepods, samples of C. ponticus were homogenized at 4 °C using an ultratorax in a buffer solution containing tris base (20 mM), EDTA (1 mM), dithiothreitol (1 mM), sucrose (500 mM), and KCl (150 mM) adjusted to pH 7.6 at a ratio of 1-4 (w/v) according to the protocol of [START_REF] Pinho | Antioxidant responses and oxidative stress after microcystin exposure in the hepatopancreas of an estuarine crab species[END_REF]. The homogenate was then centrifuged at 9000g for 30 min at 4 °C. The resulting supernatant was divided into several aliquots, which were stored at -80 °C until total protein and biomarker analysis. Total proteins were quantified by the Bradford method [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of Protein utilizing the principle of protein-dye binding[END_REF]) using 50 µL sample protein and bovine serum albumin as the standard. The assay was performed in a microplate reader using 96 well plates. Protein concentrations were expressed in mg/L after spectrophotometric quantification at 595 nm.

Superoxide dismutase (SOD) activity measurement

Superoxide dismutase activity was measured based on the method of McCord and Foridovich (1996). In brief, 1960 µL of Na 2 CO 3 /NaHCO 3 (50 mM) (pH=10.2) were combined with 10 µL of catalase bovine, 10 µL of sample, and 20 µL of epinephrine.

Total SOD activity was measured at 480 nm using a spectrophotometer, and was expressed as SOD U/mg protein.

Catalase (CAT) activity measurement

Catalase is an enzyme that catalyzes the decomposition of H 2 O 2 formed by SOD in molecular water (H 2 O) and molecular oxygen (O 2 ). Here, CAT activity was estimated according to Clairborne (1985), where the rate of disappearance of H 2 O 2 is monitored by measuring the rate of decrease in H 2 O 2 at 240 nm for 20 min. In brief, 50 µL of sample were added to 50 µL of 30% H 2 O 2 and 950 mL of 75 mM phosphate buffer at pH=7. The results were expressed as CAT U/mg protein.

Glutathione S-transferase (GST) activity measurement

Glutathionine activity was determined by measuring the conjugation of 1-chloro-2-4dinitrobenzene (CDNB) with GSH at 37 °C. The rate of GSH decrease is directly proportional to the level of GST activity in the sample. A total of 50 µL of sample was added to 50 µL of CDNB and 100 µL glutathione in 100 mM phosphate buffer (100 mM) at pH 7.4 [START_REF] Habig | Glutathione S-transferases. The first enzymatic step in mercapturic acid formation[END_REF]. The results were expressed as GST nmol/min/mg protein.

Acetylcholinesterase measurement

Measurement of AChE activity was carried out according to [START_REF] Ellman | A new and rapid colorimetric determination of acetylcholinesterase activity[END_REF], with 3 mM acetylthiocholine iodide (AcSCh) as substrate and 0.1 Mm dithiobisnitrobenzoate (DTNB) as reagent, at a controlled temperature of 20 °C.

Absorbance was recorded at 410 nm for 15 min using 20 µL of sample according to [START_REF] Fossi | Preliminary results of biomarker responses in zooplankton of brackish environments[END_REF]. AChE activity was expressed in nmol/min/mg protein.

Data analysis

The results from the lethal and sub-lethal tests are presented as means ± their standard deviation. Statistical analyses of the data were performed using SPSS (version 18).

The effects of the concentrations tested in each experiment were compared with those of controls using two-way ANOVA (time and treatment), followed by post-hoc Tukey's test. Data were log-transformed, if necessary, to meet the ANOVA assumption of normality and variance homogeneity. The level of significance was set at p<0.05. The median lethal concentration (LC 50 ) was calculated by probit analysis using SPSS.

Results

Acute lethal toxicity test and aggregation behavior

There was no mortality in the controls during the whole incubation period. The results of two-way ANOVA indicated a strong influence of both time and concentration on survival rate (p<0.05) (Table 1). They also showed a significant interaction between exposure time and NiO NP concentrations (p<0.05). The survival rate at concentrations ranging from 0.5 to 1 mg/L of NiO NPs was ≥ 90 % over the exposure period. After 24 h, the survival rate was > 90% at a concentration of 5 mg/L and <50 % at a concentration ≥10 mg/L relative to the control groups (p<0.05). After 48 h of incubation, NiO NPs at concentrations greater than 1 mg/L induced a significant (p<0.05) reduction in survival rate, which decreased from 90±1.7 % at an NiO NPs concentration of 1 mg/L to 0 % at 50 mg/L. The calculated LC 50 value for 24 h and 48 h of exposure was 13.83 ± 2.3 mg/L and 4.07 ± 0.5 mg/L, respectively (Table 2).

During the acute toxicity tests, black aggregates visible to the naked eye appeared at the bottom of the beaker at NiO NPs concentrations ranging from 2 to 50 mg/L after 48 h of incubation. Microscope observations revealed similar aggregates of NiO NPs on the exoskeleton of C. ponticus (Fig. 1a,b).

Effect on reproductive success

The effects of NiO NPs on reproductive success in C. ponticus were assessed by monitoring EPR over 48 h and then determining HS (Fig. 2). For both parameters, no significant (p>0.05) effect was observed at the lowest concentration of NiO NPs (0.002 mg/L) over 48 h.

A significant (p<0.05) influence of time exposure on EPR was observed for all NiO NPs treatments (Table 1). However, no significant (p>0.05) interaction was observed between time exposure and NiO NPs concentration. For the control groups, EPR varied between 11.3±0.6 eggs female -1 day -1 at 24 h and 8.3±1.1 eggs/female/day at 48 h. After 24 h, a significant reduction in EPR (p<0.05) was observed, to 7.5±1.5 eggs/female/day at 0.02 mg/L and 3.3±0.3 eggs/female/day at 2 mg/L of NiO NPs (Fig. 2a). After 48 h, EPR showed a continuous decrease, reaching 1.5±0.2 eggs/female/day at the highest concentration (2 mg/L).

There was no significant effect of time exposure (p>0.05) on HS for the different concentrations tested (Table 1). However, HS for eggs spawned after 24 h and 48 h decreased significantly (p<0.05) with increasing NiO NPs concentrations ≥0.02 mg/L.

For the control groups, HS varied between 85.0±2.1% for eggs spawned after 24 h and 81.3±4.0% for eggs spawned after 48 h. The lowest values of HS, observed at the highest NiO NPs concentration tested (2 mg/L), were 24±7 % and 16±2 % for eggs spawned after 24 h and 48 h, respectively (Fig. 2 b). For this endpoint measurement, no significant interaction was observed between time exposure and NiO NPs concentration (Table 1).

Effect of NiO NPs on oxidative stress

In order to assess the mechanisms behind NiO NPs toxicity at cellular level, some oxidative enzymes (CAT, SOD, GST) and the neurotoxicity biomarker AChE were measured in C. ponticus after exposure to 50%, 5%, 0.5%, and 0.05% of LC 50 (48 h) (Fig. 3). The activity of all enzymes analyzed was significantly (p<0.05) impacted by time exposure and NiO NPs concentrations (Table 1). In addition, a significant interactive effect was observed for exposure time and NiO NPs concentration (p<0.05) (Table 1).

SOD activity

The level of SOD activity in C. ponticus after NiO NP exposure showed significant stimulation (p<0.05) with increasing exposure time and NiO NP concentrations (Fig. 3a). The maximum SOD activity was 31±0.7 U/mg protein and was observed for the highest NiO NPs concentration (2 mg/L) after 48 h. For the control groups, SOD activity was 4.0±0.3 U/mg protein.

CAT activity

After 24 h of incubation, a significant (p<0.05) difference was observed between the control groups and copepods exposed to NiO NPs concentrations (Fig. 3b). The level of CAT activity was 71.3±2.5 U/mg protein at the highest concentration (2 mg/L), compared with 31.4±0.8 U/mg protein in control groups. After 48 h, CAT activity decreased significantly (p<0.05) at 0.2 mg/L (19.2±1.7 U/mg protein) and 2 mg/L (12.8±3.0 U/mg protein) compared with the control groups (32.3±2.5 U/mg protein).

GST activity

After 24 h, exposure to NiO NPs led to a significant (p<0.05) increase in GST activity in a dose-dependent manner (Fig. 3c). At 2 mg/L, GST activity reached 65.8±5.2 nmol/min/mg protein, in comparison with 20.4±1.7 nmol/min/mg protein for the control groups. A significantly (p<0.05) higher level of GST activity was also observed after 48 h relative to the values measured after 24 h of incubation.

Acetylcholinesterase activity

Exposure to NiO NPs for 24 h resulted in significant (p<0.05) stimulation of AChE activity at all concentrations, with a minimum activity level of 67.4±4.4 nmol/min/mg protein at 0.002 mg/L and a maximum of 296.4±6.4 nmol/min/mg protein at 2 mg/L (Fig. 3d). After 48 h at NiO NPs concentrations ranging from 0.002 to 0.2 mg/L, AChE activity was higher than the levels observed after 24 h. However, at the highest NiO NPs concentration tested (2 mg/L), AChE activity was significantly reduced (34.1±3.3 nmol/min/mg protein) in comparison with the control group (52.37.3±2.7 nmol/min/mg protein) (p<0.05).

