Effects of in vivo chronic exposure to pendimethalin on EROD activity and antioxidant defenses in rainbow trout (Oncorhynchus mykiss).

Pendimethalin, an herbicide active substance frequently used in terrestrial systems, has detected in European aquatic ecosystems. Reliable indicators still need to be found in order to properly assess the impact of pesticides in fish. After an in vivo chronic exposure to pendimethalin, the detoxification process and the antioxidant defense system were assessed in 120 adult rainbow trout, Oncorhynchus mykiss. Four nominal exposure conditions were tested: control (C), 500 ng L(-1) (P500), 800 ng L(-1) (P800) and the commercial formulation Prowl(®) at 500 ng L(-1) (Pw500). Fish samples were made after a 28 day exposure period (D28) and after a fifteen day recovery period in clean fresh water (D43). At D28, ethoxyresorufin-O-deethylase (EROD) activity was not activated in liver in spite of the pendimethalin uptake in fish. At D43, EROD activity in fish exposed to the commercial product was lower than in control fish, which may be explained by the high presence of herbicide in fish (613±163 ng g bile(-1)). Furthermore, antioxidant defense responses were set up by trout in gills and liver following chronic exposure to 800 ng L(-1) of pendimethalin concentration. While the glutathione content (GSH) decreased in gills, it increased in liver associated with higher activities of glutathione peroxidase (GPx) and superoxide dismutase (SOD). These disturbances could lead to reactive oxygen species production and oxidative stress in the vital organs in fish. After fifteen days in clean water, while the SOD activity was restored, the GSH content and GPx activity were still significantly disturbed in fish exposed to pendimethalin in comparison with control. These significant differences between treatments in antioxidant defenses parameters measured, attesting to the irreversibility of the effects.

Effect of chronic exposure to pendimethalin on the susceptibility of rainbow trout, Oncorhynchus mykiss L., to viral hemorrhagic septicemia virus (VHSV).

In this study, the in vivo effects of chronic pollution by the active substance (AS) pendimethalin, a dinitroaniline herbicide, on the susceptibility of rainbow trout, Oncorhynchus mykiss L., to an experimental challenge with viral hemorrhagic septicemia virus (VHSV) were assessed. After four weeks of exposure to fresh water (C group) or 500 ng L(-1) of AS (P500 group), the fish were challenged by immersion in water containing 10(4) TCID(50) mL(-1) of VHSV. While exposure to pendimethalin was maintained throughout the experiment, mortalities were recorded during the 40 days post-infection (dpi) and organs were collected from dead fish for virological examination. At the end of the experiment, anti-VHSV antibodies and the classical pathway of complement activity were assessed in trout plasma. Exposure to pendimethalin significantly affected the distribution of cumulative mortality accelerating death in fish infected by VHSV. Pendimethalin appeared to decrease the Mean Time to Death (MTD) after virus treatment from 14.9 days (C-VHSV) to 10.2 days (P500-VHSV). Nevertheless, by the end of the experiment, differences in cumulative mortality were no longer observed between the two groups, which had reached the same stage (50 percent). Furthermore, a higher concentration of the virus was recovered from the pools of organs from the P500-VHSV group than the C-VHSV group. Moreover, at 40 dpi, although no significant difference was observed in the immune response between the two groups, more fish in the P500-VHSV group had set up an immune response by secreting antibodies than in the control viral group (C-VSHV).

Effects of the total replacement of fish-based diet with plant-based diet on the hepatic transcriptome of two European sea bass (Dicentrarchus labrax) half-sibfamilies showing different growth rates with the plant-based diet.

