Chemosphere 144 (2016) 1729e1737

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Acute toxicity, uptake and accumulation kinetics of nickel in an invasive copepod species: Pseudodiaptomus marinus ne Tlili a, b, c, *, Julien Ovaert a, b, Anissa Souissi a, Baghdad Ouddane b, Sami Souissi a Sofie Univ. Lille, CNRS, Univ. Littoral Cote d'Opale, UMR 8187 LOG, Laboratoire d'Oc eanologie et de G eosciences, F-62930 Wimereux, France Universit e de Lille1, Laboratoire LASIR, UMR CNRS 8516, Equipe Physico-Chimie de l'Environnement, F-59655 Villeneuve d'Ascq Cedex, France c University of Sousse, Research Unit in Biochemistry and Environmental Toxicology, ISA Chott-Mariem, 4042 Sousse, Tunisia a

b

h i g h l i g h t s
Lethal concentrations were determined in Pseudodiaptomus marinus exposed to nickel.
Accumulation kinetics and nickel uptake were studied in P. marinus in presence and absence of food.
The uptake of nickel in P. marinus depends from the pathways of entrance (direct absorption or into food).
Isochrysis galbana has an important bioaccumulation capacity and a rapid uptake of nickel.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 August 2015 Received in revised form 23 September 2015 Accepted 13 October 2015 Available online xxx

Pseudodiaptomus marinus is a marine calanoid copepod originating of the Indo-Pacific region, who has successfully colonized new areas and it was recently observed in the European side of the Mediterranean Sea as well as in the North Sea. Actually, many questions were posed about the invasive capacity of this copepod in several non-native ecosystems. In this context, the main aim of this study was to investigate the tolerance and the bioaccumulation of metallic stress in the invasive copepod P. marinus successfully maintained in mass culture at laboratory conditions since 2 years. In order to study the metallic tolerance levels of P. marinus, an emergent trace metal, the nickel, was chosen. First, lethal concentrations determination experiments were done for 24, 48, 72 and 96 h in order to calculated LC50% but also to select a relevant ecological value for the suite of experiments. Then, three types of experiments, using a single concentration of nickel (correspond the 1/3 of 96 h-LC50%) was carried in order to study the toxicokinetics of nickel in P. marinus. Concerning lethal concentrations, we observed that P. marinus was in the same range of sensitivity compared to other calanoid copepods exposed to nickel in the same standardized experimental conditions. Results showed that the uptake of nickel in P. marinus depends from the pathways of entrance (water of food), but also that Isochrysis galbana, used as a food source, has an important bioaccumulation capacity and a rapid uptake of nickel. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Pseudodiaptomus marinus Nickel Isochrysis galbana LC50% Uptake Bioaccumulation kinetics

1. Introduction Pseudodiaptomus marinus (Sato, 1913) is a marine calanoid copepod originating of the Indo-Pacific region, but in the last 50 years it has successfully colonized new areas and it was recently observed in the European side of the Mediterranean Sea as well as in the North Sea (De Olazabal and Tirelli, 2011; Pansera, 2011;

 de Lille 1 Sciences et Technologies, UMR * Corresponding author. Universite CNRS-Lille1-ULCO 8187 LOG, Marine Station, 28 Avenue Foch, F-62930 Wimereux, France. E-mail address: sofi[email protected] (S. Tlili). http://dx.doi.org/10.1016/j.chemosphere.2015.10.057 0045-6535/© 2015 Elsevier Ltd. All rights reserved.

