Relevance of flounder caging and proteomics to explore the impact of a major industrial accident caused by fire on the Seine estuarine water quality.

On September 26th 2019, a major fire occurred in the Lubrizol factory located near the Seine estuary, in Rouen-France. Juvenile flounders were captured in the Canche estuary (a reference system) and caged one month in the Canche and in the Seine downstream the accident site. No significant increases of PAHs, PCBs and PFAS was detected in Seine vs Canche sediments after the accident, but a significant increase of dioxins and furans was observed in water and sewage sludge in the Rouen wastewater treatment plant. The proteomics approach highlighted a dysregulation of proteins associated with cholesterol synthesis and lipid metabolism, in fish caged in the Seine. The overall results suggested that the fire produced air borne dioxins and furans that got deposited on soil and subsequently entered in the Seine estuarine waters via runoff; thus contaminating fish preys and caged flounders in the Seine estuary.

Avoidance behaviour and toxicological impact of sunscreens in the teleost Chelon auratus.

There is increasing evidence that sunscreen, more specifically the organic ultra-violet filters (O-UVFs), are toxic for aquatic organisms. In the present study, we simulated an environmental sunscreen exposure on the teleost fish, Chelon auratus. The first objective was to assess their spatial avoidance of environmental concentrations of sunscreen products (i.e. a few µg.L-1 of O-UVFs). Our results showed that the fish did not avoid the contaminated area. Therefore, the second objective was to evaluate the toxicological impacts of such pollutants after 35 days exposure to concentrations of a few µg.L-1 of O-UVFs. At the individual level, O-UVFs increased the hepatosomatic index which could suggest pathological alterations of the liver or the initiation of the detoxification processes. At the cellular level, a significant increase of malondialdehyde was measured in the muscle of fish exposed to O-UVFs which suggests a failure of antioxidant defences and/or an excess of reactive oxygen species.

Seasonal variations of low pesticides contamination and biomarker responses in marine bivalves from French estuaries.

Biomarkers involved in detoxification process (GST), oxidative stress (SOD and MDA), immune response (Laccase) and neurotoxic disorders (AChE) were analysed in Pacific oysters and blue mussels collected from 4 locations within the Pertuis sea (France). Seasonal variations of total pesticide mean concentrations were found in seawater with metolachlor being the main pesticide measured (up to 32 ng/L). The majority of pesticide concentrations in sediment were below the LOD. Seasonal contamination differences were evidenced for chlortoluron, especially in mussels where concentrations reached 16 ng/g (wet weight) during the winter, in the Charente estuary, but no relationships with any of the biomarkers selected arisen. Actually, low concentrations of alpha-, beta-BHC and alachlor were correlated to GST activity, and low levels of hexachlorobenzene were linked to the AChE activity and MDA content in oysters. In mussels, low concentrations of methylparathion, parathion and beta-BHC were correlated to laccase.

Integration of environmental signatures and omics-based approaches on the European flounder to assist with health assessment of estuarine ecosystems in Brittany, France.

This study aimed to develop a multidisciplinary approach to assess the ecological status of six moderate-sized French estuaries. For each estuary, we gathered geographical information, hydrobiological data, chemistry of pollutants and fish biology, including integration of proteomics and transcriptomics data. This integrative study covered the entire hydrological system studied, from the watershed to the estuary, and considered all the anthropogenic factors that can impact this environment. To reach this goal, European flounder (Platichthys flesus) were collected from six estuaries in September, which ensures a minimum residence time of five months within an estuary. Geographical metrics are used to characterize land use in each watershed. The concentrations of nitrite, nitrate, organic pollutants, and trace elements were measured in water, sediments and biota. All of these environmental parameters allowed to set up a typology of estuaries. Classical fish biomarkers, coupled with molecular data from transcriptomics and shotgun proteomics, highlighted the flounder's responses to stressors in its environment. We analysed the protein abundances and gene expression levels in the liver of fish from the different estuaries. We showed clear positive deregulation of proteins associated with xenobiotic detoxification in a system characterized by a large population density and industrial activity, as well as in a predominantly agricultural catchment area (mostly cultures of vegetables and pig breeding) mainly impacted by pesticides. Fish from the latter estuary also displayed strong deregulation of the urea cycle, most probably related to high nitrogen load. Proteomic and transcriptomic data also revealed a deregulation of proteins and genes related to the response to hypoxia, and a probable endocrine disruption in some estuaries. Coupling these data allowed the precise identification of the main stressors interacting within each hydrosystem.

