Karine Pichavant-Rafini

Impacts of mixtures of herbicides on molecular and physiological responses of the European flounder Platichthys flesus.

The widespread use of pesticides results in a growing contamination of the aquatic environment. The effects of (1) a simple mixture of a glyphosate-based formulation and AMPA (Aminomethylphosphonic acid--a primary metabolite of glyphosate) and of (2) a more complex mixture of herbicides (glyphosate/AMPA/mecoprop/acetochlor/2,4D) were explored on the molecular and physiological responses of the European flounder Platichthys flesus, considering a long-term and environmentally realistic contamination. Molecular responses were identified using suppression subtractive hybridization on liver samples: the level of gene transcription was significantly different between contaminated fishes vs control ones for 532 sequences, after a 62-day contamination. Among them, 222 sequences were identified by homology with data-based sequences; they encoded several metabolic pathways including: methionine and lipid metabolism, immunity, protein regulation, coagulation and energetic metabolism. Expression pattern of nine transcripts in the liver was confirmed by real-time PCR. The molecular study underlined that potential markers of liver injury were expressed for both mixtures, in particular betaine homocysteine methyl transferase and chemotaxin. Physiological responses were analysed considering blood parameters and condition factor; after the two months contamination period; no significant physiological difference was detected between contaminated and control fish.

Innate immunity and antioxidant systems in different tissues of sea bass (Dicentrarchus labrax) exposed to crude oil dispersed mechanically or chemically with Corexit 9500

The aim of the study was to evaluate effects of chemically dispersed oil by the dispersant Corexit 9500 on innate immunity and redox defenses in a marine model fish. Sea bass (Dicentrarchus labrax) were exposed 48h to four experimental conditions: a control group (C), a group only exposed to the dispersant (D; 3.6mg/L) and two groups exposed to 80mg/L oil mechanically or chemically dispersed (MD; CD). Alternative pathway of complement activity and lysozyme concentration was measured in plasma in order to evaluate the general fish health status. Total glutathione, glutathione peroxidase (GPX) and superoxide dismutase (SOD) were analyzed in gills, liver, brain, intestine and muscle. The chemical dispersion induced a significant reduction of lysozyme concentration when compared to the controls, and the hemolytic activity of the alternative complement pathway was increased in mechanical and chemical dispersion. The analysis of SOD, GPX and total glutathione showed that antioxidant defenses were activated in liver and reduced in intestine and brain. Dispersant was also responsible for an SOD activity inhibition in these two last tissues, demonstrating a direct effect of this dispersant on reactive oxygen species homeostasis that can be interpreted as a signal of tissue toxicity. This result should raise concern about the use of dispersants and show that they can lead to adverse effects on marine species.

Effect of dispersed crude oil on cardiac function in seabass Dicentrarchus labrax.

In this study, the impact of dispersed oil was assessed in Dicentrarchus labrax, a fish frequently used as an oil contamination indicator species. Fish were exposed for 48h to (mechanically and chemically) dispersed oil and dispersant alone. The impact of these exposure conditions was assessed on cardiac function by measuring (i) the contraction strength, the contraction and the relaxation speeds (ii) the cardiac energy metabolism using respirometry on permeabilized cardiac fibers. Compared to control, the increase of polycyclic aromatic metabolites observed in the bile indicated oil contamination in our fish. Following 48h of oil exposure at realistic oil concentrations, alterations of cardiac performances were observed. A decrease in contraction strength, contraction and relaxation speeds was observed in the presence of oil without effect of dispersant on these three parameters. Looking at cardiac energy metabolism, dispersant alone decreases all the activity of the respiratory chain and increases the proton leak. From these results, it appears that the observed decrease in cardiac performance in fish exposed to oil was not linked to a decrease in energy availability.

Acute toxicity of chemically and mechanically dispersed crude oil to juvenile sea bass (Dicentrarchus labrax): Absence of synergistic effects between oil and dispersants

The goal of the present experiment was to assess the relative acute toxicities of mechanically and chemically dispersed oil (crude Arabian Light) in controlled conditions. Juvenile sea bass (Dicentrarchus labrax) were exposed to 4 commercial formulations of dispersants (Corexit EC9500A, Dasic Slickgone NS, Finasol OSR 52, Inipol IP 90), to mechanically dispersed oil, and to the corresponding chemical dispersions. Acute toxicity was evaluated at 24 h, 48 h, 72 h, and 96 h through the determination of 10%, 50%, and 90% lethal concentrations calculated from measured total petroleum hydrocarbon (TPH) concentrations; Kaplan-Meyer mortality analyses were based on nominal concentrations. Animals were exposed to the dissolved fraction of the oil and to the oil droplets (ranging from 14.0 μm to 42.3 μm for the chemical dispersions). Kaplan-Meyer analyses demonstrated an increased mortality in the case of chemical dispersions. This difference can be attributed mainly to differences in TPH, because the chemical lethal concentrations were not reduced compared with mechanical lethal concentrations (except after 24 h of exposure). The ratios of lethal concentrations of mechanical dispersions to the different chemical dispersions were calculated to allow direct comparisons of the relative toxicities of the dispersions. The results ranged from 0.27 to 3.59, with a mean ratio close to 1 (0.92). These results demonstrate an absence of synergistic effect between oil and chemical dispersants in an operational context.

