Effects of estuary sediment contamination on physiology, biochemical biomarkers and immune parameters in juvenile European sea bass (Dicentrarchus labrax, L., 1758)

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Choice of European sea bass (Dicentrarchus labrax, L., 1758) as a biological model

European Sea bass is a euryhaline key species in estuarine and coastal environments and a valuable commercial aquaculture species, economically and ecologically, in the Mediterranean countries (Figure 13). Juveniles of this species spend most of their first year of life in estuaries or lagoons and can survive in a wide variety of temperature and salinity (usually lower than in the open sea: 10–20‰ vs. 30–40‰) levels in the open sea, the brackish river deltas, the estuaries and the Mediterranean lagoons (Saillant et al., 2003; Varsamos et al., 2006). In addition, there is considerable knowledge about its physiology and biology and protocols for maintaining this species in captivity in good condition are well established. With the technical development of artificial spawning and larval rearing over the past two decades in Greece, D. labrax has proved to be a suitable species for commercial fish culture. In addition, knowledge of the pattern of early larval development and the effects of environmental parameters on reproduction, particularly in reared fish, is important as it facilitates aquaculture research and fish resources management. However, difficulties mainly in the early stages of its cultivation relating to skeletal deformities, feeding and rearing conditions restricted the number of available fish for further development (Conides and Glamuzina, 2001; Hatziathanasiou et al., 2002 ; Conides and Glamuzina, 2006; Moreira et al., 2008). This species has, for those reasons received an important scientific interest over the last 35 years, but aquaculture-related stress appears to have major negative impacts on the culture of this fish. Finally, D. labrax is commonly used in biomarker studies previously (Sabourault et al., 2001; Varò et al., 2003; Bado-Nilles et al., 2009; Tovar-Ramírez et al., 2010), environmental monitoring and toxicology studies and stress responses to environmental parameters on the early life of development or juveniles (Lemaire-Gony et al., 1995; Cattani et el., 1996; Romeo et al., 2000; Giari et al., 2007; Faucher et al., 2008).

Microcosm experience on sea bass juveniles (Dicentrarchus labrax, L., 1758) exposed to estuary sediment contamination

