Partial characterization of the lipidome of the cold-water scallop, Chlamys islandica.

Fingerprinting of the main lipid components of the digestive gland of the Icelandic scallop-Chlamys islandica-has been performed by ultra-high-performance liquid chromatography coupled with time of flight high-resolution mass spectrometry, UHPLC-HRMS/ToF. This method allowed the identification of 224 lipids, including phosphatidylcholines (PC), plasmanyl (PC-O)/plasmenyl (PC-P) phosphatidylcholines, lyso-phosphatidylcholines (LPC), and their plasmanyl/plasmenyl forms (LPC-O/LPC-P). Diacylglycerols (DG), triacylglycerols (TG), and cholesteryl esters (CE) were the neutral lipids (NL) analyzed. While all of the lipids showed a strong seasonal dependence in terms of quantity, only NLs presented significant qualitative changes. Principal component analysis (PCA) of TG and DG profiles evidenced a prevalence of low unsaturated TGs and DGs in spring, which were replaced by species with a higher degree of unsaturations in summer. In autumn, long and highly unsaturated TGs constitute the lipid fraction of the digestive gland of the scallop, while DG species offer a mixed profile. This study contributes to the characterization and the elucidation of the lipidome of Chlamys islandica and provides baseline data for further study of the effects of pollutants on the lipidome of the Icelandic scallop, often used as a sentinel species in biomonitoring programs.

Molecular isolation and characterization of the kisspeptin system, KISS and GPR54 genes in roach Rutilus rutilus.

The reproduction of vertebrates is regulated by endocrine and neuro-endocrine signaling molecules acting along the brain-pituitary-gonad (BPG) axis. The understanding of the neuroendocrine role played in reproductive function has been recently revolutionized since the KiSS1/GPR54 (KiSS1r) system was discovered in 2003 in human and mice. Kisspeptins, neuropeptides that are encoded by the KiSS genes, have been recognized as essential in the regulation of the gonadotropic axis. They have been shown to play key roles in puberty onset and reproduction by regulating the gonadotropin secretion in mammals while physiological roles in vertebrates are still poorly known. In order to provide new knowledge on basic reproductive physiology in fish as well as new tools to assess impacts of endocrine disrupting compounds (EDCs), the neurotransmitter system, i.e., gene/receptor, KISS/GPR54 might constitute an appropriate biomarker. This study provides new understandings on the neuroendocrine regulation of roach reproduction as well as new molecular tools to be used as biomarkers of endocrine disruption. This work completes the set of biomarkers already validated in this species.

Spatial and temporal impacts of the Skjervøy harbour diesel spill on native population of blue mussels: A sub-Arctic case study.

This work was designed to investigate biological impacts on blue mussels (Mytilus edulis spp) after being exposed to diesel spill. On December 2013, an 180,000-litre accidental acute diesel spill was reported in a small harbour of northern Norway (Skjervøy). In order to assess the biological effects on the wild population of blue mussels, bivalves were collected at three different locations: at the oil-spill spot, at the other side of the harbour (opposite the oil-spill area), and in an uncontaminated site. Body burden and seawater samples were collected from a few days up to five months after the diesel spill. Biomarkers of oxidative stress and immunological effects were assessed in the blue mussels digestive glands. Our findings reported significant modulation of GST (detoxification), SOD (antioxidant response) and MDA (lipid peroxidation) in bivalves exposed to diesel with a similar response at two and five months after the spill. Laccase-type enzyme also highlighted an important aspect in terms of biomarker response of the immune function. Overall, our study demonstrated that some biomarkers returned to basal levels a few months after the diesel spill. Consequently, it highlighted the usefulness of normalised tools and guidelines for biomonitoring strategies after a diesel spill.

Endocrine disruption effects in male and intersex roach (Rutilus rutilus, L.) from French rivers: An integrative approach based on subcellular to individual responses.