Growth and immune system performance to assess the effect of dispersed oil on juvenile sea bass (Dicentrarchus labrax)

The potential impact of chemically and mechanically dispersed oil was assessed in a model fish of European coastal waters, the sea bass Dicentrarchus labrax. Juvenile sea bass were exposed for 48h to dispersed oil (mechanically and chemically) or dispersants alone. The impact of these exposure conditions was assessed using growth and immunity. The increase observed in polycyclic aromatic hydrocarbon metabolites in bile indicated oil contamination in the fish exposed to chemical and mechanical dispersion of oil without any significant difference between these two groups. After 28 days of exposure, no significant differences were observed in specific growth rate,apparent food conversion efficiency and daily feeding). Following the oil exposure, fish immunity was assessed by a challenge with Viral Nervous Necrosis Virus (VNNV). Fish mortality was observed over a 42 day period. After 12 days post-infection, cumulative mortality was significantly different between the control group (16% p≤0.05) and the group exposed to chemical dispersion of oil (30% p≤0.05). However, at the end of the experiment, no significant difference was recorded in cumulative mortality or in VNNV antibodies secreted in fish in responses to the treatments. These data suggested that in our experimental condition, following the oil exposure, sea bass growth was not affected whereas an impact on immunity was observed during the first days. However, this effect on the immune system did not persist over time.

Activation of the MAPKs ERK1/2 by cell swelling in turbot hepatocytes

Background information. Activation of MAPKs (mitogen‐activated protein kinases), in particular ERK1/2 (extracellular‐signal‐regulated kinase 1/2), has been reported to take place in a large variety of cell types after hypo‐osmotic cell swelling. Depending on cell type, ERK1/2 phosphorylation can then serve or not the RVD (regulatory volume decrease) process. The present study investigates ERK1/2 activation after aniso‐osmotic stimulations in turbot hepatocytes and the potential link between phosphorylation of these proteins and RVD.

Acid-Base Balance and Ammonia Loading in the Siberian Sturgeon Acipenser baerii, Exposed to High Concentrations of Ammonia

The ammonia concentration was studied in the blood of the Siberian sturgeon, Acipenser baerii, with and without high concentration in water. In control conditions, the ammonia excretion was mainly due to NH3 diffusion according to the partial pressure gradient. So the distribution of the total ammonia followed the pH gradient between water and blood. After exposure to high concentration of ammonia, the blood concentration increased quickly during the early hours and a metabolic alkalosis appeared. PNH3 was stabilized after less than 24-h exposure, while the total ammonia concentration increased continuously as blood pH returned to initial values. If we take into account the real pH in the branchial boundary layer and not in the water of the circuit to calculate PNH3, we could conclude that the evolution of the blood ammonia concentration could be explained by NH3 diffusion along its partial pressure and that the ammonia exchanges by simple diffusion were dominant. However, whatever the ammonia concentration in water, the fish quickly reached a PNH3 equilibrium with the water and seemed incapable of preventing the invasion of the blood by ammonia. That also explained the initial metabolic alkalosis. But these results did not allow to attribute the ammonia toxicity to NH3.

Oxygen Demand in Sturgeon Farming

O2 is paramount in aquaculture to ensure growth and welfare in fish. But the diffusion of O2 in water layers is very difficult, and suffocation may threaten the fishes continuously in intensive farming.

Some Basic Methods in Respiratory Physiology Studies Applied in the Siberian Sturgeon

Numerous studies have shown that extracellular acid-base status in fish is greatly sensitive to small variations of temperature, PCO2, and bicarbonate system in water. Whereas control of temperature and oxygenation of the environmental water is a usual practice in physiological studies on fish and even though water is directly in contact with the gills, the main site of acid-base and ionic regulations, the environmental acid-base status (pH, alkalinity, PCO2) has rarely been paid much attention.

Effects of Exposure to Ammonia on Plasma, Brain and Muscle Concentrations of Amino Acids and Adenyl Nucleotides in the Siberian Sturgeon, Acipenser baerii

Neurological disorders appear extremely rapidly for elevated doses of ammonia in Siberian sturgeon baerii. To explain this phenomenon, the effects of ammonia during a 72-h exposure in control conditions and for a lethal and a sublethal doses were examined on the concentration of free amino-acids and adenyl nucleotides in the Siberian sturgeon plasma, muscle and brain.