Discussion

Acute toxicity test and aggregation behavior of NiO NPs

The results demonstrated the lethal acute toxicity of NiO NPs on adult copepod C. ponticus under short-term exposure (up to 48 h), with an LC 50 value of 13.83 mg/L and 4.07 mg/L for 24 and 48 h, respectively. To our knowledge, this is the first study to report on the lethal effect NiO NPs on marine planktonic copepods. Available data regarding the lethal effect of NiO NPs on marine organisms in general are scarce and the majority of previous research has focused on freshwater species (Table 3). To our knowledge, only one previous study has investigated the mortality effect of NiO NPs on the marine amphipod Leptocheirus plumulosus [START_REF] Hanna | Accumulation and toxicity of metal oxide nanoparticles in a soft-sediment estuarine amphipod[END_REF]. The results from this study indicated that NiO NPs, tested at concentrations from 500 to 2000 µg/g dry weight, provoked no mortality in the amphipod over 10 days [START_REF] Hanna | Accumulation and toxicity of metal oxide nanoparticles in a soft-sediment estuarine amphipod[END_REF].

In freshwater crustacean species including Daphnia magna, [START_REF] Gong | Acute and chronic toxicity of nickel oxide nanoparticles to Daphnia magna: The influence of algal enrichment[END_REF] observed a nine-fold higher value of LC 50 (48 h) (36.79 mg/L for neonates) than found here for C. ponticus (Table 3). However, similar values of LC 50 (24 h) as the 48 h LC 50 found in the present study for C. ponticus were reported by [START_REF] Nogueira | Assessing the ecotoxicity of metal nano-oxides with potential for wastewater treatment[END_REF] for the same life stage of D. magna exposed to NiO NPs (Table 3). Very high LC 50 values for NiO NPs have been recorded in other biological models at higher trophic levels, such as in adult zebrafish under acute (LC 50 (48 h) = 760 mg/L) and chronic exposure (LC 50 (30 days) = 45 mg/L) [START_REF] Kovrižnych | Acute toxicity of 31 different nanoparticles to zebrafish (Danio rerio) tested in adulthood and in early life stagescomparative study[END_REF][START_REF] Kovrižnych | Long-term (30 days) toxicity of NiO nanoparticles for adult zebrafish Danio rerio[END_REF]. Other studies on the toxic effects of NiO NPs have shown that exposure of the freshwater alga Chlorella vulgaris to a concentration of 13.7 mg/L, i.e., close to our LC 50 (24 h), reduces the viability of exposed cells to 50% after 72 h [START_REF] Oukarroum | Toxicity of nickel oxide nanoparticles on a freshwater green algal strain of Chlorella vulgaris[END_REF]. [START_REF] Kanold | Metallic nickel nanoparticles and their effect on the embryonic development of the sea urchin Paracentrotus lividus[END_REF] did not observe mortality effects of nickel nanoparticles (Ni NPs) on the early life stage of the sea urchin Paracentrotus lividus exposed to concentrations up to 3 mg/L of Ni NPs after 48 h, which correspond to 88 µg/L of Ni 2+ .

In this study, NiO NPs displayed aggregation in seawater during the acute lethal and sublethal tests at concentrations ranging from 2 to 50 mg/L, after 48 h. This in line with findings observed by Sousa et al. (2018b) and [START_REF] Zhou | Toxicity of nickel in the marine calanoid copepod Acartia tonsa: nickel chloride versus nanoparticles[END_REF], who observed similar behavior for NiO NPs and Ni NPs at concentrations ranging from 4 to 10 mg/L after 72 h. Like other nanoparticles (titanium oxide (TiO2) NPs, silver (Ag) NPs), NiO NPs tend to aggregate independently of the aqueous medium (freshwater, seawater, deionized water, etc.), forming larger aggregates in seawater than in freshwater due to seawater characteristics favoring aggregation [START_REF] Ates | Toxicity of engineered nickel oxide and cobalt oxide nanoparticles to Artemia salina in seawater[END_REF]. The agglomerates increased in size with time varying from few hundred nanometers to several microns both in seawater or freshwater (Wang et al. 2014;[START_REF] Ates | Toxicity of engineered nickel oxide and cobalt oxide nanoparticles to Artemia salina in seawater[END_REF]Sousa et al. 2018a). Additionally, NiO NP aggregation does not exclude the possibility of release of free dissolved Nickel ions (Ni 2+ ) in the aqueous medium test, which may also impact the physiology of the copepod [START_REF] Gong | Biotoxicity of nickel oxide nanoparticles and bio-remediation by microalgae Chlorella vulgaris[END_REF]Sousa et al. 2018b). Unfortunately, NiO NPs dissolution behavior was not examined here to get a clear idea of the amount of dissolved Ni 2+ released into seawater test medium that can act in NiO NPS toxicity. Previous studies indicated a low solubility of these NPs in an aqueous medium test which is higher in freshwater (7%) than in seawater (0.14 %) under short-term periods [START_REF] Ates | Toxicity of engineered nickel oxide and cobalt oxide nanoparticles to Artemia salina in seawater[END_REF][START_REF] Oukarroum | Toxicity of nickel oxide nanoparticles on a freshwater green algal strain of Chlorella vulgaris[END_REF][START_REF] Gong | Biotoxicity of nickel oxide nanoparticles and bio-remediation by microalgae Chlorella vulgaris[END_REF][START_REF] Gong | Stability of nickel oxide nanoparticles and its influence on toxicity to marine algae Chlorella vulgaris[END_REF]. The availability of Ni 2+ released into the seawater from NPs increased with time and NiO NPs concentrations [START_REF] Hanna | Accumulation and toxicity of metal oxide nanoparticles in a soft-sediment estuarine amphipod[END_REF][START_REF] Zhou | Toxicity of nickel in the marine calanoid copepod Acartia tonsa: nickel chloride versus nanoparticles[END_REF][START_REF] Oukarroum | Toxicity of nickel oxide nanoparticles on a freshwater green algal strain of Chlorella vulgaris[END_REF][START_REF] Gong | Biotoxicity of nickel oxide nanoparticles and bio-remediation by microalgae Chlorella vulgaris[END_REF][START_REF] Gong | Stability of nickel oxide nanoparticles and its influence on toxicity to marine algae Chlorella vulgaris[END_REF]. [START_REF] Hanna | Accumulation and toxicity of metal oxide nanoparticles in a soft-sediment estuarine amphipod[END_REF] reported a slow dissolution of NiO NPs under a long-term period, where NPs dissolved over several weeks in seawater. The same authors reported a solubility of 21 % of NiO NPs after 28 days for a concentration of 10 mg/L of NiO NPs which is higher than solubility observed in others studies under short-term test [START_REF] Gong | Biotoxicity of nickel oxide nanoparticles and bio-remediation by microalgae Chlorella vulgaris[END_REF][START_REF] Gong | Stability of nickel oxide nanoparticles and its influence on toxicity to marine algae Chlorella vulgaris[END_REF]. For 50 mg/L of NiO NPs, a concentration which causes 100 % lethality in copepods after 48 h, a previous study has reported a concentration of 110 µg/L of Ni 2+ released in seawater after 96 h [START_REF] Gong | Biotoxicity of nickel oxide nanoparticles and bio-remediation by microalgae Chlorella vulgaris[END_REF]. Copepods are therefore exposed to both particle and dissolved forms of NiO NPs and toxicity can be linked to Ni 2+ released ion from NPs (Sousa et al. 2018b), to the particles themselves [START_REF] Capasso | Nickel oxide nanoparticles induce inflammation and genotoxic effect in lung epithelial cells[END_REF] or to the combined effect of Ni 2+ and NPs aggregates [START_REF] Gong | Biotoxicity of nickel oxide nanoparticles and bio-remediation by microalgae Chlorella vulgaris[END_REF][START_REF] Gong | Stability of nickel oxide nanoparticles and its influence on toxicity to marine algae Chlorella vulgaris[END_REF]. Further work needs to be done to better explain the chemical mechanism involved in NiO NPs copepods toxicity effects and to determine the real NiO NPs concentrations.

Settling of NiO NPs, as observed on the exoskeleton of C. ponticus in this study, has also been observed for Chlorella vulgaris and the brine shrimp Artemia salina [START_REF] Gong | Biotoxicity of nickel oxide nanoparticles and bio-remediation by microalgae Chlorella vulgaris[END_REF][START_REF] Ates | Toxicity of engineered nickel oxide and cobalt oxide nanoparticles to Artemia salina in seawater[END_REF]. Biological surface coating by NPs, is considered as the main mechanism of toxicity for no-ion releasing NPs or low soluble NPs such as NiO NPs, deemed to alter the swimming behavior of aquatic organisms which could lead to negative impacts in aquatic ecosystems [START_REF] Gong | Biotoxicity of nickel oxide nanoparticles and bio-remediation by microalgae Chlorella vulgaris[END_REF][START_REF] Noss | Three-dimensional analysis of the swimming behavior of Daphnia magna exposed to nanosized titanium dioxide[END_REF][START_REF] Oukarroum | Toxicity of nickel oxide nanoparticles on a freshwater green algal strain of Chlorella vulgaris[END_REF][START_REF] Gong | Stability of nickel oxide nanoparticles and its influence on toxicity to marine algae Chlorella vulgaris[END_REF].