BACKGROUND: Efforts towards utilisation of diets without fish meal (FM) or fish oil (FO) in finfish aquaculture have been being made for more than two decades. Metabolic responses to substitution of fishery products have been shown to impact growth performance and immune system of fish as well as their subsequent nutritional value, particularly in marine fish species, which exhibit low capacity for biosynthesis of long-chain poly-unsaturated fatty acids (LC-PUFA). The main objective of the present study was to analyse the effects of a plant-based diet on the hepatic transcriptome of European sea bass (Dicentrarchus labrax). RESULTS: We report the first results obtained using a transcriptomic approach on the liver of two half-sibfamilies of the European sea bass that exhibit similar growth rates when fed a fish-based diet (FD), but significantly different growth rates when fed an all-plant diet (VD). Overall gene expression was analysed using oligo DNA microarrays (GPL9663). Statistical analysis identified 582 unique annotated genes differentially expressed between groups of fish fed the two diets, 199 genes regulated by genetic factors, and 72 genes that exhibited diet-family interactions. The expression of several genes involved in the LC-PUFA and cholesterol biosynthetic pathways was found to be up-regulated in fish fed VD, suggesting a stimulation of the lipogenic pathways. No significant diet-family interaction for the regulation of LC-PUFA biosynthesis pathways could be detected by microarray analysis. This result was in agreement with LC-PUFA profiles, which were found to be similar in the flesh of the two half-sibfamilies. In addition, the combination of our transcriptomic data with an analysis of plasmatic immune parameters revealed a stimulation of complement activity associated with an immunodeficiency in the fish fed VD, and different inflammatory status between the two half-sibfamilies. Biological processes related to protein catabolism, amino acid transaminations, RNA splicing and blood coagulation were also found to be regulated by diet, while the expression of genes involved in protein and ATP synthesis differed between the half-sibfamilies. CONCLUSIONS: Overall, the combined gene expression, compositional and biochemical studies demonstrated a large panel of metabolic and physiological effects induced by total substitution of both FM and FO in the diets of European sea bass and revealed physiological characteristics associated with the two half-sibfamilies.

Bioconcentration and immunotoxicity of an experimental oil spill in European sea bass (Dicentrarchus labrax L.).

The effects of Polycyclic Aromatic Hydrocarbons (PAHs) resulting from a water soluble fraction (WSF) of an Arabian crude oil were tested in vivo on the bioconcentration in muscles and on immune parameters in sea bass, Dicentrarchus labrax. After 15 days of acclimation, fish were acutely exposed (48 h) to the WSF of 25 g of oil, and then returned to clean sea water for a 15 day recovery period. PAH concentration in the WSF at the beginning of the exposure was estimated to 773±187 ng L⁻¹ similar to that observed in the marine environment after an oil spill. The WSF in the experimental system was composed by lightest PAH compounds and did not remain constant during the two days of exposure. Just after exposure to the WSF, a total mean concentration of 148±46 µg kg⁻¹ of PAHs was found in contaminated fish muscle, composed of parent and alkylated naphthalene compounds (86.5%), benzo[a]pyrene (10.1%) and benzo[b+k]fluoranthene (3.4%). In addition, a decrease of leucocytes counts due to a lymphopenia and granulopenia and an increase of the haemolytic activity of the alternative pathway (ACH50) were noted. After a 15 day recovery period, haematocrit was decreased whereas effects on the blood granulocytes of fish seemed to be reversible, contrary to the specific immune system and quality of flesh. In fact, contaminated fish had still less lymphocyte cells compared to controls fish and their flesh were still contaminated by naphthalene and benzo[a]pyrene creating a risk for human consumers.

In vivo effects of the soluble fraction of light cycle oil on immune functions in the European sea bass, Dicentrarchus labrax (Linné).

Hydrocarbons are major contaminants that may affect biota at various trophic levels in estuaries and coastal ecosystems. The effects of accidental pollution by light cycle oil (LCO), a refined product of heavy fuel oil, on bioaccumulation, depuration processes and immune-related parameters in the European sea bass, Dicentrarchus labrax, were investigated in the laboratory after 7 days of exposure and a 2-week recovery period. Exposure of fish to the soluble fraction of LCO (1600ngL(-1)) for 7 days led to the bioaccumulation of some polycyclic aromatic hydrocarbons (PAHs) in muscles: naphthalene, acenaphthene, fluorene, phenanthrene and anthracene. After 7 days of recovery period, half-elimination of naphthalene was reported in fish muscles due to facilitated diffusive loss by the epithelium and a faster elimination rate proven by the presence of a high level of naphthalene biliary metabolites. The other bioaccumulated molecules displayed a slower depuration rate due to their elimination by the formation of hydrophobic metabolites excreted through bile or urine. Three days after the beginning of the recovery period, each contaminated fish showed severe external lesions (tissue necrosis, suppurative exudates, haemorrhagic area). The hypothesis of a possible link with inflammatory phenomenon was supported by (i) an inversion of the leucocyte sub-population percentage, (ii) a significant up-expression in the spleen of the tumour necrosis factor alpha gene, (iii) a significant increase in ACH(50). Moreover, the lack of C3 gene regulation in the spleen suggested a non-renewal of this component. The reduction of phagocytic activity and lysozyme concentration reflected immune suppression. Finally, LCO toxicity in this fish was clearly demonstrated to be related to inflammatory reaction and immune depletion.