Brylinski et al., 2012; Sabia et al., 2012). The invasive capacity of this copepod in different marine and estuaries ecosystems suppose that it has an important eco-physiological capacity and adaptation aptitude to different environmental parameters (e.g. temperature, salinity, etc.). However, a little is known about the reactions of the possible resistance or adaptation of this species to pollutants that it may encounter in some newly invaded habitats (e.g. harbors, estuaries, etc.). In marine ecosystems, copepods are crucial components in planktonic food webs for their function as the main transfer node between primary producers and fish, and for their role in nutrient recycling and export (Miller, 2004). Due to their ecology, aquatic invertebrates, as calanoid copepods are

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continuously exposed to variable concentrations of trace metals water (Barka, 2007) but also by other mechanisms such as food web interactions (Rainbow et al., 2006; Saiz et al., 2009; Mebane et al., 2012). In spite of their high relevance in the marine ecosystem dynamics, the number of studies that focused the potential impact of emergent pollutants in marine planktonic copepods is still limited and has concentrated mainly in acute lethal responses (Sverdrup et al., 2002; Saiz et al., 2009). Aquatic invertebrates can accumulate trace metals in their tissues at levels sometime much higher than those detected in their environment and are still able to survive (Mouneyrac et al., 2002; Barka, 2007). Among trace metals, nickel (Ni) has an important environmental relevance and has been proved to exert long-term harmful effects to aquatic organisms (Kienle et al., 2009; Attig et al., 2010). In fact, nickel is a metallic element that is naturally present in the earth's crust and due to its unique physical and chemical properties, nickel and its derivate forms are largely used in electronic electroplating, coins minting, steel alloys, batteries and many other chemical industries (Denkhaus and Salnikow, 2002). In aquatic environments, nickel pollution is principally due to industrial discharges from electroplating, smelting, mining and refining operations and other industrial emissions (Vijayavel et al., 2009). Recently, increasing attention on the ecotoxicological effects of nickel contamination in aquatic organisms has been considered due to its large industrial application and quantities released in the environment (Kienle et al., 2009; Vandenbrouck et al., 2009; Attig et al., 2014). Physiologically, trace amounts of nickel are essential for growth and reproduction functions of animals, indicating that nickel can be considered as an essential trace element (Woo et al., 2009). Considering human health, exposure to nickel compounds can cause a variety of adverse effects and damage on human health, from skin dermatitis over immune toxicity (Denkhaus and Salnikow, 2002). In aquatic crustaceans and copepods, the chronic exposure to Ni is responsible of oxidative stress, respiratory and osmoregulation disruption, cellular disorder and the reduce of naupliar viability (Pane et al., 2003; Buttino et al., 2011; Jiang et al., 2013, Blewett et al., 2015). In fact, expositions of aquatic organisms to dissolved nickel in water can contribute to the production of free radicals in cells and the depletion of some antioxidant enzymes (e.g. Catalase, Glutathione-S-Transeferases, Catalase, Glutathione Peroxydases…) (Denkhaus and Salnikow, 2002; Attig et al., 2010, 2014; Wang and Wang, 2010). Despite this ecotoxicological aspect and in comparison with other trace metals, the toxico-kinetics of nickel has not been well studied in copepods as well as other animals (both vertebrates and invertebrates). P. marinus as all calanoid copepods, are principally herbivorous and detritivorous (Uye and Kasahara, 1983; Sabia et al., 2014) and microalgae represent the major food source for them. Among microalgae widely used in aquaculture for copepod mass production, Isochrysis galbana (I. galbana) represents an important issue (Pan et al., 2014). I. galbana is a marine flagellated microalga belonging to the phylum Haptophyta, class Coccolithophyceae, subclass Prymne-siophycidae, order Isochrysidales, family Isochrysidaceae (Guiry and Guiry, 2013; Sadovskaya et al., 2014). I. galbana cells are easily and quickly assimilated by larval animals due to their small size (4e7 mm) and specially the absence of a tough cell wall (Jeffrey et al., 1994; Liu and Lin, 2001; Pan et al., 2014; Sadovskaya et al., 2014). The present research work propose three complementary objectives: i) the determination of the lethal concentration of nickel in P. marinus measured at 24, 48, 72 and 96 h; ii) the study of toxicokinetics of one selected sub-lethal concentration (corresponding to 1/3 of 96 h-LC50%) in the whole body of P. marinus for 1, 2, 3, 4, 7, 10 and 15 days; iii) the investigation of a possible transfer of nickel