This experience was performed about the sublethal effects of chemical contamination and physiological performances of European sea bass to different level of contaminated sediment. Juveniles of sea bass from Aquanord marine hatchery (Graveline, France) (genetically homogenous) were transported to the laboratory in oxygenated balloons in the same temperature of sea water where they have been cultured in the hatchery. Juvenile fish has been chosen randomly but regarding the same range of size. In the laboratory of Marine station (Wimereux), juveniles of European Sea bass (< 1 years old, ~100 days) (n=115 fish/tank) were acclimatised during two weeks before the experiment in two of 160 L (100*40*40 cm) aquaria supplied with closed seawater circuit and the water was aerated with air pumps and filtered with external filter (900l/h) (JBL, Cristal profi e900). One-third of the seawater in each tank was renewed manually every 2 days. The photoperiod was maintained at 10L:14D in a thermostated room (15 ± 1 °C) and water temperature in tanks was kept constant at 14 ± 1°C as in the hatchery condition for juveniles of sea bass during the experiment. Fish were fed 1% of the mean body weight once a day with commercial dry pellets (Biomar S.A., Bio-Optimal Start No 1.5) throughout the experiment including acclimation and exposure time. Water parameters such as temperature (°C), salinity, oxygen (mg/l), pH using a Hanna HI 9828 multiprobes and turbidity (NTU) using a waterproof turbidimeter (Eutech instruments, TN-100) were measured 2 times every day (at 10:00-17:00). Following the acclimatization, sea bass juveniles were anaesthetised (0.32 ml / l of 2-phenoxyethanol: ~3-4 min anaesthesia time, ~30sec recovery time), weighed (near to 10 mg), measured for total length (near to 0.1 mm), and individually marked (Visual Implant Tag, 1.2
mm x 2.7 mm, Northwest Marine Technology) before exposure. Afterwards, thirteen fish were sampled at the beginning of the experiment as reference (t0). Fish were then rapidly transferred to the thermostated and aerated room (15 ± 1 °C) at the Marine station of Wimereux into duplicate aquariums of 30 l (20*30*50 cm) randomly where 5 L was filled with sediment and 25 l with seawater aerated with air pumps (14 ± 1°C). For each condition, sediment let settled during 2 days to avoid release of suspended particles in the water. Three contaminated (Seine estuary: Normandie Bridge, Caudebec and Rouen), one less impacted system (Canche estuary) and one reference (Wimereux) sediment was collected with a plastic spatula from surface up to a depth of ~10 cm during low tide and stored in polyethylene bags prior to the exposure experiment. GPS coordinates and sampling dates for each sampling site were recorded: Normandie Bridge (49°26′ 44.64 » N, 0°15′ 01.94 E), Caudebec (49°31′ 25.84 » N, 0°44′ 14.32 E) and Rouen (49°23′ 48.81 » N, 1°01′ 05,82 E )) (26 January 2010), Canche estuary (50°30′ 35.57 » N, 1°38′ 30.38 E) (3 February 2010) and Wimereux as control station (50°46′ 00.87 » N, 1°36′ 04.82 E) (4 March 2010). About 10 L of sediment of each condition was transported to the laboratory. The fractions samples dedicated to chemical analysis was frozen and stored at – 20 °C in order to determine granulometry, organic matter, metals and its bioavailability, PAHs (EPA’s 16 priority PAHs) and PCBs (7 congeners) contents . Beside this, it has been analyzed also heavy metals in sea bass juvenile’s gills. The remainder of the sediment was stored at -4°C during few days until the exposure experiment.
During the exposure time, one-third of the seawater in each tank was renewed manually every day, the photoperiod was maintained at 10L:14D, fish were fed 1% of the mean body weight once a day and water parameters were measured for each aquarium. In order to monitor growth and physiological performances of juveniles, 10 individuals per aquarium were removed at the end of experiment (t21) and rapidly transferred to the laboratory (within 2 h), anaesthetised, identified (tag), weighed, and measured for total length. For biomarker analyses after one week of exposure (t7) livers of 10 fish per aquarium (20 per condition) were sampled, frozen in nitrogen liquid and stored at -80°C. Muscles and gills on t21 were stored at −20 °C, respectively, for biochemical and metal bioaccumulation analysis. Daily observations were carried out every morning before the first food supply to assess fish mortality. In order to demonstrate experimentally sublethal effects of contaminated sediment on the physiological performances of sea bass juveniles, it was examined the correlative relationships between short term responses analyzing biomarkers (detoxification enzymes: EROD, GST and oxidative stress enzymes: catalase (CAT)) and long term responses measuring growth rate in length and weight, RNA-DNA ratio and relative condition index (Fulton’s K). In order to determine the impact of contaminated sediment on the immune system of fish juveniles, two immunocompetent organs: thymus and spleen were sampled for each condition at the end of experiment and stored at -80 °C before analysis. This party were analysed by Frauke Seeman, PhD student in LEMA laboratory (Laboratoire d’Ecotoxicologie-Milieux Aquatiques) of the University of Le Havre (France). In spleen, the number of melanomacrophage centers per area (MMC/µm2*10-6) (10 random cuts per 5 fish of each treatment) was investigated as the innate immune responses. The accumulation of melanomacrophage centers (MMC) is used as a general marker for stress in fishes as well as it is an indicator for enhanced phagocytoses of particles. In thymus, it is analyzed the adaptive (comparison of mature and immature T-cells in cortex and medulla, volume of thymus, cortex and medulla, cortex / medulla ratio) immune responses for the control and the most contaminated (Rouen) condition. Both thymi per fish (n=5) were examined and a section every 100th µm was studied.