Preliminary inter-port study of the quality of environments using physiological responses of invertebrates exposed to chronic trace element and organic contamination in Corsica (Mediterranean Sea).

Port areas are socio-ecosystems impacted by chronic mixture pollution. Some marine species benefit from living there and may be studied to define the ecological state of such environments. In this study, the risks of chronic chemical contamination and its consequences on three marine molluscs were evaluated in North Corsica (France) port areas. Mediterranean mussel Mytilus galloprovincialis, tubular sea cucumber Holothuria tubulosa and Mediterranean limpet Patella sp. were sampled in three port areas and a reference location. A set of biomarkers was analysed to evaluate oxidative stress, detoxification, energetic metabolism, neurotoxicity, immunity and bioaccumulation (metallic trace elements and organic pollutants). The objectives were to assess pollution-induced effects in organisms, to determine the best bioindicator species for the selected locations and to validate a "pool" sampling technique (when the analysis is done on a single pool of samples and not on individual samples). The results validate the sampling techniques as "pool" for management purposes. St-Florent was demonstrated as the most contaminated location. All the other locations present a low contamination, below the recommended threshold values (for metallic trace elements and organic pollutants). Finally, the limpet appears to be the best bioindicator for the selected locations. Mussel and sea cucumber are inappropriate due to their absence in this oligotrophic region and the lack of responses observed, respectively.

Active and passive biomonitoring of trace elements, polycyclic aromatic hydrocarbons, and polychlorinated biphenyls in small Mediterranean harbours.

Pollution particularly affects coastal ecosystems due to their proximity to anthropic sources. Among those environments, harbours are subjected to marine traffic but also to accidental and chronic pollution. These areas are thus exposed to complex mixtures of contaminants such as trace elements and organic contaminants which can impact marine species, habitats, and ecosystem services. The monitoring of these compounds is thus a crucial issue for assessment of environmental health. In this context, the aim of the present work was to evaluate the chemical contamination of harbours in Corsica (NW Mediterranean) by measuring the bioaccumulation of trace elements, polycyclic aromatic hydrocarbons, and polychlorinated biphenyls in mussels, limpets, and sea cucumbers. The human health risks associated with seafood consumption were also assessed. Results reveal a relatively low contamination in the Corsican harbours studied compared to larger Mediterranean ports and suggest that the potential health risk for consumers eating seafood is low.

Impacts of chemical stress, season, and climate change on the flounder population of the highly anthropised Seine estuary (France).

The main objective of this study was to improve our knowledge on the responses of fish populations to multistress (diffuse pollution and warming waters) in estuaries. Adult flounders were caught in two estuaries in the Eastern English Channel: the heavily polluted Seine estuary vs the moderately contaminated Canche estuary. Fish samplings were conducted in January just before the reproduction period, and in July when gonads were at rest. The overall rise in coastal winter water temperatures detected over the Channel impairs the flounder's phenology of reproduction in the two estuaries, inducing a delay of maturation process and probably also spawning. The higher liver histopathology index in Seine vs Canche could be the consequence of the fish exposition to a complex cocktail of contaminants in a strongly industrialized estuary. Higher levels of neurotoxicity, gill lipid peroxidation, and liver EROD activity were observed in Seine vs Canche. Furthermore, a possible impairment in mitochondrial metabolism was suggested in the Seine flounder population. We confirmed in this study the potential role of two membrane lipids (sphingomyelin and phosphatidylserine) in the resistance towards oxidative stress in Seine and Canche. Finally, we suggest that the Seine flounder population (and possibly the connected Eastern English Channel flounder populations over the French Coast) could be seriously impacted in the future by multistress: higher winter temperatures and chemical contamination.

Pesticides, nonylphenols and polybrominated diphenyl ethers in marine bivalves from France: A pilot study.

The present pilot study aimed to provide an overview of organic contaminant concentration levels in the littoral ecosystems of the Pertuis seas. The study determined the concentrations of twenty-nine pesticides, six nonylphenols and seven polybrominated diphenyl ethers (PBDEs) in sediments, seawater, Pacific oysters and blue mussels. Oysters accumulated a higher number of pesticides than blue mussels. Indeed, alpha BHC (0.60-0.72 ng/g, ww), chlorfenvinphos (1.65-2.12 ng/g, ww), chlorpyrifos (0.79-0.93 ng/g, ww), chlortoluron (2.50-4.31 ng/g, ww), metolachlor (up to 0.38 ng/g, ww) and parathion (0.56-0.69 ng/g, ww) were quantified in oysters whereas only alpha BHC (0.24-0.31 ng/g, ww), was quantified in mussels. The present results also revealed that the POPs detected in water or sediments were not ultimately found accumulated in bivalves. Other molecules such as methylparathion and BDE47 were quantified in sediments. These molecules, BDE99 and one nonylphenol (OP2OE) were quantified in seawater. Finally, the comparison with the available environmental guidelines showed that the values measured were at concentrations not considered to cause adverse effects at the populations' level except for chlortoluron in seawater (15-50 ng/L).