contamination by the food web using a previously contaminated microalgae culture (I. galbana) as unique food resource for P. marinus for 1, 2, 3, 4, 7, 10 and 15 days monitoring. 2. Materiel and methods 2.1. Copepod mass culture Populations of P. marinus used in this study, has been cultured in the Marine Station of Wimereux since 2011. The original strain of P. marinus comes from The Lake Faro, Sicily (Italy) previously collected by Sabia et al. (2014). The copepod strains are maintained continuously in 2 L beakers in an incubator (SANYO model MLR351) at 18  C and a photoperiod of 12L:12D cycle under a fluorescent light with an intensity of 2500 lux. The protocol of maintaining the copepods during several generations is slightly modified from those used for the estuarine copepod Eurytemora affinis (Souissi et al., 2010, 2015). In order to obtain high number of copepods for the need of our experiments a stock culture was maintained in 20 L Nalgene flasks and 40 L transparent Plexiglas flat bottom tank with moderate aeration. The seawater used was pumped from the English Channel near Wimereux Marine Station, and was filtered several times up to 1 mm. The salinity of the water was around 33 PSU ± 1 and temperature was around 20  C ± 2, average concentrations composition are presented in Table 1. Copepods were feed with the microalgae I. galbana cultivated in Conway medium with the same method described in Sadovskaya et al. (2014). 2.2. Algae mass culture The starter culture of I. galbana was obtained from the Roscoff culture collection (Roscoff, France). The strain was maintained in the laboratory in 250 ml Erlenmeyer flasks and was shaken automatically at slow speed using an automatic shaker (KS 250 basic, IKA Labortechnik). The culture was kept in an incubator (SANYO model MLR-351) at 18  C and a photoperiod of 12L:12D cycle under a fluorescent light with an intensity of 2500 lux. Batch cultures in 2e6 L flasks were used to grow the microalgae for this study. The culture flasks were filled with autoclaved seawater of salinity 33 PSU and enriched with Conway medium. The composition of Conway medium was: each liter of autoclaved seawater contained 100 mg NaNO3, 20 mg NaH2PO4, 45 mg Na2EDTA, 33.6 mg H3BO3, Table 1 Seawater average concentration in Wimereux Station. Major element

mg/L

Sodium Magnesium Calcium Potassium Strontium Chloride Sulfate Bicarbonate Bromide Borate Fluoride

10 690 ± 110 128 ± 5 416 ± 15 390 ± 10 13.2 ± 0.5 19 295 ± 132 2701 ± 35 145 ± 12 62 ± 5 27 ± 2 1.35 ± 0.02

Trace element

mg/L

Cadmium Copper Iron Manganese Nickel Lead Vanadium Zinc

0.035 ± 0.008 0.635 ± 0.025 1773 ± 0.012 5235 ± 0.046 0.327 ± 0.012 0.045 ± 0.025 1.45 ± 0.07 0.785 ± 0.002

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0.36 mg MnCl2, 1.3 mg FeCl3, 0.021 mg ZnCl2, 0.02 mg CoCl2$6H2O, 0.02 mg CuSO4$5H2O, 0.09 mg (NH4)6Mo7O24$4H2O, 0.2 mg thiamine HCl (vitamin B1) and 0.01 mg cyanocobalamin (vitamin B12). Cultures were aerated with sterile air and incubated in the same condition as the strain inoculum. 2.3. Nickel solutions for experiments Nickel salt (NiSO4$6H2O) used for experiment was originated from Merck (99.9% pure) and was readily dissolved in MilliQ water to make stock solutions which were acidified to pH ~2 with suprapur nitric acid. Final nickel solutions at the considered concentration used in this study were obtained by adding appropriate volumes of stock solutions to filtered seawater originating from Wimereux marine station (Wimreux, France). 2.4. Lethal concentration determination experiments Lethal concentrations determination experiments were done in standard conditions as described in Lassus et al. (1984), Forget et al. (1998) and Barka et al. (2001a,b). Preliminary tests were conducted to establish a mortality range from 0% to 100%. Tests were conducted at controlled physicechemical parameters (ambient temperature ¼ 20  C ± 2; pH ¼ 8.4 ± 0.2; Dissolved oxygen ¼ 8 ± 1 mg/L) in absence of feeding as recommended in all standard lethal concentration determination experiments. Three groups of adult copepod (n ¼ 100) were placed in 140 ml glass beakers containing 100 ml of each test solution and were exposed to six nickel concentrations (0; 50; 150; 250; 350 and 500 mg/L) and mortality levels were observed every 24 h. Lethal concentrations were calculated for 24, 48, 72 and 96 h. Probit analysis was applied to estimate lethal concertation values.