P. globosa has a complex life cycle alternating free cell phase and colonial phase where cells are embedded into a mucilaginous matrix composed of polysaccharides. However, the colonial phase forms the bulk of Phaeocystis bloom. P. globosa and P. delicatissima are classified as harmful algae: P. globosa is undesirable mainly because of deleterious effects on tourism and recreational activities that it generates, due to the seafoam events characterizing the end of bloom; Pseudo-nitzschia spp. is harmful because of their ability to produce domoic acid, an amnesic shellfish poisoning (ASP) (Zingone and Enevoldsen 2000, Fehling et al., 2005; Sazhin et al., 2007). Seafoam originates from the Phaeocystis colony disruption (Lancelot 1995). However, its magnitude and intensity depends on both the Phaeocystis bloom magnitude and intensity, but also on wind speed and direction (Lancelot, 1995). Once colonial Phaeocystis cells have released from the muccopolysacccharidic matrix, this last then deteriorates and become a Transparent Exopolymer Particle (TEP, Mari et al., 2005). TEP are ubiquitous and abundant sticky substances in the oceans, gel-like particles, ranging from ∼1 to several hundred micrometers in size (Passow 2002b).
TEPs play a crucial role in aggregation of particles in the oceans (Alldredge et al., 1993; Mari et al., 2005), and therefore in flocculation processes (Seuront et al., 2006), and seafoam formation (Lancelot, 1995). Therefore, TEPs have been many studied, about their origin, distribution, and fate in the oceans (Passow, 2002a, b). However few studies have explored the effect of these “glue particles” on living organisms, especially fishes. To our knowledge, only zooplankton has been the subject of research to investigate any negative impact on phytoplankton ingestion. TEPs were detrimental for small calanoid copepod feeding (Dutz et al. 2005) whereas they were beneficial to euphausiids (Passow and Aldredge 1999). Net-clogging has been observed during Phaeocystis bloom (Savage, 1930; Hurley, 1982; Rogers and Lockwood, 1990; Huang et al., 1999), and fish mortality (Savage, 1930), as well as decline in shell fish growth and reproduction (Pieters et al., 1980; Davidson and Marchant, 1992; Prins et al., 1994; Smaal and Twisk, 1997). These negative impacts of Phaeocystis bloom on these living organisms could be due in part to the presence of TEPs.
Pseudo-nitzschia blooms are common in marine and estuarine environment and have deep socio-economic impacts on shellfish farming or harvesting and fishermen, but were recognized as being potentially toxic only 20 years ago (Bates et al. 1989; Klein et al., 2010). As it is reported in the literature, pinnate diatoms such as the small needle-shaped Nitzchia species are able to reveal abundant populations on Phaeocystis colonies and can be capable of producing the toxin as domoic acid on marine organisms such as mussels, filter-feeding shellfish or finfish (Fehling et al., 2005; Sazhin et al., 2007). Members of the genus Pseudo-nitzschia have been confirmed as producers of the neurotoxin DA which may enter the food chain from diatoms via filter-feeding shellfish or finfish. The toxin then accumulates to such levels that ingestion of the vectors by humans or other animals may lead to sickness or mortality in sea mammals, seabirds and humans due to ASP. Numerous laboratory-based toxicity studies were performed in order to characterize the neurotoxicity of DA has been observed in several animal species including humans, non-human primates, rodents, rats, fish, marine mammals and birds (Iverson et al., 1990; Tryphonas et al., 1990; Tasker et al., 1991 ; Lefebvre et al., 2001 ; Schaffer et al., 2006). To date, these harmful algae have become a focal point of numerous ecological studies and monitoring efforts in recent years and are subjected of various aspects of DA toxicology, pathology, bioaccumulation, and production in toxic diatom species. (Fehling et al., 2005; Schnetzer et al., 2007; Costa et al., 2010). However, the toxic effects of DA on the growth and condition indices of aquatic organisms, especially of fish juveniles, are scarcely investigated (Dizer et al., 2001; Tiedeken et al., 2005; Lefebvre et al., 2007).
The goal of the present study was to explore the effects either of the mucilage aggregates derived from P. globosa, predominantly under the form of Transparent Exopolymer Particles (TEP), as well as freshly foam formed, and P. pseudodelicatissima bloom on growth, survival and physiological performances of European sea bass juveniles (Dicentrarchus labrax, L., 1758), using various biological and biochemical indices.