Among the chemicals found in the aquatic environment, some have been reported to interact with the endocrine system of organisms and have been identified as endocrine disrupting compounds. In this study, we have assessed the biological effects due to environmental estrogenic compounds in adult male roach (Rutilus rutilus). Wild fish were collected in the Seine River, and one of its affluent, the Epte River in Normandy, France and impacts on endocrine and reproductive functions have been assessed. This study provides innovative data on the intersex condition in wild fish that are crucial to understand estrogenic contamination impacts and maintenance of sustainable fish population. The biomarker responses found in fish from contaminated sites included high plasmatic vitellogenin concentrations, altered steroid levels, histopathological and up to 50% intersex fish among the male. However, the overall sex ratio of the roach population indicated sign of feminization with some sites showing only 20% of males. The chemical organic contamination at the different location sites has been characterized by the levels of PAHs, PCBs and phthalates and total estrogenic activity was measured by the YES in vitro assay.

Biological effects of marine diesel oil exposure in red king crab (Paralithodes camtschaticus) assessed through a water and foodborne exposure experiment.

Shipping activities are expected to increase in the Arctic Seas. Today, the majority of vessels are using marine diesel oil (MDO) as propulsion fuel. However, there is a general lack of knowledge of how cold-water marine species respond to acute exposures to MDO. Arctic red king crabs (Paralithodes camtschaticus) were exposed to mechanically dispersed MDO in a flow-through exposure system for one week followed by three weeks of recovery. Observations of increased movements in exposed crabs were interpreted as avoidance behaviour. Further, glutathione peroxidase activity increased in high exposed crab, the catalase activity showed an insignificant increase with exposure, while no differences between groups were observed for lipid peroxidation and acetylcholinesterase activity. After three weeks of recovery in clean seawater, polycyclic aromatic hydrocarbons concentrations in the crabs were significantly reduced, with no specific biomarker responses in exposed groups compared to the control. The results suggest that effects from instantaneous MDO spill only will have short-term effects on the red king crab.

First evidence of marine diesel effects on biomarker responses in the Icelandic scallops, Chlamys islandica.

The decrease of ice cover in polar areas is expected to lead to an increase in ship traffic. In this context, the risk associated with exposure of the aquatic environment to oil-related chemicals from spills and/or accidental fuel discharges from ships will increase in the near future, potentially causing negative impacts on sensitive Arctic species. This study investigated for the first time the biomarker responses of the Icelandic scallops (Chlamys islandica) to marine diesel exposure. Antioxidant response, neurotoxic effect, gonad maturation, and lipid content were assessed in male and female scallops, Chlamys islandica after a 7-day exposure to marine diesel. At the end of exposure, results showed an inhibition of acetylcholinesterase activity in Icelandic scallops exposed to high concentration of diesel. At the end of exposure, results showed an increase in naphthalene residues in hemolymph, together with an inhibition of AChE activity in Icelandic scallops exposed to high concentration of diesel. In addition, alteration of the escape capacity was observed suggesting effects on behavior responses. Overall, this study contributes to the better knowledge of physiological effects of marine diesel in Arctic marine organisms.

Effects of chlordecone on 20-hydroxyecdysone concentration and chitobiase activity in a decapod crustacean, Macrobrachium rosenbergii.

Chlordecone (CLD) is an organochlorine insecticide abundant in aquatic environment of the French West Indies. However, few studies have investigated its impact on freshwater invertebrates. Whereas CLD is suspected of inducing endocrine disruption, this work aimed to study the effects of environmentally relevant concentrations of CLD on the 20-hydroxyecdysone (20-HE) hormone concentration and on the chitobiase activity, both having key roles in the molting process of crustaceans. In addition, the bioaccumulation of CLD was measured in the muscle tissue of Macrobrachium rosenbergii to underline potential dose-response relationship. The results have shown that CLD was bioaccumulated in exposed organisms according to a trend to a dose-response relationship. Moreover, it was observed that CLD decreased the 20-HE concentration in exposed prawns when compared to control, whatever the duration of exposure, as well as it inhibited the chitobiase activity after 30days of exposure. The present study indicates that CLD could interfere with molting process of M. rosenbergii by disturbing the 20-HE concentration and the activity of chitobiase, suggesting consequences at the long term on the shrimp development. This study also confirmed that CLD could be an endocrine disruptor in decapod crustaceans, as it was already observed in vertebrates.

Biological effects of mechanically and chemically dispersed oil on the Icelandic scallop (Chlamys islandica).