According to [START_REF] Sukhanova | Dependence of nanoparticle toxicity on their physical and chemical properties[END_REF], the lethal toxicity effect of NiO NPs on aquatic organisms depends on the biological model (size, life stage, weight) and on nanoparticle characteristics (size, shape, behavior). To our knowledge, the LC 50 (48 h) observed in the present study is lower than any LC 50 value reported previously in the literature, which suggests that copepods can be a good bio-indicator of the toxic effects of NiO NPs. Comparison of our LC 50 values with NiO NPs environmental concentrations is not possible due to the lack of data in aquatic environments. However, it is worth noting that mortality effect of NiO NPs on of C. ponticus was observed at concentrations that were much higher than predicted environmental concentrations, which do not exceed the µg range [START_REF] Zhang | Detection of engineered nanoparticles in aquatic environment: 2 state-of-art and challenges in enrichment, separation and analysis[END_REF]. Thus, the concentrations reported in this study may reflect the lethal effect upon accidental exposure to NiO NPs.

Effect on reproductive traits

Our results showed that NiO NPs did not impact the reproductive performance (EPR and HS) of C. ponticus at a concentration of 0.002 mg/L during 48 h of exposure.

However, EPR and HS were significantly affected at NiO NP concentrations ≥0.02 mg/L. Previous studies have also found that metallic nanoparticles (e.g., zinc oxide (ZnO) NPs and Ni NPs) are associated with impairment of copepod reproductive traits [START_REF] Zhou | Toxicity of nickel in the marine calanoid copepod Acartia tonsa: nickel chloride versus nanoparticles[END_REF][START_REF] Parlapiano | Chronic toxicity of ZnO nanoparticles: effects on survival and reproductive traits of the marine copepod Tigriopus fulvus[END_REF]. Time exposure and concentration had a strong influence on EPR (Table 1), which decreased with increasing NiO NPs concentration and time exposure (by 32% and 46%, respectively, at 0.02 mg/L and by 70% and 82%, respectively, at 2 mg/L after 24 h and 48 h). Similar effects have been observed in the crustacean species D. magna, with NiO NPs markedly reducing offspring production at concentrations ≥0.2 mg/L after 21 days [START_REF] Gong | Acute and chronic toxicity of nickel oxide nanoparticles to Daphnia magna: The influence of algal enrichment[END_REF].

Despite the difference in incubation duration and probably frequent renewal of test solution in that study, reproduction success in C. ponticus was impacted at much lower concentrations (≥0.02 mg/L) than those tested by [START_REF] Gong | Acute and chronic toxicity of nickel oxide nanoparticles to Daphnia magna: The influence of algal enrichment[END_REF]. Likewise, [START_REF] Zhou | Toxicity of nickel in the marine calanoid copepod Acartia tonsa: nickel chloride versus nanoparticles[END_REF] did not observe any effect on EPR in the marine calanoid copepod Acartia tonsa at Ni NPs concentrations up to 0.01 mg/L over 4 days.

The results in the present study showed significant concentration-related trends in HS, which decreased with increasing NiO NPs concentrations. At the highest concentration tested here (2 mg/L), HS was reduced by 70% and 79%, respectively, for eggs produced after 24 h and 48 h. [START_REF] Zhou | Toxicity of nickel in the marine calanoid copepod Acartia tonsa: nickel chloride versus nanoparticles[END_REF] also observed a negative effect of Ni NPs on HS of the copepod A. tonsa at a concentration of 17 mg/L, where only 9% of incubated eggs hatched. Similar responses to NiO NPs have been observed in zebrafish, including a delay in egg hatching and a reduction in HS at concentrations ranging from 100 to 800 mg/L [START_REF] Kovrižnych | Acute toxicity of 31 different nanoparticles to zebrafish (Danio rerio) tested in adulthood and in early life stagescomparative study[END_REF]. Our results provide evidence of the impact of NiO NPs on copepod reproductive success at much lower concentrations (0.02 mg/L) than those tested in previous studies on marine organisms. To our knowledge, only one study has reported similar negative effects, on reproduction of the freshwater crustacean D. magna, at similar NiO NPs concentrations (0.045-0.14 mg/L) to those tested in the present study [START_REF] Nogueira | Assessing the ecotoxicity of metal nano-oxides with potential for wastewater treatment[END_REF]. Regarding NiO NPs solubility at the sublethal concentrations tested here (0.002-2 mg/l) and according to our knowledge, there are no data in the literature on NiO NPs solubility in seawater at the same concentrations range. The study of Sousa et al. (2018b) reported a concentration of Ni 2+ of 250 µg/L released in deionized water after 72 h from a concentration of 1.6 mg/L. Based on this finding and considering the low solubility of NiO NPs in seawater in comparison to freshwater, we can suggest that the concentration of Ni 2+ released in seawater at concentration of 2 mg/L, under our experimental conditions, could be less or close to 250 µg/L. This concentration is well higher than the highest levels of nickel which are not expected to pose a significant risk in species in saltwater under acute (74 µg/L) and chronic (8.3 µg/L) exposure as fixed by US EPA Aquatic Life Criteria Water Quality Standards (EPA 2009).

Interestingly, the NiO NPs concentrations causing toxic effects on reproduction physiology in C. ponticus and D. magna are close to those measured in contaminated ecosystems, which are suspected to occur at µg range, suggesting that environmentally realistic concentrations of NiO NPs are likely to impact reproduction of both species. Reproduction is a crucial biological function in organisms to maintain existence of species. Impairment of reproduction in copepods by NiO NPs, as observed in this study, can result in alteration of marine community structure and in modification of the food trophic web.

Oxidative stress

In order to understand the mechanisms of NiO NPs toxicity at cellular level, changes in the activity of antioxidant enzymes and a neurotoxicity biomarker in C. ponticus under short-term NiO NPs exposure were investigated. The results indicated a significant modulation of the first-defense antioxidant enzymes SOD and CAT in C. ponticus in response to sublethal exposure to NiO NPs. The stimulation of antioxidant enzymes was possibly a defense mechanism for coping with the oxidative stress generated by NiO NPs. This is in line with previous studies showing ability of nanoparticles to generate oxidative stress in aquatic organisms under short-term exposure (e.g. the brine shrimp Artemia salina, the copepod Calanus finmarchirus, the fish Nile tilapia, etc.) (Abdel-khaled et al. 2015;[START_REF] Ates | Toxicity of engineered nickel oxide and cobalt oxide nanoparticles to Artemia salina in seawater[END_REF][START_REF] Farkas | Combined effects of exposure to engineered silver nanoparticles and the water-soluble fraction of crude oil in the marine copepod Calanus finmarchicus[END_REF].

Our results showed that antioxidant enzyme levels in C. ponticus were significantly influenced by time exposure and NiO NPs concentration. Expression of SOD and CAT enzymes exhibited similar patterns, with an increasing trend after 24 h that was probably linked to production of reactive oxygen species (ROS), namely O 2 and H 2 O 2 , in C. ponticus as a consequence of NiO NPs exposure, as previously observed in various freshwater microalgae and plants (e.g., Chlorella vulgaris, Pseudokirchneriella subcapitata, Lemna gibba) [START_REF] Oukarroum | Toxic effects of nickel oxide bulk and nanoparticles on the aquatic plant Lemna gibba L[END_REF][START_REF] Oukarroum | Toxicity of nickel oxide nanoparticles on a freshwater green algal strain of Chlorella vulgaris[END_REF]Sousa et al. 2018b;[START_REF] Gong | Stability of nickel oxide nanoparticles and its influence on toxicity to marine algae Chlorella vulgaris[END_REF]. After 48 h, SOD activity showed a similar trend as at 24 h, while there was a significant reduction in CAT enzymatic activity at the two highest concentrations of NiO NPs (0.2 and 2 mg/L). This decrease might be linked to high production of H 2 O 2 by SOD and limited capacity of CAT to degrade it [START_REF] Ighodaro | First line defence antioxidants-superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX): Their fundamental role in the entire antioxidant defence grid[END_REF]. Such antioxidant stress response was also reported in the marine copepods Tigriopus japonicus exposed to sunscreens containing Zinc oxide nanoparticle (ZnO NPs); with high ROS levels observed after 96 h at environmentally realistic concentrations of ZnO NPs [START_REF] Wong | Sunscreens containing zinc oxide nanoparticles can trigger oxidative stress and toxicity to the marine copepod Tigriopus japonicus[END_REF].

In combination with the antioxidant response, exposure to NiO NPs also induced activation of the multifunctional GST enzyme over the whole exposure period (48 h).