Phytoplankton strains and culture conditions

P. globosa and P. pseudodelicatissima strains were first isolated from the eastern English Channel in autumn 2008 April 2009, respectively, pippeting cells from coastal water samples by Dr. Elsa Breton (LOG, Wimereux). Cultures were maintained in monospecific conditions in f/2 medium (Guillard and Ryther 1962; Guillard, 1975) at 12 ± 0.5°C with a 12:12 h light:dark cycle under a photon flux density of 400 µmol photons m-2 s-1 (Daylight HQIT-WD 250 W F, OSRAM). For the experiments, a first culture in 10-L and in 12-L Nalgene bottles, for P. globosa and P. pseudodelicatissima, respectively, were cultivated before to decant it into a 250 L and 100 L plexiglass batches at the hatchery unit of Aquanord, filled up with sea water of UV-treated to reduce the risk of pathogenic organism. At this time, culture of P. globosa and P. pseudodelicatissima were grown at 15 ± 0.5°C (same light conditions) for 2 weeks before starting experiments with sea bass juveniles, in order to allow sufficient TEP production from the degradation of Phaeocystis bloom decaying colonies formed on the mucoid polysaccharides fragments to use in the fish tanks (Mari et al. 2005). In addition, freshly accumulated foam and associated TEP were regularly collected at the hatchery pumping site and volume was assessed by gently placing the foam in a large bucket of 46 L. Care was taken not to press the foam so as to preserve its natural aspect and its apparent density. Since, domoic acid production from the diatom species P. pseudodelicatissima often starts at the onset of stationary phase of Pseudo-nitzschia delicatissima and peaks about one week later in batch cultures (Pan et al., 2001), this species was grown separately for 1 and 2 weeks, in order to test whether P. pseudodelicatissima physiological state (exponential versus death phase) affects on juvenile sea bass. For this reason, first batch (250 L) was served as exponential phase where second batch (100 L) was served for death phase that can produce domoic acid.

Phytoplankton experimental procedure

In order to explore the effects of P. globosa derived material and P. pseudodelicatissima on the growth, survival, and physiological performances of juvenile sea bass, two mesocoms experiments in larval rearing tanks of 1m3 were conducted from April to July 2009 at the Aquanord hatchery. For the first experiment, three treatments were applied in the experimental designs for 28 days (from 17 April to 15 May 2009): (1) control (250 individuals), (2) Phaeocystis and TEP at starting concentration of 1.16 108 cells L-1 and 7634.3 µg XG eq.l-1 (250 individuals in duplicate), (3) freshly 46 L of formed dense seafoam (250 individuals in duplicates). Seafoam was collected with plastic planter at the hatchery pumping station which generates seafoam through turbulence. For the second experiment, 3 treatments were also applied: (1) control (250 individuals), (2) P. pseudodelicatissima in exponential phase (Ps1 and Ps2) (250 individuals in duplicate), and (3) P. pseudodelicatissima in death phase (Ps3) (250 individuals), all cultures at an initial concentration of ~108 cell L-1. These three last treatments were run for 21 days (from 10 to 31 July 2009), except for treatment (3), which started one week later (from 17 to 31 July 2009).
The juvenile sea bass provided by Aquanord were 113 and 103 days old at the start of the experiments for P. globosa and P. Pseudodelicatissima, respectively. All tanks were supplied with sand-filtered and UV sterilized running seawater, thus permitting to keep mesocosm at in situ temperature (15 ±1°C) throughout the experiment. Tanks were illuminated according to the natural photoperiod at a photon density of 400 µmol m-2 s-1 (Daylight HQIT-WD 250 WF) in a 12:12 h light:dark cycle and gently aerated (compressed air), thus preventing settling of particles and maintaining oxygen saturation above 80%. Fish were fed daily ad libitum in the morning with commercial dry pellets (Skretting Ltd., France, Gemma PG 1.0), which contain 56% protein, 18%oil, 10%ash, 0.6% fibre, 1.3% total phosphorus, copper (8 ppm CuSO4), vitamin A (15000 IU/kg), vitamin D3 (1125 IU/kg), and vitamin E (225 IU/kg). In each tank, 25% of the seawater was renewed every two days, therefore, 50 L of P. globosa and P. pseudodelicatissima cultures added into the each tank to maintain high and stable TEP and P. pseudodelicatissima concentrations.