This study aimed to simulate conditions in which dispersant (Dasic NS) might be used to combat an oil spill in coastal sub-Arctic water of limited depth and water exchange in order to produce input data for Net Environmental Benefit Analysis (NEBA) of Arctic and sub-Arctic coastal areas. Concentration dependent differences in acute responses and long-term effects of a 48h acute exposure to dispersed oil, with and without the application of a chemical dispersant, were assessed on the Arctic filter feeding bivalve Chlamys islandica. Icelandic scallops were exposed for 48h to a range of spiked concentrations of mechanically and chemically dispersed oil. Short-term effects were assessed in terms of lysosomal membrane stability, superoxide dismutase, catalase, gluthatione S-transferases, glutathione peroxidases, glutathione reductase, glutathione, total oxyradical scavenging capacity, lipid peroxidation and peroxisomal proliferation. Post-exposure survival, growth and reproductive investment were followed for 2 months to evaluate any long-term consequence. Generally, similar effects were observed in scallops exposed to mechanically and chemically dispersed oil. Limited short-term effects were observed after 48h, suggesting that a different timing would be required for measuring the possible onset of such effects. There was a concentration dependent increase in cumulative post-exposure mortality, but long-term effects on gonadosomatic index, somatic growth/condition factor did not differ among treatments.

Sensitivity of the deep-sea amphipod Eurythenes gryllus to chemically dispersed oil.

In the context of an oil spill accident and the following oil spill response, much attention is given to the use of dispersants. Dispersants are used to disperse an oil slick from the sea surface into the water column generating a cloud of dispersed oil droplets. The main consequence is an increasing of the sea water-oil interface which induces an increase of the oil biodegradation. Hence, the use of dispersants can be effective in preventing oiling of sensitive coastal environments. Also, in case of an oil blowout from the seabed, subsea injection of dispersants may offer some benefits compared to containment and recovery of the oil or in situ burning operation at the sea surface. However, biological effects of dispersed oil are poorly understood for deep-sea species. Most effects studies on dispersed oil and also other oil-related compounds have been focusing on more shallow water species. This is the first approach to assess the sensitivity of a macro-benthic deep-sea organism to dispersed oil. This paper describes a toxicity test which was performed on the macro-benthic deep-sea amphipod (Eurythenes gryllus) to determine the concentration causing lethality to 50% of test individuals (LC50) after an exposure to dispersed Brut Arabian Light (BAL) oil. The LC50 (24 h) was 101 and 24 mg L(-1) after 72 h and 12 mg L(-1) at 96 h. Based on EPA scale of toxicity categories to aquatic organisms, an LC50 (96 h) of 12 mg L(-1) indicates that the dispersed oil was slightly to moderately toxic to E. gryllus. As an attempt to compare our results to others, a literature study was performed. Due to limited amount of data available for dispersed oil and amphipods, information on other crustacean species and other oil-related compounds was also collected. Only one study on dispersed oil and amphipods was found, the LC50 value in this study was similar to the LC50 value of E. gryllus in our study. Since toxicity data are important input to risk assessment and net environmental benefit analyses, and since such data are generally lacking on deep-sea species, the data set produced in this study is of interest to the industry, stakeholders, environmental management, and ecotoxicologists. However, studies including more deep-sea species covering different functional groups are needed to evaluate the sensitivity of the deep-sea compartments to dispersed oil relative to other environmental compartments.

Biomarker modulation associated with marine diesel contamination in the Iceland scallop (Chlamys islandica).

The decrease of ice cover in the Arctic will lead to an increase of ship traffic in the upcoming decades. Consequently, oil pollution is expected. In this context, the goals of this study were to evaluate the biological impact of marine diesel contamination and, on this basis, to determine analytical tools of interest (biomarkers) for future biomonitoring of diesel spills. Using a 7-day contamination protocol, this study investigated biochemical modulations in the digestive gland of the Iceland scallop (Chlamys islandica). Incorporation of contaminants was verified assessing haemolymph metabolites. Results showed a response of glutathione-S-transferase to contamination suggesting detoxification processes and the suitability of such a tool for diesel spill biomonitoring. The lack of modulation of superoxide dismutase activity and lipid peroxidation suggests no oxidative stress and the unsuitability of these molecular tools for biomonitoring.

Acute and long-term biological effects of mechanically and chemically dispersed oil on lumpsucker (Cyclopterus lumpus).