In line with our results, [START_REF] Farkas | Combined effects of exposure to engineered silver nanoparticles and the water-soluble fraction of crude oil in the marine copepod Calanus finmarchicus[END_REF] reported that exposure to silver nanoparticles (Ag NPs) triggered an increase in gene expressions of antioxidant enzymes GST in the copepod Calanus finmarchirus after 96 h. GST enzyme plays a key role in biotransformation of endogenous and exogenous toxic compounds present in the cell and is also recognized as an indicator of lipid damage [START_REF] Prione | GST activity and membrane lipid saturation prevents mesotrione-induced cellular damage in Pantoea ananatis[END_REF][START_REF] Dasari | Glutathione s-transferases detoxify endogenous and exogenous toxic agents-mini review[END_REF]. NiO NPs has previously been reported as inductor of lipids peroxidation at concentrations of 1 to 50 mg/L in Artemia salina and Gracilaria lemaneiformis [START_REF] Han | Bioaccumulation and toxicity of NiO nanoparticles in Gracilaria lemaneiformis[END_REF][START_REF] Ates | Toxicity of engineered nickel oxide and cobalt oxide nanoparticles to Artemia salina in seawater[END_REF]. Results of the present study are in accordance with the results observed for the copepod Eucyclop sp. exposed to titanium dioxide nanoparticle (TiO 2 ) combined to lead (Pb), where Glutathione peroxidase (GPx), Glutathione reductase (GR), and CAT significantly increased after contaminant accumulation confirming that the exposed copepods had suffered from oxidative stress triggered by TiO 2 and Pb [START_REF] Matouke | Bioaccumulation and physiological effects of copepods sp. (Eucyclop sp.) fed Chlorella ellipsoides exposed to titanium dioxide (TiO 2 ) nanoparticles and lead (Pb 2+)[END_REF].

Neurotoxicity is one manifestation of oxidative stress damage induced by chemicals.

The enzyme AChE hydrolyzes the neurotransmitter acetylcholine (Ach) to acetate and choline, in order to prevent constant stimulation of the synapse. The present study showed a stimulating effect on AChE activity of NiO NPs (0.002-2 mg/L) after 24 h, but we observed a significant reduction in AChE activity at 2 mg/L of NiO NPs after 48 h. Under metals exposure, AChE exhibited the same pattern in living organisms, depending on time exposure; AChE increased during the earliest hours of metal exposure and then decreased with time [START_REF] Bainy | In vivo effects of metals on the acetylcholinesterase activity of the Perna perna mussel's digestive gland[END_REF][START_REF] Emadeldeen | Biochemical response of the cyclopoida copepod Apocyclops borneoensis exposed to nickel[END_REF][START_REF] Ensibi | Toxicity assessment of cadmium chloride on planktonic copepods Centropages ponticus using biochemical markers[END_REF].

The high level of AChE activity in C. ponticus observed in this study may be related to interactions between NiO NPs and acetylcholine receptors causing accumulation of free unbound acetylcholine at cholinergic receptor sites. Some authors considered the excess production of AChE activity as a compensatory mechanism related to an initial reduction in this enzyme by pollutants [START_REF] Bainy | In vivo effects of metals on the acetylcholinesterase activity of the Perna perna mussel's digestive gland[END_REF][START_REF] Ferreira | L-Tyrosine administration increases acetylcholinesterase activity in rats[END_REF].

Inhibition of AChE activity is commonly used as an indicator of neurotoxicity in living organisms such as copepods and cladoceran species under exposure to metal and pesticide [START_REF] Forget | Acetylcholinesterase activity in copepods (Tigriopus brevicornis) from the Vilaine River estuary, France, as a biomarker of neurotoxic contaminants[END_REF][START_REF] Cailleaud | Effects of salinity and temperature on the expression of enzymatic biomarkers in Eurytemora affinis (Calanoida, Copepoda)[END_REF][START_REF] Ensibi | Toxicity assessment of cadmium chloride on planktonic copepods Centropages ponticus using biochemical markers[END_REF].

Inactivation of AChE activity at a concentration of 2 mg/L suggests that NiO NPs can affect the brain of aquatic species and cause neurotoxicity through disturbance of cholinergic neurotransmission. Similar inhibitory effects of metal nanoparticles (silicon dioxide (SiO 2 ), TiO 2 , aluminum oxide (Al 2 O 3 ), and aluminum (Al) NPs) on AChE have been observed previously and are reported to depend on nanoparticle concentration [START_REF] Wang | Adsorption and inhibition of acetylcholinesterase by different nanoparticles[END_REF]. This reduction can be explained by the excess production of AChE in C. ponticus ceased at a NiO NPs concentration of 2 mg/L. Previous research has suggested, not confirmed, that modulation of AChE enzyme in copepods can result in a disorder in swimming behavior, tetany, and paralysis [START_REF] Forget | Acetylcholinesterase activity in copepods (Tigriopus brevicornis) from the Vilaine River estuary, France, as a biomarker of neurotoxic contaminants[END_REF][START_REF] Ferreira | L-Tyrosine administration increases acetylcholinesterase activity in rats[END_REF]. Swimming behavior response of aquatic organisms, such as fish and cladocerans (Daphnia sp.), under contaminant exposure, has been reported to be strongly related to AChE activity. Further, it increases as the levels of AChE enzyme increase while the opposite occurs when AChE enzyme levels decrease, impacting, therefore, the survival of living organisms [START_REF] Bonansea | Behavioral swimming effects and acetylcholinesterase activity changes in Jenynsia multidentata exposed to chlorpyrifos and cypermethrin individually and in mixtures[END_REF][START_REF] Ren | The role of ache in swimming behavior of Daphnia magna: Correlation analysis of both parameters affected by deltamethrin and methomyl exposure[END_REF]. Therefore, alteration of swimming movement in copepods may have severe impacts in the copepods community which affect other communities of the ecosystem because it is associated with numerous vital functions like perception and acquisition of food, mating and reproduction, predator-prey interaction, respiration rate, and social behavior as stated by Mazzocchi and Paffenhofer (1999).

Changes in these biomarkers reflected the ability of NiO NPs to trigger oxidative stress and highlighted the significant contribution of time exposure and NiO NPs concentration in the response of C. ponticus to oxidative stress. Our results provide evidence that oxidative stress is one of the main mechanisms of nanoparticle toxicity in copepods under short-term exposure, as previously reported for other species (Sousa et al. 2018b;[START_REF] Gong | Stability of nickel oxide nanoparticles and its influence on toxicity to marine algae Chlorella vulgaris[END_REF].

Conclusions

This is the first study to examine the potential toxic effects of NiO NPs on a marine planktonic calanoid copepod. A LC 50 (48 h) of around 4 mg/L was found for adult No mortality at ≥2000 µg/g [START_REF] Hanna | Accumulation and toxicity of metal oxide nanoparticles in a soft-sediment estuarine amphipod[END_REF]

Fig. 1

 1 Fig. 1 Aggregates of nickel oxide nanoparticles (NiO NPs) visible by microscopy after 48 h in (a) on urosome and furca and (b) on the antennules of the marine copepod Centropages ponticus exposed to 2 mg/L.

Fig. 2

 2 Fig. 2 (a) Effect of nickel oxide nanoparticles (NiO NPs) on egg production rate (eggs/female/day) of Centropages ponticus females after 24 h and 48 h and (b) egg hatching success rate (%) of eggs produced by C. ponticus after 24 h and 48 h. Values shown are mean ± standard deviation. Letters on bars indicate significant difference between groups (Tukey's post-hoc p <0.05).

Fig. 3

 3 Fig. 3 Effects of nickel oxide nanoparticles (NiO NPs) on activity of the enzymes (a) superoxide dismutase (SOD), (b) catalase (CAT), (c) glutathione S-transferase (GST), and (d) acetylcholinesterase (AChE) in adult Centropages ponticus after 24 h and 48 h. Values shown are mean ± standard deviation. Letters on bars indicate significant difference between groups (Tukey's post hoc test, p <0.05).

Fig. 1

 1 Fig. 1 Aggregates of nickel oxide nanoparticles (NiO NPs) visible by microscopy after 48 h (a) on the urosome and furca and (b) on the antennules of the marine copepod Centropages ponticus exposed to 2 mg/L.

Fig. 2

 2 Fig. 2 (a) Effect of nickel oxide nanoparticles (NiO NPs) on egg production rate (eggs/female/day) of Centropages ponticus females after 24 h and 48 h and (b) egg hatching success rate (%) of eggs produced by C. ponticus after 24 h and 48 h. Values shown are mean ± standard deviation. Different letters on bars indicate significant difference between exposed C. ponticus females and controls (Tukey's post-hoc p <0.05).

Fig. 3

 3 Fig. 3 Effects of nickel oxide nanoparticles (NiO NPs) on activity of the enzymes (a) superoxide dismutase (SOD), (b) catalase (CAT), (c) glutathione S-transferase (GST), and (d) acetylcholinesterase (AChE) in adult Centropages ponticus after 24 h and 48 h. Values shown are mean ± standard deviation. Different letters on bars indicate significant difference between groups (Tukey's post hoc test, p <0.05).