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(HNO3) (65%)/Hydrochloric acid (HCl) (30%) (1:3 v/v) to mineralize the remaining solid fraction. The obtained solution was then filtered to eliminate the carbon residue (Lesven et al., 2010) and diluted to final volume of 20 ml with deionized distilled water and stored at 4  C for analysis (Priadi et al., 2011). For water, samples of 5 ml were directly taken from the experimental tanks, carefully filtered and stoked in analytic tubes with 50 mL of HNO3 until analysis. Nickel concentrations in organism tissues and water samples were determined in triplicate using inductively coupled plasma atomic emission spectrometry (ICP-AES, Varian, Vista-PRO axial view) and/or inductively coupled plasma mass spectrometry (ICPMS; Thermo Electron Corporation, Element X7 Series) depending on the concentration level. The determinations were validated using certified reference materials IAEA-452 (the marine scallop Pecten maximus, certified from the International Atomic Energy Agency). A good agreement was observed between the obtained and the certified values for nickel (data non-shown). 2.7. Uptake kinetic model The uptake kinetic model, expressing the rate of metal uptake from solution (mg g1 d1), was calculated following the equation (Luoma and Rainbow, 2005):

Cin ¼ Ku Cw t where “Cin” is the Nickel concentration in tissues (mg/g dry weight), “ku” the uptake rate constant (g dry weight/L/day), “Cw” the Ni concentration in water (mg/L) and “t” the exposure time (days). 3. Results

2.5. Accumulation kinetics experiments

3.1. Lethal concentration determination

The selected dose correspond to the 1/3 of 96 h-LC50% (45.39 mg/) was chosen in order to test realistic environmental concentration and to study the sub-lethal effects of nickel. For accumulation kinetics monitoring, three kinds of experiments were done. First, adult copepods were exposed to one selected lethal concentration of Ni (45 mg/L) for 1, 2, 3, 5, 7, 10 and 15 days without feeding. A group of P. marinus (n ¼ 1000) were transferred into a 2 L glass beaker containing the selected dose dissolved with filtered seawater. Experimental tanks were maintained at constant temperature (20  C ± 2) with a very moderate artificial bulling and shaking. Copepods were not fed during this experiment. For the second experiment, a group of P. marinus (n ¼ 1000) were transferred into a 2 L glass beaker containing filtered seawater and a precontaminated algae solution (during 24 h) used as a the unique source of food. For the third experiment, an aliquot of I. galbana, from the exponential phase of growth, were placed in 200 ml glass beaker containing the selected dose dissolved with filtered seawater with continuous artificial bulling and shaking as recommended for sample of copepods or algae were taken, carefully filtered, weighted and directly dried at 60  C during 72 h for total nickel determination.

Results concerning lethal concentration determination of nickel in adult P. marinus were summarized in Table 2. Our results showed that the 96 h-LC50% of nickel in adult P. marinus was 136 mg/L.

2.6. Nickel concentration determination Total nickel concentration in P. marinus bodies and I. galbana cells was determined according to the protocol described by Amiard et al. (1987) and Lesven et al. (2010). Samples of dry copepods (approximately 200 mg) was digested for 24 h at 140  C with 10 ml of Hydrofluoric acid (HF) (40%) in Teflon tubes until evaporation. Then, samples were attacked by adding nitric acid