Concentration dependent differences in acute and long-term effects of a 48 h exposure to mechanically or chemically dispersed crude oil were assessed on juvenile lumpsucker (Cyclopterus lumpus). Acute or post-exposure mortality was only observed at oil concentrations representing higher concentrations than reported after real oil spills. Acute mortality was more apparent in chemically than mechanically dispersed oil treatments whereas comparable EC50s were observed for narcosis. There was a positive correlation between EROD activity and muscle PAH concentration for the lower oil concentrations whereas higher concentrations inhibited the enzyme activity. The incidence of gill tissue lesions was low with no difference between dispersion methods or oil concentrations. A concentration dependent decrease in swimming- and feeding behavior and in SGR was observed at the start of the post-exposure period, but with no differences between corresponding oil treatments. Three weeks post-exposure, fish from all treatments showed as high SGR as the control fish.

Impact of dispersed fuel oil on cardiac mitochondrial function in polar cod Boreogadus saida.

In this study, impact of dispersed oil on cardiac mitochondrial function was assessed in a key species of Arctic marine ecosystem, the polar cod Boreogadus saida. Mature polar cod were exposed during 48 h to dispersed oil (mechanically and chemically) and dispersants alone. The increase observed in ethoxyresorufin-O-deethylase activity and polycyclic aromatic hydrocarbon metabolites in bile indicated no difference in contamination level between fish exposed to chemical or mechanical dispersion of oil. Oil induced alterations of O2 consumption of permeabilised cardiac fibres showing inhibitions of complexes I and IV of the respiratory chain. Oil did not induce any modification of mitochondrial proton leak. Dispersants did not induce alteration of mitochondrial activity and did not increase oil toxicity. These data suggest that oil exposure may limit the fitness of polar cod and consequently could lead to major disruption in the energy flow of polar ecosystem.

Brain cytochrome P450 aromatase activity in roach (Rutilus rutilus): seasonal variations and impact of environmental contaminants.

P450 aromatase catalyses the conversion of C19 androgens to C18 estrogens which is thought to be essential for the regulation of the reproductive function. In this study, brain aromatase activity (AA) was measured monthly over a reproductive cycle in wild roach (Rutilus rutilus) sampled in a reference site in Normandy. AA peaked during the breeding season, reaching 35 fmol mg(-1)min(-1) in both male and female fish, and was low during the rest of the year except for a significant rise in October. AA was correlated with ovary maturation (measured either as gonado-somatic index or by histological analysis of the gonads) and plasma sex-steroid levels (11-ketotestosterone in males and 17-ß-estradiol in females). Measurements of AA in polluted sites showed that activity was significantly upregulated in sites with fish showing high levels of plasma vitellogenin and large proportion of intersexuality (20-50%) thus suggesting the occurrence of estrogenic compounds and their involvement in AA modulation.

A new in vitro screening bioassay for the ecotoxicological evaluation of the estrogenic responses of environmental chemicals using roach (Rutilus rutilus) liver explant culture.

There is growing evidence that many chemicals released in the environment are able to disturb the normal endocrinology of organisms affecting the structure and function of their reproductive system. This has prompted the scientific community to develop appropriate testing methods to identify active compounds and elucidate mechanisms of action. Of particular interest are in vitro screening methods that can document the effects of these endocrine disrupting compounds on fish. In this study, an in vitro bioassay was developed in the roach (Rutilus rutilus) for evaluating the estrogenicity or antiestrogenicity potency of environmental pollutants by measuring vitellogenin (VTG) induction in cultured liver explants. The cell viability was assessed by the measurement of nonspecific esterase activity using a fluorescein diacetate hydrolysis assay. Results showed that explants could be cultured for 72 h without any significant loss of activity. Dose-dependent responses have been measured with estrogenic model compounds such as 17-ß-estradiol (E2) and 17-α-ethynylestradiol (EE2) or antiestrogenic compounds such as tamoxifen. Lowest observable effective concentrations were 1 nM for E2, 1 nM for EE2, and 100 nM for tamoxifen, showing a good sensitivity of the test system. Estrogenicity of butyl 4-hydroxybenzoate, 4-nonylphenol, and bisphenol A was tested. bisphenol A (100 µM) or butylparaben induced a twofold increase in VTG production when compared with 100 nM E2, whereas this production was only 20% with 100 µM 4-nonylphenol. Overall, this study shows that the bioassay could provide valuable information on endocrine disrupting chemicals including metabolites and mixtures of compounds.