Effect of an experimental oil spill on vertebral bone tissue quality in European sea bass (Dicentrarchus labrax L.).

In order to identify biomarkers of oil pollution in fish we tested the effects of an experimental Light Cycle Oil (LCO) exposure on vertebral bone of sea bass, Dicentrarchus labrax L. A total of 60 adult fish were acclimated for fifteen days, then twenty were collected as controls (Day 0) while 40 were exposed to a soluble fraction of LCO (1136 ng L(-1) of ten Polycyclic Aromatic Hydrocarbons, PAHs) for seven days. Twenty of them were sampled at the end of the exposure period and the twenty last after a recovery period of fourteen days in clean seawater. Vertebral abnormalities were counted and bone mineralization, total bone area and bone density profiles were established for several post-cranial and caudal vertebrae. In sea bass, seven days of LCO exposure did not affect the frequency and severity of the vertebral abnormalities. No significant differences were observed in bone density and bone repartition (parameters of bone area profiles) between unexposed (Day 0), exposed (D7) and decontaminated (D21) fish. In contrast, bone mineralization of the vertebrae decreased in contaminated sea bass, but in a reversible way, which confirms a previous study in trout showing that this parameter is an early stress indicator. Our results suggest that vertebral bone mineralization could be used as a biomarker of PAH pollution in sea bass. It would be interesting to check this new biomarker in other teleost species exposed to various xenobiotics.

Immune effects of HFO on European sea bass, Dicentrarchus labrax, and Pacific oyster, Crassostrea gigas.

The European sea bass, Dicentrarchus labrax, and the Pacific oyster, Crassostrea gigas, were exposed to a soluble fraction of heavy fuel oil for 5 and 9 days, respectively. The organisms were then transferred to non-contaminated seawater for 1 month. The bioaccumulation and elimination of PAHs in contaminated tissues were dissimilar between species. In fish, acenaphthene and naphthalene were detected and naphthalene was still detectable 30 days after the beginning of the recovery period. In oysters, on the other hand, pyrene and phenanthrene were bioaccumulated and 14 days after exposure no more PAHs were detected. Concerning innate immune parameters, the increase of haemolytic activity of the alternative complement pathway in fish and the reduction of phenoloxidase activity in oysters endured, respectively, 1 and 2 weeks in contaminated organisms. This indicates that these two enzymatic cascades could be quite useful for monitoring pollution by oil.

EROD activity and antioxidant defenses of sea bass (Dicentrarchus labrax) after an in vivo chronic hydrocarbon pollution followed by a post-exposure period.

Chronic concentrations of polycyclic aromatic hydrocarbons (PAHs) have been commonly detected in international estuaries ecosystems. Reliable indicators still need to be found in order to properly assess the impact of PAHs in fish. After an in vivo chronic exposure to hydrocarbons, the enzymatic activity of 7-ethoxyresorufin O-deethylase (EROD) and the antioxidant defense system were assessed in sea bass, Dicentrarchus labrax. A total of 45 fish were exposed to the water-soluble fraction of Arabian crude oil, similar to a complex pollution by hydrocarbons chronically observed in situ, while 45 other control fish sustained the same experimental conditions in clean seawater. Fish samples were made after a 21-day exposure period and after a 15-day recovery period in clean fresh water. Throughout the experiment, liver EROD activity was significantly higher in contaminated fish than in control fish. In addition, nonenzymatic (total glutathione) and enzymatic (GPx, SOD, and CAT) antioxidant defense parameters measured in liver were not significantly different in fish. Furthermore, in gills, glutathione content had significantly increased while SOD activity had significantly decreased in contaminated fish compared to controls. On the other hand, CAT and GPx activities were not affected. Chronic exposure to PAHs disturbing the first step (SOD) and inhibiting the second step (GPx and CAT) could induce oxidative stress in tissues by the formation of oxygen radicals. After the postexposure period, there was no significant difference between control and contaminated fish in any of the antioxidant defense parameters measured in gills, attesting to the reversibility of the effects.

Effects of in vivo chronic exposure to pendimethalin/Prowl 400® on sanitary status and the immune system in rainbow trout (Oncorhynchus mykiss).