Sampling

Fifty juvenile sea bass were sampled at the start of the experiment to determine the initial fitness condition of fishes (t0) and to compare those with treatment conditions. Dissolved oxygen concentration (mg/L), temperature (°C), salinity, pH (with Hanna HI 9828 multiprobe) and turbidity (NTU) (with Eutech instruments, TN-100) were measured every 2 days before before seawater renewal and fish feeding. A 40 ml seawater was collected every two days before renewing seawater into the each tank to determine phytoplankton cell abundance for both experiences. TEP and P. pseudodelicatissima samples were preserved with formaldehyde (2% final concentration) and with Lugol-gluteraldehyde (1% final concentration), respectively. All samples were stored at 4°C in the dark until analysis (within one month). In order to monitor growth and physiological performances of juveniles, 30 individuals from each tank were removed on each week and at the end of experiments after being anaesthetised within 3-4 minutes by immersion in 0.32ml/l of 2-phenoxyethanol and stored at -20 °C for further analysis. Finally, fishes remaining in the experimental tanks were observed every two days early in the morning before the first food supply to assess fish mortality. In addition, daily mortality rate was calculated for each treatment during the experiment. In the case of any morphological abnormality or lesion indicating cannibalism, fish were photographed with a digital camera. At the end of Phaeocystis experiment, the X-rays were taken of some frozen fish for control and foam conditions. The fish were transferred to plastic bags prior to radiography, performed in a radiology clinic with mammography X-ray equipment. The radiographies of sea bass juveniles were analyzed using TNPC (5.0. NOESIS). The detection of skeletal abnormalities was carried out by visual inspection of the radiographies (Figure 14).
At the laboratory, juveniles were defrosted, measured for SL (standard length) and TL (total length) (near to 0.1 mm) and weighed (wet weight) (near to 0.001 g) to determine growth performance. Beside this, it is calculated the growth rate in length and weight, Fulton’s K condition index, RNA-DNA ratio and TAG-ST ratio for sea bass juveniles.

Table of contents :