Endocrine disruption in wild populations of chub (Leuciscus cephalus) in contaminated French streams.

The aim of this study was to assess endocrine disruptive effects in wild population of fish in five French rivers selected to represent different pollution contexts at two seasons (summer and fall). For that purpose, a panel of biometrical parameters (length, weight, and gonado-somatic index: GSI) and biochemical (ethoxyresorufin-O-deethylase: EROD, vitellogenin: VTG, and brain aromatase) and histological biomarkers (gonads histology) were used in chub (Leuciscus cephalus), a common cyprinid fish species. In fish from the reference site, EROD activity and VTG levels were low at the two seasons. Brain aromatase activities (AAs) were similar to other species and increased with increasing GSI and gonad maturation. Among the four contaminated sites, the Jalle d'Eysines River was the most impacted site. At this site, fish were exposed to estrogenic substances as demonstrated by the VTG induction in males and the arrest of development of the gonads that led to lower brain AA compared to fish from the reference site. In fish from other contaminated sites, EROD activity was induced as compared to fish from the reference site and some males had elevated concentrations of VTG. Moreover, the presence of aromatase-inhibiting compounds was demonstrated in the sediments of three contaminated sites, even if the precise nature of contaminants is not known. This study provides new data concerning endocrine disruption in wild fish populations inhabiting French rivers and demonstrates that measurements of in vivo and in vitro aromatase could be used as biomarkers of endocrine disruption in field studies.

Seasonal variations and alterations of sex steroid levels during the reproductive cycle of male roach (Rutilus rutilus).

Seasonal variations of plasma steroid concentrations i.e. progesterone (P), 11-ketotestosterone (11-KT) and 17-ß-estradiol (E2) were determined immunoenzymatically during a whole reproductive cycle in male roach (Rutilus rutilus) caught in a reference site. Plasma 11-KT concentrations were significantly correlated with gonad growth, expressed as the gonado-somatic index (R² =0.60; p<0.05) and highest concentrations (757 ± 99 pg ml⁻¹ ) coincided with the final testis maturation in March. E2 and P concentrations peaked once during the reproductive cycle. E2 synthesis was significantly induced during the spawning period while P concentration peaked at the very start of the gametogenesis (June) thus suggesting specific roles of these steroids in the reproductive cycle. Comparison of reference levels were then made with plasma steroid concentrations from male roach sampled in polluted areas in the North of France. A significant decrease of E2 (50-60%) and 11-KT (76-84%) was measured, indicating that endocrine disrupting compounds may have interfered with the normal sex steroid synthesis. Contrary to the E2 and 11-KT sex steroids, plasma P concentration was not significantly affected in fish inhabiting impacted areas.

Roach (Rutilus rutilus) reproductive cycle: a study of biochemical and histological parameters in a low contaminated site.

Fish reproduction is subjected to worrying trends in many aquatic environments. In this study, we report the absence of histological and biochemical alterations in fish sampled in a low contaminated site (characterised by the absence of detectable oestrogenic activity and mutagenicity in sediment extracts). A total of 474 roach (Rutilus rutilus) were monthly sampled during 18 months, and no intersex fish were recorded after careful histological examination, thus indicating that the incidence of this phenomenon may be very low under natural conditions. Furthermore, mean male plasma vitellogenin concentration was 24 ng ml(-1) and was only slightly elevated during the spawning period (up to 120 ng ml(-1)) indicating that these low values may be characteristic of a low contaminated site. Of the male roach, 45.3% were sampled, a sex-ratio that did not significantly deviated from the expected 1:1 ratio between male and female. Results also showed that natural conditions can greatly affect the reproductive cycle of roach. Gametogenesis showed a biphasic pattern with first gonad maturation between September and December and a final maturation occurring at the end of winter/early spring. Under decreasing temperatures, particularly below 6 degrees C, gametogenesis was stopped or even regressed with secondary oocytes becoming rare under histological observation. Conversely, elevated temperatures during the winter lead to an earlier gonad maturation.