3.2. Starved experiments The assessment of Ni accumulation in adult P. marinus tissues and water were presented in Fig. 1(a and b). After 24 h of exposure, the concentration of Ni in P. marinus tissues was 4.58 mg/g dry weight, slightly decreased after 48 h of exposure then increased gradually to attend 8, 20.2 and 23 mg/g dry weight after 3, 4 and 7 days respectively. The concentration became then stable until the end of experiment. By the other hand, the concentration of Ni in water was 40.2 mg/L at 24 h and gradually decreased to attend 13.3 and 12.2 mg/L at 10 and 15 days of experiment, respectively. 3.3. Feeding experiments Nickel accumulation kinetics in adult P. marinus tissues and water were presented in Fig. 2(a and b). After 24 h of exposure, the Table 2 Lethal concentration determination (LC25%, LC50%, LC75% and LC100%) of nickel in adult P. marinus calculated at 24, 48, 72 and 96 h. Lethal concentration (mg/L)

24 h

48 h

72 h

96 h

LC25% LC50% LC75% LC100%

259.65 531.39 803.04 1074.86

328.06 313.84 494.64 675.63

72.116 182.51 292.97 403.38

39.62 136.17 232.7 328.81

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Fig. 1. Nickel accumulation kinetics in P. marinus measured in tissues (a) and water (b).

concentration of Ni in P. marinus tissues was 2.85 mg/g dry weight, slightly decreased after 48 h of exposure then increase gradually to attend 4.3 and 7.6 mg/g dry weight after 4 and 7 days respectively. The concentration of Ni became then relatively stable until the end of experiment to attend 24.6 mg/g dry weight after 15 days of exposure. For water, the concentration of Ni was 35.3 mg/L at 24 h and gradually decreased to attend 8.2 and 3.2 mg/L at 10 and 15 days of experiment, respectively. 3.4. Pre-contaminated algae experiments Nickel accumulation kinetics in I. galbana cells and water were presented in Fig. 3(a and b). After 24 h of exposure, the concentration of Ni was 0.9 mg/g dry weight, and then an increase was recorded after 48 h and 72 h of exposure (1.4 and 3.9 mg/g dry weight respectively). The clear increase was observed from day 7 to 15 (7.5 and 15.5 mg/g dry weight respectively). Concerning the concentration profile of Ni in water, the concentration was 40.1 mg/L and gradually decreased until the end of the experiment to attend 1.4 mg/L at 15 days. 3.5. Uptake kinetics model Results of the experimental calculation of the uptake rate

constant (Ku) from aqueous solution (seawater in our case) as represented in Table 3. Results showed that I. galbana have clearly the most important uptake coefficient Ku, in comparison with P. marinus singly exposed to a sub-lethal concentration of Ni and P. marinus feed by pre-contaminated algae.

4. Discussion Due their ecology and physiology, copepods are known to be especially sensitive to environmental pollutants, and for this reason, they have been proposed as a target group for ecological testing of environmental threats (Barka et al., 2001a,b; Cailleaud et al., 2007; Zhang et al., 2011; Kwok et al., 2015). Calanoid copepods have a widespread biogeographic distribution and are an important trophic source for both macro-invertebrates and fish (Beaugrand et al., 2003). The calanoid copepod, P. marinus is one of them, having entered the Mediterranean Sea in the last few years (De Olazabal and Tirelli, 2011; Delpy et al., 2012; Zenetos et al., 2012; Sabia et al., 2014) and reached the south of the North Sea very recently (Brylinski et al., 2012). Ecologically, P. marinus is a typical estuarine-coastal copepod, living only in shallow inshore waters, often highly eutrophicated and it is a herbivorous and detritivorous species (Uye and Kasahara, 1983). From a practical point of view, it was indicated that P. marinus was easy to rear

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Fig. 2. Nickel accumulation kinetics in P. marinus feed by pre-contaminated algae measured in tissues (a) and water (b).