Chapter I: General introduction
I.1. Coastal zones and its importance
I.2. Context of the Eastern English Channel
I.3. The quality of ecosystems and effects on organisms; the use of biological indicators
I.4. Fish as bioindicator of aquatic habitats
I.5. Thesis objectives and organisation
Chapter II: Methodology
II.1. In situ approach
II.1.1. Canche, Authie, Somme and Seine estuaries
II.1.2. Choice of European flounder (Platichthys flesus, L., 1758) as a biological model
II.1.3. Sampling strategies
II.1.4. Measurement of environmental parameters
II.1.4.1. Physicochemical parameters
II.1.4.2. Sediment sampling
II.1.4.3. Sediment analysis
II.1.4.3.1. Macrobenthos
II.1.4.3.2. Granulometry
II.1.4.3.3. Organic matter
II.1.5. Feeding analysis
II.2. Experimental approaches
II.2.1. Choice of European sea bass (Dicentrarchus labrax, L., 1758) as a biological model
II.2.2. Microcosm experience on sea bass juveniles (Dicentrarchus labrax, L. 1758) exposed to estuary sediment contamination
II.2.3. Mesocosm experiences on the effects of two toxic algal blooms: Phaeocystis globosa and Pseudo-nitzschia pseudodelicatissima on the physiological performance of sea bass juveniles (Dicentrarchus labrax, L., 1758)
II.2.3.1. Phytoplankton strains and culture conditions
II.2.3.2. Phytoplankton experimental procedure
II.2.3.3. Sampling
II.3. Other analysis of in situ, microcosm and mesocosm experiences
II.3.1. Sediment analysis
II.3.1.2. Metal analysis
II.3.1.3. Polycyclic aromatic hydrocarbons and Polychlorinated biphenyls analysis
II.3.1.4. Metal analysis: a) in fish and b) in fish gills
II.3.2. Biomarkers
II.3.2.1. Standard and samples preparations
II.3.2.2. Biotransformation (detoxification) enzymes
II.3.2.3.Antioxidant enzymes (oxidative stress biomarkers)
II.3.3. Fish mortality and physiological performance indicators
II.3.3.1. Daily mortality
II.3.3.2. Biological analysis
II.3.3.3. Specific growth rate in length and weight
II.3.3.4. Morphological condition index
II.3.3.5. Growth index
II.3.3.6. Nutritional indices
II.3.3.6.1. TAG/ST ratio
II.3.3.6.2. RNA/DNA ratio
II.3.4. Histology
II.3.5. Analysis of two algal blooms: Phaeocystis globosa and Pseudo-nitzschia pseudodelicatissima
II.3.5.1. Colorimetric method analysis for transparent exopolymer particles (TEP)
II.3.5.2. Sampling and determination of Pseudo-nitzschia pseudodelicatissima total abundances
II.4. Statistical analysis
Chapter III: Pollution impact on fish
III.1. Relating biological responses of juvenile flounder to environmental characteristics and sediment contamination of estuarine nursery areas
III.1.2. Introduction
III.1.3. Materials and Methods
III.1.3.1. Study area and sampling
III.1.3.2. Environmental variables
III.1.3.3. Sediment contaminant analysis
III.1.3.3.1. Metal analysis
III.1.3.3.2. PAHs and PCBs analysis
III.1.3.4. Fish metal analysis
III.1.3.5. Biological analysis
III.1.3.5.1. Growth and condition indices
III.1.3.6. Feeding analysis
III.1.3.7. Statistical analysis
III.1.4. Results
III.1.4.1. Environmental variables
III.1.4.2. Fish biological responses
III.1.5. Discussion
III.2. Effects of estuary sediment contamination on physiology, biochemical biomarkers and immune parameters in juvenile European sea bass (Dicentrarchus labrax, L., 1758)
III.2.1. Introduction
III.2.2. Materials and Methods
III.2.2.1. Sediment collection
III.2.2.2. Fish and experimental set up
III.2.2.3. Sediment analysis
III.2.2.4. Physiological parameters
III.2.2.5. Molecular biomarker analysis
III.2.2.6. Metal analysis in gills
III.2.2.7. Histology
III.2.2.8. Statistical analysis
III.2.3. Results
III.2.3.1. Environmental parameters
III.2.3.2. Physiological parameters
III.2.3.3. Metal concentrations in gills
III.2.3.4. Biomarker responses
III.2.3.5. Immune system responses
III.2.3.6. Correlation between parameters
III.2.4. Discussion
III.2.4.1. Estuarine sediment contamination and metal accumulation in fish gills
III.2.4.2. Physiological indicators
III.2.4.3. Biomarker responses
III.2.4.4. Immune system alterations
III.2.5. Conclusion
Chapter IV: Effects of algal bloom
IV.1. Effects of transparent exopolymer particles (TEP) derived from Phaeocystis globosa bloom on the physiological performance of European sea bass juveniles
IV.1.1 Introduction
IV.1.2. Materials and Methods
IV.1.2.1. TEP production from decaying algal cultures and foam
IV.1.2.2. Experimental set up and sampling strategy
IV.1.2.3. Determination of TEP concentrations
IV.1.2.4. Fish mortality and physiological performance
IV.1.2.5. Statistical analysis
IV.1.3. Results
IV.1.3.1. Physico-chemical variables
IV.1.3.2. TEP concentrations
IV.1.3.3. Fish mortality and physiological performance
IV.1.4. Discussion
IV.2. Does Pseudo-nitzschia pseudodelicatissima can be deleterious to the growth and condition of European sea bass juveniles?
IV.2.1 Introduction
IV.2.2. Materials and Methods
IV.2.2.1. Pseudo-nitzschia pseudodelicatissima algal cultures
IV.2.2.2. Experimental set up and sampling strategy
IV.2.2.3. Sampling and determination of Pseudo-nitzschia pseudodelicatissima total abundances
IV.2.2.4. Fish mortality and physiological performance
IV.3.2.5. Statistical analysis
IV.2.3. Results
IV.2.3.1. Physico-chemical variables
IV.2.3.2. Pseudo-nitzschia pseudodelicatissima total abundance
IV.2.3.3. Fish mortality and physiological performance
IV.2.4. Discussion
Chapter V: General Conclusion
V.1. Pollution impact on fish
V.2. Effects of algal bloom
V.3. Perspectives
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