Table 2

 2 Lethal concentrations (LC 10 , LC 50 , LC 70 , LC 90 , mg/L) in Centropages ponticus adult females exposed to nickel oxide nanoparticles (NiO NPs) for 24 h and 48 h, with associated 95% confidence intervals (CI).

	Exposure duration	LC 10 (95% CI)	LC 50 (95%)	LC 70 (95% CI)	LC 90 (95% CI)
	24 h	4.29 (2.64-6.96)	13.83 (8.52-22.44)	22.33 (13.762-36.2)	44.6 (27.59-72.33)
	48 h	1.13 (0.69-1.84)	4.07 (2.49-6.63)	6.86 (4.21-11.29)	41.45 (25.43-67.59)

Table 3

 3 Median lethal concentration (LC 50 ) of nickel oxide nanoparticles (NiO NPs) in the marine copepod Centropages ponticus and in some other aquatic species.

	Species	Freshwater /	Endpoint Test	LC 50	References
		marine
		species
	Chlorella vulgaris		96 h EC50 (NiO NPs 30	13.7 mg/L	Oukarroum et al.
	(microalga)		nm)		(2017)
	Danio rerio		48 h LC 50 (NiO NPs <50	760 mg/L	Kovrižnych et al.
	(zebrafish)		nm)		(2013)
	adults			420 mg/L
			96 h LC 50 (NiO NPs <50
			nm)
	Danio rerio		30 day LC 50 (NiO NPs	45 mg/L	Kovrižnych et al.
	adults		<50 nm)		(2014)
		Freshwater
	Danio rerio	species	96 h LC 50 (NiO NPs <50	1300 mg/L Kovrižnych et al.
	eggs		nm)		(2013)
	Daphnia magna		Immobilization (NiO		Gong et al. (2016)
	(cladoceran)		NPs <50 nm)
	neonates (<24 h)		48 h LC 50	36.79 mg/L
			Immobilization		Nogueira et al. (2015)
			24 h LC 50 (NiO NPs 100	14.6 mg/L
			nm)	9.74 mg/L
			48 h LC 50 (NiO 100 nm)	13.98 mg/L
			24 h LC 50 (NiO NPs 10-	9.76 mg/L
			20 nm)
			48 h LC 50 (NiO NPs 10-
			20 nm)
	Centropages		24 h LC 50 (NiO NPs <50	13.85 mg/L	Present study
	ponticus		nm)	4.07 mg/L
	(copepod)		48 h LC50
	adults	Marine
	Leptocheirus	species	10 day LC 50 (NiO NPs13
	plumulosus		nm)
	(amphipod)

Acknowledgements

The authors would like to thank the members of the Laboratory for Environmental Biomonitoring (Faculty of Sciences of Bizerte; Tunisia), especially Professor Mohamed Dalleli and Ms Wiem Saidani, for their help in biomarker analysis. We are also grateful to the Institute of Fishing and Aquaculture (ISPA) (Bizerte, Tunisia), especially Professor Mohammed Chalghaf, for algae supply. Special thanks to Professor Abdelhak Othmani for providing nanoparticles. We would like to thank Mary McAfee for the English correction and improvement. This research is part of the PhD work of Ms Emna Djebbi, which is co-funded by the University of Carthage (Bourse d'Alternance) and IRD-Tunisia (Institut de Recherche pour le Développement).

List of table legends:

Table 1 Synthesis of results of two-way factorial analysis of variance (ANOVA) showing the effects of concentration of nickel oxide nanoparticles (NiO NPs) and time exposure on the different parameters studied in Centropages ponticus: EPR = egg production rate, HS = hatching success, SOD = superoxide dismutase activity, CAT = catalase activity, GST = glutathione S-transferase activity, AChE = acetylcholinesterase.

Table 2 Lethal concentrations (LC 10 , LC 50 , LC 70 , LC 90 , mg/L) in Centropages ponticus adult females exposed to nickel oxide nanoparticles (NiO NPs) for 24 h and 48 h, with associated 95% confidence intervals (CI).

Table 3 Median lethal concentration (LC 50 ) of nickel oxide nanoparticles (NiO NPs) in the marine copepod Centropages ponticus and in some other aquatic species.


Anais Barasinski
email: anais.barasinski@univ-pau.fr

Chady Ghnatios
email: chadyghnatioscghnatios@ndu.edu.lb

Erik Abenius
email: erik.abenius@esi-group.com

Stephane Bechtel
email: stephane.bechtel@airbus.comfranciscochinestafrancisco.chinesta@ensam.eu

Francisco Chinesta

Electromagnetic field propagation in a composite laminate and induced thermal field Application to microwave composites processing

Keywords: Composite, Thermoplastic, Electromagnetic heating, Microwaves, Coupling Anais Barasinski

Microwave (MW) technology relies on volumetric heating, where thermal energy is induced from an electromagnetic field. Nowadays, the main drawback of this technology is that the complex physics involved in the conversion of electromagnetic energy into thermal energy is not entirely understood and controlled. The main objective of this work is to model, simulate and validate the interactions of microwaves with a composite laminate consisting of a stack of unidirectional layers composed of a resin matrix and carbon fibers, to predict its heating. Once validated, this simulation tool will serve to predict, control and optimize composites forming processes.

Introduction

Composites are major protagonists in many industries and activity sectors, among them transport industries (aeronautic and automotive). This is mainly due to the combination ESI Chair @ Arts et Métiers Institute of Technology, PIMM Laboratory, 151 Boulevard de l'Hôpital, 75013 Paris, France of appealing properties and low weight, despite their long curing cycle time considered as a disadvantage. The production using conventional processing methods for polymer composite parts usually involve the application of heat to the material by convection or conductive heating, both implying surface heat transfer.

Microwave -MW-technology relies on volumetric heating, which means thermal energy is transferred through electromagnetic fields to materials that can absorb it at specific frequencies. Volumetric heating enables better process temperature control [START_REF] Mishra | Microwave-material interaction phenomena: Heating mechanisms, challenges and opportunities in material processing[END_REF] and less overall energy use, which can results in shorter processing cycles [START_REF] Mgbemena | Accelerated microwave curing of fibre-reinforced thermoset polymer composites for structural applications: a review of scientific challenges[END_REF]. Furthermore, comparable mechanical properties can be shown between parts made with the MW technology and parts made with a traditional curing system (autoclave in the case of [START_REF] Maenz | Mechanical properties of microwave cured glass fibre epoxy composites prepared by resin transfer moulding[END_REF][START_REF] Li | A new process control method for microwave curing of carbon fibre reinforced composites in aerospace applications[END_REF]). These virtues of the MW technology attracted industrial production companies as well as researchers in developing the associated heating technology and adopting it for the production of thermoset as well as thermoplastic composite structures.

The main drawback of this technology is that the complex physics involved in the conversion of the electromagnetic energy to thermal energy (heating) is not entirely understood and controlled [START_REF] Zhou | A multi-pattern compensation method to ensure even temperature in composite materials during microwave curing process[END_REF].

The present work aims at describing the interactions of microwaves with a composite laminate composed of a sequence of unidirectional prepreg composite layers (in that case carbon fibers impregnated of a polymeric matrix), by simulating the way electromagnetic energy propagates within the material and is converted in thermal energy.

Our previous work [START_REF] Tertrais | On the Proper Generalized Decomposition applied to microwave processes involving multilayered components[END_REF] addressed the calculation of the propagation of an electromagnetic field in a composite laminate composed of plies whose characteristic in-plane dimension is orders of magnitude higher than the one related to the thickness (typical aspect ratio are of tens of thousands). The present work represents a step forward by numerically coupling the electromagnetic and thermal fields. Therefore, a set up is defined to heat up a laminate plate in a microwave oven and temperature measurements are performed in order to validate experimentally the simulation results.

Materials

The study is carried out on a composite laminate plate exposed to an electromagnetic field induced in a microwave oven cavity, concretely a Hephaistos 100/100. The dimensions of the cavity is 1050 × 1470 × 1800 mm, equipped with 12 magnetrons of 1.2 kw each.

The plate is composed of unidirectional -UD-prepregs involving 64% (volume fraction) of carbon fibers preimpregnated by a thermoplastic resin. The thickness of one ply is approximately 180 micrometers, and the laminate consists of 25 plies with the following stacking sequence: 11 plies at 0 • and 14 plies at 0 • /90 • . The stacking was chosen asymmetric to evaluate its impact on the electromagnetic wave penetration.

The whole is surrounded by a thermalimide layer of 60 micrometers, and put into a ceramic mould, coated by an enameled ferrite layer (100 micrometers thick). The inplane dimension of the prepreg plate is 300 × 300 mm and the dimension of the mould is 600 × 600 × 100 mm. Several optic fibers are positioned in the system as depicted in Fig. 1, to record the temperature evolution inside the laminate plate plate as well as on its top surface during the thermal cycle.

Electromagnetic calculation and heat transfer modeling

The modeling and simulation consists of three calculations:

1. Macroscopic electromagnetic simulation in the volume of the oven cavity, referred as macro-EM-simulation, 2. Microscopic electromagnetic simulation affecting the composite laminate and its surrounding mould, referred as micro-EM-simulation, 3. Thermal calculation associated with the micro-EMsimulation.