The in vivo effects of the herbicide active substance (AS) pendimethalin (alone and with Prowl 400® adjuvant) were evaluated on sanitary status i.e. the health status with regard to chemical pollution and on the physiological state via the immune system in rainbow trout, Oncorhynchus mykiss. Four nominal exposure conditions were tested: i) control (C), ii) AS at 500 ng L(-1) (P500), iii) AS at 800 ng L(-1) (P800) and iv) Prowl 400® at 500 ng L(-1) (Pw). After a 28 day exposure period (D28), 10 fish were sampled for each condition and 10 other after a 15 day recovery period in clean fresh water (D43). Pendimethalin concentrations in the exposure water and muscles were followed. White blood cell counts, differential leucocyte counts, cell mortality and phagocytosis activity were measured. Haemolytic alternative complement activity, lysozyme concentration and stress parameters were analyzed. The resulting concentration of pendimethalin in the exposure water was lower than the expected concentration. At D28, the concentration quantified in the contaminated fish was negligible in comparison with the Reference Dose for Oral Exposure estimated by US-EPA's Integrated Risk Information System. Leucopenia was noted in all contaminated fish. A decrease in phagocytosis activity and ACH(50) was also observed in contaminated fish by P800 and Pw. Disturbed lysozyme activity was noted only in fish exposed to Pw. Furthermore, during exposure to a similar concentration of pendimethalin, the commercial product seemed to be more immunotoxic than the AS alone. Finally, at D43, the effects proved reversible for sanitary status while immunity was still disturbed in contaminated fish by P800 and Pw.

Effects of in vivo chronic hydrocarbons pollution on sanitary status and immune system in sea bass (Dicentrarchus labrax L.).

Following the development of an experimental system to expose adult fish to low and stable concentration of pollutant over a prolonged period, the in vivo effects of hydrocarbons on sanitary status, i.e. the health status of fish with regard to chemical pollution, and immune system in sea bass, Dicentrarchus labrax were assessed. A total of 90 fish were acclimated for 15 days, then 45 fish were exposed to the water soluble fraction (WSF) of Arabian crude oil, similar to a complex pollution by hydrocarbons chronically observed in situ in estuaries, while the 45 other control fish sustained the same experimental conditions in clean seawater. After 21 days of exposure, 30 contaminated and control fish were sampled, then 30 other fish were collected after a 15 day recovery period in clean sea water. PAH concentrations in crude oil, WSF, muscles and bile were measured by gas chromatography coupled with mass spectrometry analysis. White blood cell counts and differential leucocyte counts were determined by classical haematology methods. Cell mortality and phagocytosis activity of leucocytes were analyzed by flow cytometry. Haemolytic alternative complement activity and stress parameters were analyzed in blood plasma by spectrophotometry. After a 21 day exposure period to a mixture of 41 parent/alkylated-PAHs (835 ± 52/85 ± 1 5 ng L(-1)). Fish flesh was contaminated by a bioconcentration of naphthalene very closed to the Reference Dose for Oral Exposure estimated by US-EPA's Integrated Risk Information System, causing a potential risk for human consumers. A leucopenia due to a lymphopenia, a rise in leucocyte mortality and a decrease in phagocytosis activity were noted in contaminated fish compared to controls. All these results may be explained by the damage to membrane cells integrity by uptake of PAHs and suggested an impairment of specific and nonspecific immune systems. After a 15 day recovery period, effects were reversible for sanitary status and an offset in immunity was noted by a significant increase in leucocytes in contaminated fish compared to controls.

In vivo effects of LCO soluble fraction on immune-related functions and gene transcription in the Pacific oyster, Crassostrea gigas (Thunberg).

The effects of a soluble fraction of light cycle oil (LCO) on haemocyte parameters, phenoloxidase (PO) activity and mRNA expression of immune-related genes, in the Pacific oyster, Crassostrea gigas, were tested after seven days of exposure and two weeks of recovery period. Five polycyclic aromatic hydrocarbons (PAHs) out of ten detected in tank water had bioaccumulated at the end of the contamination period. The concentration of PAHs in oyster tissues decreased during the recovery period and 14 days after the exposure, 69% of bioaccumulated PAHs were detected in contaminated oysters. The exposure induced severe oyster mortality (21%), external and internal green colouration of the shell and a significant decrease of PO activity. The mRNA expression of several genes was altered. As a conclusion, a modulation of immune-related parameters was demonstrated using three different approaches, namely cellular (flow cytometry), biochemical (spectrophotometry) and genomics (gene transcription) in oysters after contact with soluble fraction of LCO.