Subchronic exposure to high-density polyethylene microplastics alone or in combination with chlortoluron significantly affected valve activity and daily growth of the Pacific oyster, Crassostrea gigas.

Nowadays, pesticides and microplastics (MPs) are commonly found in coastal waters worldwide. Due to their widespread use, their persistence and toxicity, they may induce adverse effects on physiology and behaviour of marine organisms such as the Pacific oyster (Crassostrea gigas). This study explored the growth and valve activity of juvenile oysters exposed for 24 days to two frequently detected pollutants in the Pertuis Charentais (South West, France): a herbicide (chlortoluron, 85 µg.L-1) and high-density polyethylene microparticles (HDPE 20-25 µm, 112 MP.mL-1) alone or in combination (cocktail condition; 97 µg.L-1 of chlortoluron + 108 MP. mL-1). The valve activity of juvenile oysters recorded by using a High Frequency and Non-Invasive valvometer (HFNI) was characterized by three parameters: the number of valve micro-closures (VMC), the Valve Opening Amplitude (VOA), and the Valve Opening Duration (VOD). Additionally, daily shell growth and the oyster daily rhythm were assessed. The exposure to MPs of oysters led to a significant increase of VMC and a decrease of VOD and shell growth. The exposure to chlortoluron showed a significant increase of VOA and a decrease of VMC. In combination with MPs, chlortoluron still increased VOA and decreased VMC but also reduced the shell growth. Chronobiological analysis did not reveal any effects on the daily rhythm of both contaminants. This work highlighted significant effects of high environmental concentrations of MPs and Chlortoluron on the behaviour and growth of the Pacific oyster.

Coastal ecosystem inventory with characterization and identification of plastic contamination and additives from aquaculture materials.

In the early 1970s, studies of marine litter first appeared in the scientific literature. Fifty years later, knowledge of several coastal areas of the Atlantic, the driving forces of oyster farmers and aquaculture, is lacking. This work documents a pilot study on an Atlantic coastal area (France). This study aims to (i) characterize the abundance of macroplastics related to aquaculture tools; (ii) microplastics present in beach sediments and (iii) characterization of pollutants present on aquaculture plastics collected. First, it was observed that 70% of the plastics collected on the beach were characteristic of aquaculture materials. In sediments, MPs most found were Polyamide between 10 and 20 µm, with a total MP concentration of 397-457 MPs.kg-1. Pipes collectors (PVC), frequently used in aquaculture, have been shown to have concentrations of dimethylphthalates and naphthalene. Waste management and support policies can then base their actions on such studies, in order to improve their knowledge.

Effects of dispersant treated oil upon exploratory behaviour in juvenile European sea bass (Dicentrarchus labrax).

Accidental spills are pervasive pollution in aquatic ecosystems. Resorting to chemical dispersant is one of the most implemented strategies in response to oil spills, but it results in an increase in the bio-availability of oil compounds known to disturb fish neurosensory capacities and hence fish habitat use. While it has become well established that acute oil exposure can cause a range of physiological defects, sub-lethal consequences on animal behaviour have only received recent attention. Here we investigated the effect of an exposure to a 62 h- dispersant treated oil on the exploration tendency (exploratory activity, and avoidance of unfamiliar open areas) of juvenile European sea bass. Three different concentrations of chemically dispersed oil were tested, low and medium conditions bracketing the range of likely situations that fish encounter following an oil spill, the high dose representing a more severe condition. Fish recovery capacities were also evaluated during 2 weeks post-exposure. Our results suggest a dose-response relationship; the low dose (0.048 ± 0.007 g L-1 of total petroleum hydrocarbons ([TPH])) had no effect on sea bass behavioural response to a novel environment while medium (0.243 ± 0.012 g L-1 [TPH]) and high (0.902 ± 0.031 g L-1 [TPH]) doses altered fish exploratory activity and their typical avoidance of unfamiliar open areas. Our experiment also suggest signs of recovery capacities in the first 10 days following oil exposure even if fish might need more time to fully recover from observed alterations. We discuss the possibility that observed alterations may result from a neurosensory or physiological known defects of oil exposure, causing anaesthetic-like sedative behaviours. Altogether, this study shows that juvenile sea bass exposed to oil spill exhibit transient behavioural impairments that may have major population-level consequences given the high mortality experienced by juveniles.