Electromagnetic modeling

The macro-EM-simulation is first performed by using CEMOne ® software by ESI Group. The model related to the visualization-) is calculated in the single element disposed along the composite laminate thickness by using the homogenized electromagnetic properties reported in Table 1 the experimental set-up is depicted in Fig. 1(right) where it can be noticed that most of elements are appropriately modelled and included in the numerical model, among them the magnetron waveguides and the table where the mould and part are positioned. However the software resolution is unable to represent the plies involved in the composite laminate, as well as the ferrite layer, ... The characteristic thicknesses are much smaller than the other in-plane characteristic dimensions avoiding the use of an adequate mesh to resolve the rich fields existing at these scales. Thus the numerical model is unable to resolve the ferrite layer and considers a single element along the laminate thickness. Thus, the volumes occupied by the mould and the composite part are considered as homogenous materials in the macroscopic simulation, with the homogenized material parameters defined in Table 1, that almost correspond to a mixture rule, and whose temperature dependences were ignored because their effects on the EM calculation have an incidence of second order. The macro-EM-simulation is carried out by using a FTDT formulation, by considering Fig. 3 Volume of interest where the mico-EM-simulation will be performed by considering the electromagnetic field resulting from the macro-EM-simulation on its surface more than 34 million of elements with characteristic length of about 1 mm.

The macro-EM-simulation output is the electromagnetic field ampitude everywhere in the oven as depicted in Fig. 2, and more importantly the electromagnetic field amplitude on the surface of the volume of interest depicted in Fig. 3 that will be used in the micro-EM-simulation (that considers a space-frequency formulation [START_REF] Tertrais | On the Proper Generalized Decomposition applied to microwave processes involving multilayered components[END_REF]) and then in the thermal calculation for evaluating the composite heating. It is important to note that the volume of interest, V, encloses the composite laminate and its surface, ∂V, is comprised within the ceramic mould where the composite laminate is placed. The thickness of V is 6 cm whereas the one of the mould enclosing the composite laminate is 10 cm. The composite laminate is centered into V.

As just indicated, the micro-EM-simulation applies in the volume of interest V. The 25 plies laminate previously described is simulated by using the electromagnetic properties reported in Table 2. The composite laminate is surrounded by a ceramic mould coated by a ferrite layer, whose properties are also reported in Table 2. To take into account the extremely degenerated domain (characteristic in-plane length to thickness ratio) the inplane-out-of-plane separated representation at the heart of the so-called Proper Generalized Decomposition -PGDis applied as deeply reported in [START_REF] Tertrais | On the Proper Generalized Decomposition applied to microwave processes involving multilayered components[END_REF]. Thus, the in-plane solutions are achieved by using a mesh consisting of 3960 nodes while 8000 nodes are considered for describing the through-the-thickness solution richness. Such a fine mesh along the thickness direction is compulsory due to the electric field discontinuity at the plies interfaces as well as the boundary layer induced by the attenuation of the electromagnetic wave when penetrating in a conductive material, as it is the case when the electromagnetic wave penetrates the carbon fibers.

The use of a separated representation allows the computation of the fully 3D solution while solving 2D and 1D problems with few thousands degrees of freedom each, with a combined equivalent accuracy of an hypothetical 3D calculation involving tens of millions degrees of freedom.

The micro-EM-simulation solution, as described before, uses as boundary conditions the electromagnetic field coming from the macro-EM-simulation solution previously described.

Thermal modeling

The computed electric and magnetic fields allow the calculation of the induced thermal sources in the domain occupied by the composite material according to

S = 1 2 (E • H * ), ( 1
)
acting as source term in the heat equation

ρC p ∂T ∂t -∇ • (K∇T ) = S, ( 2
)
where T is the temperature, ρ the density, C p the heat capacity, K the anisotropic conductivity tensor (refer to Table 3 for the values considered in the numerical applications), E the electric field intensity and H * the magnetic field intensity (the star superscript refers to the complex conjugation) and refers to the complex real part.

In the proposed thermal model the crystallization effect is ignored, as well as its impact on the material thermal properties.

Standard convection boundary conditions are enforced on the mould surfaces ∂V, according to

-∇T • n = h(x) (T -T amb ) , x ∈ ∂V, ( 3
)
with n the unit outwards vector defined on ∂V and T amb the ambient temperature (the one of the air occupying the oven).

The values of the convection coefficients (h in Eq. ( 3)) are given in Table 4.

The numerical solution of the thermal problem is computed by using again the Proper Generalized Decomposition -PGD-method [START_REF] Chinesta | The Proper Generalized Decomposition for advanced numerical simulations[END_REF] using again the in-plane-out-of-plane separated representation that reads

T (x, y, z) ≈ N i X i (x, y) • Z i (z). ( 4 )
This separated representation proved its efficiency for computing thermal and structural problems in such a degenerated geometries [START_REF] Bognet | Advanced simulation of models defined in plate geometries: 3D solutions with 2D computational complexity[END_REF][START_REF] Bognet | Separated representations of 3D elastic solutions in shell geometries[END_REF][START_REF] Chinesta | First steps towards an advanced simulation of composites manufacturing by Automated Tape Placement[END_REF], where it allows the use of a very fine mesh in the thickness direction while ensuring reasonable computing times.

To facilitate the electromagnetic/thermal coupling, the thermal problem is solved using the same meshes than the ones considered for solving the in-plane and throughthe-thickness electromagnetic problems of the micro-EMsimulation.

The four hours process is discretized with a time step of one second and takes few hours calculation by employing a standard laptop.

Results

The solution of the micro-EM-smulation is shown in Fig. 4 where the electromagnetic field distribution is depicted. The corresponding thermal field is represented in Fig. 5 for two different time instants t = 1000 s and t = 2500 s. We can notice the hot spots in the middle plane (composite material) Figure 6 represents the minimal and maximal temperature evolution in different case. The left graph shows the time evolution of the hottest and coldest points in the composite domain (top surface and middle plane). It can be noticed that the temperature distribution is almost the same across the composite laminate thickness. The temperature difference between the hottest and coldest points is extremely high, with more than one hundred degrees difference for the highest temperatures. It seems that the inplane thermal conduction does not suffice for ensuring a temperature homogenization.

Comparison with experiments

For validating the simulation procedures just described Fig. 7(right) compares the temperature recorded by the different optic fibers and the simulated temperatures for the applied power (left). The agreement seems reasonable, with a quite good representation of the three process stages: the heating, the plateau and the cooling. The accuracy seems reasonable when compared with the very large difference of temperatures between hottest and coldest points just discussed. The noticed deviations could also be due to underestimation of the thermal diffusion as well as to the perturbations induced by the presence of the thermocouples their-self.

A more detailed analysis constitutes a work in progress, that also considers a fully micro-macro electromagnetic coupling to better estimate the induced thermal sources.

Conclusion

In this work we proposed a simulation tool for coupling the electromagnetic and thermal fields in a composite laminate when subjected to microwave-based processing.

A macroscopic electromagnetic simulation performed by the commercial software CEMOne ® (by ESI Group) at the oven level allowed computing the electromagnetic fields on the surface of the domain of interest occupied by the laminated surrounded by the ceramic mould. This electromagnetic field is then used as boundary condition by the so-called micro-EM-simulation that by using the PGD-based in-plane-out-of-plane separated representation calculates with an extremely high resolution all the electromagnetic fields in the domain of interest (mould and laminate). Finally, the electromagnetic induced thermal source is applied as source term in the PGD thermal solver enabling a high-resolution calculation of the temperature field in the composite laminate.

The solution was validated experimentally where a reasonable agreement between predicted and recorded temperatures was noticed.

Fig. 1 (Fig. 2

 12 Fig.1(Left) Optic fiber sensors inserted in the laminate as well as placed on its top surface during its microwave heating: FO1 is placed inside the laminate, in the interface between the unidirectional plies and the ones with the 0/90 sequencing, FO2, FO3 and FO4 are placed

Fig. 4 Fig. 5 Fig. 6 Fig. 7

 4567 Fig. 4 Snapshot of the micro electromagnetic field in the domain of interest V (left) and on its middle plane (right)

Table 1

 1 Electromagnetic material properties used in the macro-EMsimulation

	Material	r	σ	μ r
	composite	20	100	1.0
	mould	3.54	0.28	1.0

Table 2

 2 Electromagnetic material properties used in the micro-EMsimulation

	Material	r	σ	μ r
	UD ply -fiber direction	20 + i	1000	1.0
	UD ply -other plane dir	20 + i	100	1.0
	UD ply -out of plane dir	20 + i	1	1 . 0
	Ferrite layer	10 + 0.05i	50	1.2 + 0.17i
	Ceramic mould	5 + 0.03i	0.28	1.0

Table 4

 4 Thermal convection coefficientsConvection coefficient with the air (W/m 2 K)

Conduction coefficient with the support (W/m 2 K)

Acknowledgements This work was partially done in the framework of the Simutool H2020 Project. The authors would like to thank Jasmin Stein from TWI (UK) for the thermal measurements done in the microwave oven, and Remi Chauveau from Loiretech (France) for the tooling and experimental set up.