under laboratory conditions in comparison to other calanoid species, and was amenable to laboratory testing (Uye et al., 1982; Huang et al., 2006). In order to understand the invasion capacity of P. marinus in the Mediterranean Sea and the North Sea, some recent research works were focused on its spatial and temporal distribution (De Olazabal and Tirelli, 2011; Pansera, 2011), Bio-ecology (Brylinski et al., 2012) and feeding and swimming behavior (Sabia et al., 2014). However, at our knowledge, the present work is the first focused on the study of lethal concentrations determination and the accumulation kinetics of some selected trace metals in P. marinus. The use of standardized monoculture of P. marinus under controlled conditions allowed to obtain the initial stock mass cultures required to realize all experiments. Moreover cultured copepods provide high number of individuals that are in the same physiological status, which contrasts with earlier studies based on field collected individuals (Cailleaud et al., 2009). 4.1. Lethal concentration determination Lethal concentrations determination is the first step of each toxico-kinetics study and aims to characterize the mean concentration of a selected pollutant that causes mortality, corresponding

to the final irreversible toxicological damage. Our results showed that in adult P. marinus the LC50-96 h was about 136 mg/L. This value could be considered as relatively lower than reported in other copepods such as Tigriopus brevicornis, Acartia tonsa, Amphiascus tenuiremis (Taylor, 1981; Barka et al., 2001a,b; Hagopian-Schlekat et., 2001). The lethal concentration of Ni causing the mortality of the whole population of P. marinus tested (LC100-96 h) was evaluated at 328.8 mg/L. Such all-essential metals, Ni can be toxic and/or lethal depending from its concentration but also its bioavailability. In fact, at low concentration, Ni is an essential element for normal growth and reproduction of various species (Woo et al., 2009). However, at elevated concentrations, Ni is toxic and can change intracellular calcium levels, disturb the cellular homeostasis, bind to protein involved in oxygen sensors and produce oxidative stress (Refvik and Andreassen, 1995; Salnikow et al., 2000; Jiang et al., 2013). Recently, Jiang et al. (2013) found that, in Pseudodiaptomus annandalei, a calanoid copepod, exposure to high doses of Ni (8.86 mg/L for 24 h) is responsible of the induce of an important generation of reactive oxygen species (ROS) and distribution of metabolic signaling pathways. As a consequence, the induction of DNA methylation and the suppression of histone acetylation caused gene alteration and cellular disorders. In the same context, it was demonstrated that in the calanoid copepod A. tonsa, the exposure

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Fig. 3. Nickel accumulation kinetics in I. galbana measured in cells (a) and water (b).

Table 3 Uptake rate constant (Ku) calculated from accumulation kinetics of Ni in P. marinus, I. galbana and P. marinus feed by contaminated algae. Experience

P. marinus

I. galbana

P. marinus feed by contaminated algae

Uptake rate constant Ku (mg/L/day) R2

0.1459 0.9058

0.5143 0.7332

0.1661 0.8512

to sub-lethal concentration of Ni (0.025 L1) during 7 days strongly reduced naupliar viability to 35% with 85% of the surviving nauplii marked positively for apoptosis (Buttino et al., 2011). 4.2. Starved, feeding and pre-contaminated experiments Several studies have been devoted to trace metal accumulation or its uptake by aquatic invertebrates from the dissolved phase (Rainbow and Luoma, 2011) but studies concerning the accumulation and toxico-kinetics Ni in copepods were very limited (Jiang et al., 2013). Our results showed that, in absence of food, P. marinus has a speed uptake of dissolved Ni in water especially between 4 and 7 days of experiments. These results are confirmed by the gradually decrease of nickel measured in water. These results can be explained by the availability of Ni in water. In fact, metals

accumulated by organisms can be classified into two components metabolically soluble and available metals and stored detoxified metals. All metals have the potential to be deleterious to cellular mechanisms, even though some of them are essential to normal metabolic processes, and their toxic effects are related to the metabolically soluble available form (Rainbow, 2002, 2006). In fact, metal speciation and physicochemical changes in seawater influenced both metal bioavailability and its uptake by aquatic invertebrates (Rainbow and Luoma, 2011). The assessment of the Ni accumulation in P. marinus (Fig. 1), showed a slight decrease at 48 h. This result could be explained by the adaptation phase of copepods to a new chemical environment (the addition of Ni in the experimental tanks). After the speed uptake of Ni dissolved in water (from day 4 to 7), Ni content in P. marinus tissues remains constant until the end of the experiment. This could be related to the intracellular mechanism of metals

