Copper and cadmium effects on growth and extracellular exudation of the marine toxic dinoflagellate Alexandrium catenella: 3D-fluorescence spectroscopy approach
Introduction
Dinoflagellates are responsible for 75% of the marine harmful algal blooms (HABs) (Chan et al., 2002). They may induce various syndromes such as: The Neurotoxic Shellfish Poisoning (NSP), the Paralytic Shellfish Poisoning (PSP) and the Ciguatera Fish Poisoning (CFP) (Glibert et al., 2005). Over the last decades, the frequency and the distribution of HABs have increased in marine coastal ecosystems (Sellner et al., 2003). As a result, although it has not yet been really demonstrated, eutrophication forced by anthropogenic inputs could partially explain the HAB expansion (Glibert et al., 2005). Among the dinoflagellates, Alexandrium genus is one source of HABs. Blooms of Alexandrium have been reported in various marine waters, such as the northwest Mediterranean (Penna et al., 2005), the coastal waters of Chile (Cordova and Muller, 2002), Tunisia (Turki and Balti, 2005), Algeria (Frehi et al., 2007), and in France (Thau lagoons) (Lilly et al., 2002).
In a polluted coastal marine ecosystem (Toulon Bay, France), the dinoflagellates were predominant relatively to other phytoplankton groups like diatoms (Jean et al., 2005). In this ecosystem, where metal contamination has been reported (Tessier et al., 2011), Alexandrium bloomed at some periods of the year (Jean et al., 2006), suggesting that this genus could be tolerant to metal contamination.
In marine ecosystems, metal cations are complexed by natural inorganic and organic ligands. This complexation leads to a competition between natural ligands present in organic matter, inorganic anions, and membrane transport proteins for binding metal cations, which change the bioavailability of the metal for the cell (Sunda and Hunstman, 1998). As a result, complexation of metals by natural organic ligands often decreases their toxicity in aquatic environments (Moffett et al., 1990). Hence, metal toxicity towards organisms does not only depend on the total metal concentration, but essentially on the metal cation chemical speciation, which can be calculated on the basis of the free ion activity model (Sunda and Guillard, 1976). The natural organic ligands capable of metal complexation can have a biological origin, as the phytochelatine, which is produced by phytoplankton for the metal detoxication (Le Faucheur et al., 2005). Moreover, organic ligands released by phytoplankton can modify trace metal speciation (Vasconcelos et al., 2002). Therefore, the studies characterizing the DOM released by phytoplankton under stress metal conditions should bring a better knowledge of the DOM implication in the metal availability for phytoplankton. This DOM can be monitored by 3D-fluorescence spectroscopy approach.
The fluorescence spectroscopy technique has been applied to characterize DOM coming from various environments. Successive advances in fluorescence analysis have improved the method, from 1-dimension (Vodacek et al., 1997), and then, 2-dimensions (Lloyd, 1971), to a powerful technique based on the Excitation–Emission Matrices of fluorescence (EEMs). Studies on Fluorescent Dissolved Organic Matter (FDOM) have been carried out from samples collected in many types of aquatic ecosystems, such as rivers (Mounier et al., 1999), estuaries (Jaffé et al., 2004), benthic (sediment pore water) or polluted areas (Burdige, 2004), biological cultures (Parlanti et al., 2000) and marine ecosystems (Kowalczuk et al., 2005). To improve identification of fluorescent compounds, the PARAFAC algorithm is applied as a statistical EEMs treatment (Bro, 1997). Thanks to PARAFAC, the FDOM can be mathematically identified, leading to the optimal separation of fluorescent components. Other algorithms have helped to non linear corrections: the elimination of Rayleigh and Raman scatterings (Zepp et al., 2004), the statistical method Concordia (Bro and Kiers, 2003), and an inner filter effect correction using the Controlled Dilution Approach (CDA) (Luciani et al., 2009). PARAFAC has been successfully used to investigate the FDOM origins in aquatic samples from estuarine (Luciani et al., 2008), marine (Murphy et al., 2008) and freshwater ecosystems (Holbrook et al., 2006), and it also has been applied to soil-extracted DOM (Banaitis et al., 2006). However, to our knowledge, no study mentions the use of PARAFAC to characterize FDOM released by phytoplankton grown in cultures contaminated by metals.
The first objective of this study is to examine the effects of the two trace metals, copper and cadmium, on the growth of Alexandrium catenella, in order to explore the physiological response of this dinoflagellate to metal stress. A second objective is to characterize the FDOM released by A. catenella cultures submitted to increasing Cu and Cd concentrations. This characterization, based on DOC analysis, and on FDOM 3D-fluorescence results treated by PARAFAC, aimed at investigating the links potentially existing between FDOM exudates and metal contamination as a result of A. catenella stress reaction.
Section snippets
Alexandrium catenella cultures
A strain of A. catenella (ACT03) was isolated from the Thau lagoon (France) in 2003, by the Laboratory Ecologie des Systèmes Marins Côtiers (ECOSYM UMR 5119, Université Montpellier 2, CNRS, Ifremer and IRD). The cultures from this strain were maintained in f/2 medium prepared in 0.2 μm polycarbonate filtered and sterilized natural low contaminated seawater. The cultures were grown at +20 °C in sterile 250 mL flasks exposed to 135 μmol photons m−2 s−1 during light:dark cycle of 12 h:12 h.
To study the
Effects of metals on A. catenella growth kinetics
Growth of the metal contaminated cultures showed quantitative perturbations in comparison with the control (Fig. 3). For low Cu2+ concentration, ΔD was decreased from 7% at pCu2+ = 9.00 (Cu3) to 38% at pCu2+ = 8.15 (Cu12) (Fig. 1). At higher concentrations, beyond pCu2+ = 7.96 (Cu16), ΔD decreased drastically (100%), indicating that no growth was observed. For Cd, ΔD was about 30% at pCd2+ = 7.88 (Cd3). Concentration of free metals reaching around 1.30 × 10−8 M induced 100% of decrease in cell
Growth of A. catenella and DOC/Cell release
The growth of A. catenella in f/2 culture medium followed a similar pattern to that described for other dinoflagellate species (Juhl, 2005). Reduction of the A. catenella growth after metal contamination, recorded in this study, was in agreement with previous studies conducted on other dinoflagellates (Lage et al., 1994). Here, we have expressed the decrease in cell concentration through some variables such as the net growth rate (μ) and the specific doubling rate (K) (Landry and Hassett, 1982
Conclusions
The main objective of this study was to determine, for the first time, the characterization of exudates from the toxic dinoflagellate A. catenella grown in metal contaminated medium. Results revealed that under metal stress conditions, the development of A. catenella was perturbed from the ion free concentration of: 10−8 M for Cu and 10−7 M for Cd.
We report an increase in organic matter exudates by cells under metal stress, at the end of the exponential phase. The release of organic molecules
Acknowledgments
This research was supported by the Conseil Général du Var (CG), Toulon Provence Méditerranée (TPM) and ARCUS CERES project (Région PACA-MAE). We are deeply indebted to Yves COLLOS and Estelle MASSERET from the Laboratory ECOSYM UMR 5119 of the Montpellier 2 University, for providing us with the A. catenella strain ACT03 used in the study.
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