Compliance with Ethical Standards

Conflict of interests The authors declare that they have no conflict of interest.


Louis Delannoy

Salil Puri

A T D Perera
email: dasun.perera@epfl.ch

Silvia Coccolo

Dasaraden Mauree

Jean-Louis Scartezzini

Climate impact and energy sustainability of future European neighborhoods

Keywords: urban energy systems, energy efficiency, renewable energy integration, energy supply optimization I

This study presents a comprehensive analysis of typical European city centers energy demand and supply for present time and 2050, according to the Intergovernmental Panel for Climate Change (IPCC) climatic scenario A1B. Two multifamily houses are modeled, adapted and simulated considering local thermal characteristics and climate conditions of twentytwo European countries. An energy hub system is optimized in order to investigate how the micro-grid responds to the renewable energy integration for ten countries. The European heating demand is evaluated to be reduced by half by 2050 (49%) due to the climate change (6%) and refurbishment (43%) effects. Integration of renewable energy sources in most European countries is set to surpass the EU renewable energy targets given proper implementation of appropriate governmental polices coupled with emerging technological advancements in the renewable energy sector.

INTRODUCTION

Climate change, temperature increase, demographic explosion aligned with the scarcity of our raw resources and the decrease in biodiversity threatens our society model. This so called Grand Challenge [START_REF] Beniston | Grand challenges in climate research[END_REF] forces many countries to develop an alternative attitude paving the way towards a sustainable future. Being responsible for 60% of world greenhouse gas emissions and 78% of its total energy consumption while they cover less than two percent of the Earth's surface [START_REF]Cities and climate change[END_REF], cities are primary actors of climate change. Driven by the exponential growth of urban population [START_REF]The World's Cities in 2016 -Data Booklet[END_REF], urban energy management is a priority to ensure a proper transition of our energy model. More precisely, three key drivers are pivotal: energy efficiency, renewable energy integration and smart energy management [START_REF] Guen | Improving the energy sustainability of a Swiss village through building renovation and renewable energy integration[END_REF].

Integrated energy systems such as energy hub [START_REF] Geidl | Energy hubs for the future[END_REF] [6] [START_REF] Perera | Electrical hubs: An effective way to integrate non-dispatchable renewable energy sources with minimum impact to the grid[END_REF] are promising prospects to integrate renewables for small urban areas and villages. And while European policy makers are seeking ways to address energy transition to large urban areas and respect their planned country specific targets [START_REF]Energy 2020 A strategy for competitive, sustainable and secure energy[END_REF], an extension of such system shall serve them as a basis to compare renewable energy integration potential per country. Adding energy demand forecast provides with a double-sided tool to estimate actual urban energy and its future in Europe.

The European database for buildings forms and energy systems, Tabula, has just been finalized [START_REF]TABULA Web Tool[END_REF], making this process possible and could help assess energy sustainability in Europe.

II. PROBLEM DEFINITION

A. Scope and steps

This study aims to compare (i) energy demand projections for present and future time horizon and (ii) renewable energy integration to European standards and targets per country, using firstly computed actual energy demand simulations. Five steps are designed to assess a global representation of urban energy in Europe, chronologically:

• Collect climatic data

B. Main asumptions

To carry out this study, two multi-family houses of five floors are chosen as representative of city centers based on typical archetypes that compose inner urban areas of large cities. Building envelopes are adapted to match historical thermal characteristics for present time simulation and retrofitting state for the 2050 timeframe. On Tabula, this decision lead to select attributes of the oldest multi-family house, latter updated to the usual refurbishment state. Climate projection follows Swiss "Office Fédéral de Météorologie et de Climatologie MéteoSuisse" [START_REF]Scénarios climatiques suisse -un aperçu régional[END_REF] A1B scenario, establishing a growth of greenhouse effect gases until 2050, followed by a slight decline. This scenario presents the closest temperature rise to the main target of Paris agreement (2°C) -the further objective of 1.5°C being today highly questionable -and is selected as the representative climatic scenario. For renewable energy integration, an Energy Hub system is modeled and results are then compared to the Renewable Energy Directive 2020 targets [START_REF]Energy strategy and energy union[END_REF].

III. METHODOLOGY AND TOOLS

A. Overview

Assessing a comparative energy demand study and renewable energy integration on the European level is difficult as no 978-1-5386-5517-7/18/$31.00 ©2018 IEEE single software or single database allows a direct analysis. A computational platform has thus been developed to meet the requirements of this study, in which each software plays a unique role (Figure 1). Firstly, building data -architectural design and U-valueshave been collected on Tabula for each multi-family house per country. This information is secondly transformed through the software Lesosai [12], which defines materials layers, thickness, conductivity, density and heat capacity for each component of the house: floor, roof, walls and windows. An empirical process is used to match Tabula building specifications and Lesosai materials. The physical characteristics of the dwelling are implemented in a 3D building model proper to each country. Thirdly, climatic data and horizon file of every European capital are gathered by means of the software Meteonorm [13]. Fourthly, the hourly profile of the energy demand is computed through CitySim software [START_REF] Kampf | Optimisation of building form for solar energy utilisation using constrained evolutionary algorithms[END_REF]. The same process is applied for 2050 energy projections, where the usual refurbishment state offered in Tabula is selected as the representative future of thermal characteristics and assuming A1B climatic scenario on Meteonorm. Given the energy demand results for present time and 2050 horizon, a first ad-hoc energy demand comparative analysis is made possible. Renewable energies integration and optimization of the energy supply is assessed through the software Homer based on the present hourly energy demand computed in CitySim and a nationally modified electrical load. Theses optimization are carried for present time only as representative of the actual energy transition.

B. Energy demand simulation

CitySim is a decision support tool for urban energy planners and stakeholders, here to help reducing environmental impact of buildings by either improving renewable energy use or reducing carbon emissions gases. It uses several thermal zones resistance capacity models to provide hourly energy demand profiles of imported dwellings. Interactions between buildings are taken in consideration through an algorithm of shadowing and radiation effects. CitySim requires climatic data (temperature, precipitation, horizontal diffuse and direct radiation, relative humidity, wind speed and direction, cloud cover fraction), geographic data (horizon topographical description), the 3D building model (space arrangement, thermal properties for walls, roof, floor and windows, openings, glazing ratio, energy systems) and the occupancy building pattern description, which can be obtained from the Swiss norms SIA 2024. No cooling system is modelled in this study. The final energy demand comprises the simulated heating demand and the electrical demand, modelled building on the Swiss norms SIA 2024 and later on converted for every country based on the national share of final energy consumption in the residential sector by type of end-use [START_REF]Share of final energy consumption in the residential sector by type of end-use[END_REF].

C. Energy supply simulation

To comply with hourly demand calculated via CitySim, energy supply is modelled through the microgrid software called Homer -abbreviated for Hybrid Optimization Model for Multiple Energy Resources. The concept of Energy Hub applied combines decentralized energy systems and suitable renewable sources in a bid to efficiently optimize the energy supply (Figure 2). The main criteria behind using the Energy Hub concept is to comply with the EU goals and reduce the dependence on conventional sources of energy by utilizing renewable energy sources in a cost-effective manner without causing environmental degradation, by minimizing carbon dioxide emissions. For the purpose of improving the share of renewable sources towards energy generation, this study primarily focuses on wind power generation using wind turbines and solar power generation using flat plate PV panels. This model of Energy Hub shows that in addition to using conventional energy supply from the grid, renewable sources particularly PV panels and wind turbines can also be used to effectively produce electricity and therefore be utilized to meet a certain part of the demand. However, due to the nondispatchable and stochastic nature of renewable energy production, several lead acid batteries are also employed that are utilized during periods of climatic fluctuations when the renewable sources are unable to produce electricity meeting the current demand. Based on the net energy demand computed through CitySim, the simulations were conducted for the major cities of ten European countries in Homer that aimed to enhance the overall efficiency of the micro-grid system modelled for each country and maximize the net renewable energy fraction under realistic conditions. To achieve this, special focus revolved around the following output parameters:

• Net Present Cost (NPC)

• Cost of Energy (COE)

• Renewable Energy Fraction (Ren Frac %)

• Grid Purchases • Grid Sales The sensitivity variables or input parameters considered for simulations included Grid Sale Capacity, Grid Purchase Price, Net Grid Purchase Limit, Annual Grid Purchase Capacity, and PV Panel Efficiency. The net present cost is used to estimate the total life cycle cost of the system. It takes into account all the various revenues, investments and costs that occur during the lifetime of the system and reduces into a single lump sum in year-zero dollars whereas the cash flows in the future are discounted back to year zero with the help of discount rate. The various costs that are taken into account include capital and replacement costs of the components as well as their operating and maintenance costs and costs involving grid electricity purchases. Revenues are calculated from the income generated by selling power back to the grid along with the salvage value of system equipment at their end of life. While simulating, HOMER uses the net present cost to rank different system combinations in the output section [START_REF]Total net present cost in HOMER[END_REF]. The renewable energy fraction ) pertaining to the micro-grid is calculated using the following equation [START_REF]7.133 Renewable Fraction[END_REF]:

 8760 8760 11 1 nonren ren served tt E f E 
where Enonren is the Nonrenewable Electricity Production (kWh/yr) and Eserved is the Net Electricity Served by the Grid (includes grid sales). The sale capacity of the grid refers to the maximum amount of energy that can be sold back to the grid in each time step [START_REF]Purchase and Sale Capacities[END_REF]. The Net Grid Purchase Limit refers to the maximum value of net grid purchases less the grid sales allowed to the micro-grid [START_REF] Kim | Lecture notes on Design and Simulation of Micro-Power Systems of Renewables[END_REF] and the annual purchase capacity is defined as the maximum power that can be purchased from the grid in each time step [START_REF]Purchase and Sale Capacities[END_REF]. The levelized cost of Electricity (COE), as defined by Homer, is the average cost per kWh of useful electrical energy produced by the system [START_REF]Levelized Cost of Energy[END_REF]. It is defined as follows:

, ann tot served C COE E  
where Cann,tot is the total annualized cost of the system ($/yr).