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storage and complexation in order to detoxify and eliminate exceed metals out of copepods bodies. In fact, copepods can accumulate trace metals, such as Ni, by storage into granules of the lysosomal system (Viarengo et al., 1987; Barka, 2007; Rainbow, 2007) or/and by complexation of metals by cytosolic compounds including metallothioneins (MTs) or metallothionein-like proteins (MTLPs) (Amiard et al., 2006; Rainbow, 2006). Metallothioneins, are a family of low-molecular-weight cysteine-rich metal binding proteins, and they are involved in the homeostatic regulation of intracellular metals and the detoxification of excess amounts of both essential and non-essential trace metals (Mouneyrac et al., 2002; Amiard et al., 2006). Although the fact that Ni has a very high affinity for amino acids, specially histidine and cysteine (Costa et al., 1994), it was demonstrated that, in calanoid copepods, such as T. brevicornis, Ni was generally as weak inducer of MTLPs in comparison with other trace metals such as cadmium, zinc and copper (Barka et al., 2001a,b). In other hand, testing toxic effects on copepods under starvation may severely bias the threshold toxic concentrations eliciting negative effects, because food intake may represent the major path for toxic incorporation in copepods, and because of a weakening effect under starving for copepods with low lipid reserves (Saiz et al., 2009). In fact, experiments done with P. marinus fed by a pre-contaminated culture of I. galbana showed that the uptake of Ni is higher in presence of a food source. Under real environmental conditions; waterborne pollutants, such as trace metals, may transfer to copepods by directly absorption from water or ingested after consumption of contaminated phytoplankton (Sobek et al., 2006; Magnusson et al., 2007). Uptake of trace metals into the invertebrate's body from water is possible through permeable body surfaces and from the gut (Wang et al., 2002). However, uptake of trace metals from the diet may be the major source of metals for many aquatic invertebrates and this trophic exposure usually resulting in higher accumulation and stronger effects (Hook and Fisher, 2001; Wang et al., 2002; Magnusson et al., 2007). Actually, many studies are devoted to metal accumulation by phytoplankton and its subsequent transfer to marine copepods (Wang et al., 2007). In this context, I. galbana exposed a sub-lethal concentration of Ni showed a quick uptake of Ni dissolved in water especially from day 4 to 10 of the experiment. In fact, as the majority of microalgae, I. galbana is characterized by an important capacity of water pumping in the purpose to extract minerals needed for growth and survival (Jeffrey et al., 1994; Marchetti et al., 2012). Results showed also, that this microalga has an important capacity for accumulation of Ni in their tissues. In fact, Yap et al. (2004) reported that I. galbana has a great capacity for the bioaccumulation of Cd, Pb, Zn and Cu and supposed that is the result of phytochelatin induction. Metals are generally taken up into cells by membrane transport proteins and differences in the structure and composition of cellular membranes could explain the inter-specific strategies and speed of Ni (Yap et al., 2004; Wang et al., 2007). In microalgae, the majority of Ni accumulated is distributed in the soluble substances or the protein fraction and this can imply that algal-bound Ni could be easily assimilated by the predator such copepods and consequently tends to be transferred along the food chain. Especially, that previous studies find that copepods only assimilate the cytoplasmic pool of intracellular metal storage in the algal cells (Reinfelder and Fisher, 1991; Hutchins et al., 1995). 5. Conclusion The assessment of the accumulation kinetics of nickel in P. marinus demonstrated that this species has a rapid uptake of Ni, especially in the presence of food source. This information confirmed that the food web is the major pathway for trace metals

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accumulation and consequently the important role of copepods in the biomagnification of metallic pollutants in marine ecosystems. The accumulation of Ni in P. marinus could be summarized into three principal steps: uptake, stabilization and detoxification. Further studies concerning the pathways of detoxification and elimination of trace metals in P. marinus should be undertaken in the future to understand and characterize metal pollution fate in this species as well as in calanoid copepods in general. In the same context, experiments showed that the tested algae, I. galbana has an important uptake and accumulation capacity of Ni dissolved in water. This particularity may be useful in the use of micro-algae as powerful tools of bio-stabilization of metal polluted sites. Results from this work showed that P. marinus is relatively resistant to metallic stress, and has a rapid uptake and tissular capacity to storage metals, that can explain in part the resistance of this copepod and its eco-physiological plasticity. However, further complementary studies are needed to characterize the toxicokinetics but also the biological effects cascade induced by Ni in P. marinus. Acknowledgments This study was funded by an Erasmus Mundus (Fatima Al Fihri Lot 2) Post-Doctoral fellowship accorded to S. Tlili. Thanks are due to the technical staff of the research team “Marine and Analytic Chemistry” and as well to past and present team members of S. Souissi for their help in maintaining continuous cultures of microalgae and a collection of several species of copepods including P. marinus used here. We thank L. Sabia for providing the initial strain of P. marinus present in Wimereux Marine station. We thank LOG COPEFISH team for their help in keeping P. marinus cultures in the laboratory since 2011 in the framework of the COPEFISH project (Young Researcher (A.S.) Emerging Project of the Nord-Pas de Calais Regional Council).This work is a contribution to the Interdisciplinary Environmental Institute of Lille 1 University (IREPSE) as well as to Lille 1 grant (BQR-Convergence-2014) targeting to reinforce the multidisciplinary studies around copepods. References Amiard, J.C., Amiard-Triquet, C., Barka, S., Pellerin, J., Rainbow, P.S., 2006. Metallothioneins in aquatic invertebrates: their role in metal detoxification and their use as biomarkers. Aquat. Toxicol. 76, 160e202. http://dx.doi.org/10.1016/j. aquatox.2005.08.015. Amiard, J.C., Pineau, A., Boiteau, H.L., Metayer, C., Amiard-Triquet, C., 1987. Applitrie d'absorption atomique Zeeman aux dosages de huit cation de la spectrome  le ments traces (Ag, Cd, Cr, Cu, Mn, Ni, Pb et Se) dans des matrices biologiques e solides. Water Res. 21, 693e697. http://dx.doi.org/10.1016/0043-1354(87) 90081-9. Attig, H., Dagnino, A., Negri, A., Jebali, J., Boussetta, H., Viarengo, A., Dondero, F., Banni, M., 2010. Uptake and biochemical responses of mussels Mytilus galloprovincialis exposed to sublethal nickel concentrations. Ecotoxicol. Environ. Saf. 73 (7), 1712e1719. Attig, H., Kamel, N., Sforzini, S., Dagnino, A., Jebali, J., Boussetta, H., Viarengo, A., Banni, M., 2014. Effects of thermal stress and nickel exposure on biomarkers responses in Mytilus galloprovincialis (Lam). Mar. Environ. Res. 94, 65e71. http://dx.doi.org/10.1016/j.marenvres.2013.12.006. Barka, S., 2007. Insoluble detoxification of trace metals (copper, zinc, nickel, cadmium, silver and mercury) in a marine crustacean Tigriopus brevicornis (Müller). Ecotoxicology 16, 491e502. Barka, S., Pavillon, J., Amiard, J., 2001a. Influence of different essential and nonessential metals on MTLP levels in the Copepod Tigriopus brevicornis. Comp. Biochem. Physiol. C 128 (4), 479e493. http://dx.doi.org/10.1016/S15320456(00)198-8. Barka, S., Pavillon, J.F., Amiard, J.C., 2001b. Influence of different essential and nonessential metals on MTLP levels in the copepod Tigriopus brevicornis. Comp. Biochem. Physiol. C 128, 479e493. Beaugrand, G., Brander, K.M., Lindley, J.A., Souissi, S., Reid, P.C., 2003. Plankton effect on cod recruitment in the North Sea. Nature 426, 661e664. Blewett, T.A., Glover, C.N., Fehsenfeld, S., Lawrence, M.J., Niogi, S., Goss, G.G., Wood, C.M., 2015. Making sense of nickel accumulation and sub-lethal toxic effects in saline waters: fate and effects of nickel in the green crab, Carcinus

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