The power generated by the PV array is calculated by the following equation [START_REF]How HOMER Calculates the PV Array Power Output[END_REF]:

 
where YPV is the rated capacity of the PV array (kWh), xPV is the PV derating factor (%), GT is the solar radiation incident on the PV array per time step (kW/m 2 ). GT,STC corresponds to the incident radiation at standard test conditions (1 kW/m2), θ is the temperature coefficient of power (%/°C), TC is the PV cell temperature in the current time step (°C), TC,STC is the PV cell temperature under standard test conditions (25°C). In order to compute the power produced by the wind turbine, Homer first calculated wind speed at the hub height of the wind turbine using the following equation [START_REF]How HOMER Calculates Wind Turbine Power Output[END_REF]:



Uhub is the wind speed at hub height of wind turbine (m/s), Uanem is the wind speed at anemometer height (m/s), zhub is the hub height of wind turbine (m), zanem is the anemometer height (m) and zo the surface roughness length (m).

After calculating the wind speed at the hub, the software then takes into account the wind turbine's power curve to calculate the expected power output from the wind turbine at that wind speed under standard conditions of temperature and pressure [START_REF]How HOMER Calculates Wind Turbine Power Output[END_REF], as presented in Figure 3.

. , ,

(

)[1 ( ) Since the power is predicted at standard test conditions, Homer makes use of the air density ratio to adjust to the actual conditions using the following equation [START_REF]How HOMER Calculates Wind Turbine Power Output[END_REF]:

T PV PV PV C C STC T STC G P Y x T T G     ln( / ) . ln (
 
where PWTG (kW) is the wind turbine power output at actual conditions, PWTG,STP corresponds to the wind turbine power output at STP (kW), ρ is the actual air density (kg/m 3 ) and ρo is the air density at STP (kg/m 3 ).

IV. RESULTS AND ANALYSIS

A. Energy demand Figures 4 and5 show the net gross heating demand, in current and future climatic conditions. Northern countries have a higher heating demand than Southern countries due to climatic differential. On the same hand, Western countries have a lower heating demand compared to Eastern countries which can be explained by the economical gradient: Western countries historically have a heavier economical weight and have been able to prevent heating demand by refurbishment strategies over the past decades. Net gross energy demand simulations for 2050 decreased for every country compared to present time simulation. Additionally, due to the renovations strategies, the difference of heating demand is lower between the counties, from 147 kWh/m 2 today, to 99 kWh/m 2 in 2050.

The mean net gross energy heating in 2050 is estimated to 70.8 kWh/m 2 , representing a mean heating demand reduction of 48.75%. This important decline is caused simultaneously by a refurbishment strategy and a temperature increase due to climate change. Importing Tabula net gross energy demand for usual refurbishment state (78.9 kWh/m 2 on average), climate change impact over heating demand is estimated as the difference between the projections: 8.1 kWh/m 2 , or 11.44% of the 2050 simulated heating demand. Similarly, refurbishment strategy is responsible of 88.56% of heating demand downturn.

B. Energy supply

For the countries under study, it is imperative to define a Renewable Energy Potential that aids in measuring the attainable renewable energy penetration under realistic conditions. For this purpose, the Renewable Energy Potential (R.E.P) was defined as follows: Re .. Re

i i Max nFrac R E P nFrac  
where R.E.Pi is the R.E.P for country i, RenFraci is the Renewable Energy Fraction obtained for country i under sale capacity of 50% peak load and Net Grid Purchase Limit set to 50% yearly demand. RenFracMax is the Maximum Renewable Energy Fraction obtained among all countries at sale capacity of 50% peak load and Net Grid Purchase Limit of 50% yearly demand. The R.E.P ranges between 0 and 1 with higher R.E.P value indicating higher potential for renewable energy integration. However, it was also necessary to define a Cost Index (C.I) which compares the net costs involved in attaining the R.E.P for various countries. The Cost Index (C.I) is defined as follows:

.

V. CONCLUSION

Energy demand for heating is set to decrease in Europe due to global warming and refurbishment strategy. This downturn is estimated to nearly a half of today's present heating demand, as calculated by the proposed computational platform. Thanks to the proposed methodology, a database of European archetypes is defined, assuming several urban configurations and buildings properties, as function of each country. If today's present heating demand is following increasing South-North and West-East paths, the future heating demand will be unevenly distributed due to differential refurbishment strategies. The close results obtained between the European web tool Tabula and the CitySim exemplify a good behavior and adaptation of the process used. Moreover, the important number of buildings present in Tabula database sets a high commitment for the basis of the method and carries in general a reliable initial data. Further development is needed to ensure a more precise modeling where an idea of city's representation is suggested by dividing inner and outer city and adding several building forms. This path would allow to set housings parameters closer to reality on top of lowering the inputs data and output results by diversification of inputs.

The integration of renewable energy is estimated to grow on fairly positive scale. Given the potential for solar and wind energy production throughout Europe coupled with increasing initiatives by the EU towards building a sustainable economy, it seems evident that the contribution of renewable energies towards total energy generation will expectedly grow and most probably exceed the targets set by the European Union. However, switching towards renewable energy production does have its own set of drawbacks. A well-balanced mix of resources essentially becomes important in order to minimize the effects of intermittency and allow for higher renewable energy market shares. Also, the cost aspects cannot be neglected when considering renewable energy production. In the present scenario, costs of installing, operating and maintaining solar panels and wind turbines by far exceed the costs of current technologies and power plants that thrive on conventional or fossil-based resources. As a result, emerging technologies should also take the cost factor into account that would help bridge this gap and make renewable energy production commercially viable and essentially profitable, consequently, generating interest and attracting more investments in the future.

Fig. 1 .

 1 Fig. 1. Chart flow of the global methodology, in chronological order.

Fig. 2 .

 2 Fig. 2. Schematic of the micro-grid designed for simulations in Homer

Fig. 3 .

 3 Fig.3. Power Curve for a wind turbine at STP[START_REF]How HOMER Calculates Wind Turbine Power Output[END_REF]

Figures 4 and 5 :

 5 Figures 4 and 5: Net gross heating demand (kWh/m 2 ) per country, present time (4) and 2050 simulation (5).

  Ii is the Cost Index (C.I) for country i, NPCi is the Net Present Cost of the System for country i at sale capacity of 50% peak load and Net Grid Purchase Limit set to 50% yearly demand. NPCMax is the Maximum value of the Net Present Cost among all countries at sale capacity of 50% peak load and Net Grid Purchase Limit set to 50% yearly demand. The , values are calculated for all the countries under study. A higher value of C.I implies higher costs involved in integrating renewable energy into the country. Therefore, a higher C.I value imposes greater barriers for implementing renewable energies which is not considered desirable. Combining the R.E.P and C.I values for all the cities, as shown in Figure6, underlines that Amsterdam has the highest R.E.P amongst the lot which can be attributed to highly effective and efficient wind power generation due to reasonably high annual average wind speeds at 7.07 m/s. Cities like Budapest witnessed fairly low R.E.P which can be attributed to comparatively less favorable climatic conditions in the region which could be seen from its average solar global horizontal irradiance at 3.38 kWh/m 2 /day and the average annual wind speeds was just 3.90 m/s. Paris and Amsterdam also showed the least C.I values which signifies lower restrictions and economic barriers towards integrating renewable energy generation in these regions. Since the R.E.P and C.I are indices measured relatively with each country under study, Switzerland had the highest C.I value which could be attributed to the fact that the costs of capital, installing and maintaining and replacing solar panels and wind turbines is comparatively more in Switzerland than other countries under study as Switzerland boasts a very high overall cost of living.

Fig. 6 .

 6 Fig. 6. Representation of R.E.P and C.I values for different European cities

Authorized licensed use limited to: INRIA. Downloaded on April 21,2021 at 17:47:00 UTC from IEEE Xplore. Restrictions apply.

VI. ACKNOWLEDGEMENTS

This research project is financially supported by the Swiss Innovation Agency Innosuisse and is part of the Swiss Competence Center for Energy Research SCCER FEEB&D.