The effects of hypoxia on aerobic metabolism in oil-contaminated sea bass (Dicentrarchus labrax).

Hypoxia and petrogenic hydrocarbon contamination are two anthropogenic stressors that coexist in coastal environments. Although studies have estimated the impact of each stressor separately, few investigations have assessed the effects of these stressors in interaction. We therefore investigated the impact of these combined stressors on sea bass, (Dicentrarchus labrax) physiology. After experimental contamination with physically dispersed oil, fish were exposed to hypoxia or normoxia, and active/standard metabolic rates (AMR and SMR, respectively), and metabolic scope (MS) were estimated. At the protocol's end, the uptake of polycyclic aromatic hydrocarbons (PAHs) was estimated by evaluating relative concentrations of bile metabolites. In terms of bile metabolites, our results validated the uptake of PAHs by contaminated fish in our experimental settings, and further suggest that the hypoxic period after contamination does not reduce or increase compound metabolization processes. Our data showed significant effects of hypoxia on all metabolic rates: a significant drastic AMR reduction and significant SMR diminution led to decreased MS. We also found that oil contamination significantly impacted AMR and MS, but not SMR. These results suggested that when evaluated separately, hypoxia or oil affect the metabolic rate of sea bass. On the other hand, when evaluated in combination, no cumulative effects were observed, since fish exposed to both stressors did not show a stronger impact on metabolism than fish exposed to hypoxia alone. This suggests that oil impacts fish metabolism when fish occupy normoxic waters, and that oil does not magnify hypoxia-induced effects on fish metabolism.

Combined effects of high hydrostatic pressure and dispersed oil on the metabolism and the mortality of turbot hepatocytes (Scophthalmus maximus).

Since the DeepWater Horizon oil spill and the use at 1450 m depth of dispersant as a technical response, the need of relevant ecotoxicological data on deep-sea ecosystems becomes crucial. In this context, this study focused on the effect of high hydrostatic pressure (10.1 MPa) on turbot hepatocytes isolated from fish exposed either to chemically dispersed oil, mechanically dispersed oil or dispersant alone. Potential combined effects of oil/dispersant and hydrostatic pressure, were assessed on cell mortality (total cell death, necrosis and apoptosis), cell viability and on hepatocyte oxygen consumption (MO2). No change in cell mortality was observed in any of the experimental conditions, whereas, the results of cell viability showed a strong and significant increase in the two oil groups independently of the pressure exposure. Finally, oil exposure and hydrostatic pressure have additive effects on oxygen consumption at a cellular level. Presence of dispersant prevent any MO2 increase in our experimental conditions. These mechanistic effects leading to this increased energetic demand and its eventual inhibition by dispersant must be investigated in further experiments.

Deep-sea versus shallow conditions: a comparative ecobarotoxicological study.

In the context of new oil exploration/production areas, knowledge of the biological impact of dispersed oil in the deep-sea environment is essential. Hence, the aim of this study was to perform a comparison, at atmospheric pressure (0.1 MPa) and at a high hydrostatic pressure corresponding to 1000 m depth (10.1 MPa), of lethal concentrations (LC) on a model fish, Scophthalmus maximus, exposed to chemically dispersed oil. Fish were exposed concomitantly at 0.1 and 10.1 MPa using two exposure tanks connected to the same source tank thanks to a closed circuit. Acute toxicity was evaluated at 24 h through the determination of LC10 and LC50 (respectively, 10 and 50% of mortality) calculated from measured total petroleum hydrocarbon concentrations in the water. No statistical differences were observed between the LC10 at 0.1 MPa (46.1 mg L- 1) and the LC10 at 10.1 MPa (31.0 mg L- 1), whereas the LC50 of fish exposed to 0.1 MPa (90.8 mg L- 1) was significantly higher than the LC50 at 10.1 MPa (50.9 mg L- 1). These results clearly show an increase in oil toxicity under high hydrostatic pressure. This effect may be due to synergistic effects of pressure and oil contamination on fish energetic metabolism.

An Integrated Biomarker Approach Using Flounder to Improve Chemical Risk Assessments in the Heavily Polluted Seine Estuary.

The objective of this study was to develop an integrative approach in ecotoxicology (from biomarkers to population genetics) to assess the ecological status of fish populations. Flounders (Platichthys flesus) collected after the spawning season in the heavily polluted Seine estuary were compared with the moderately polluted Bay of Douarnenez. The muscle energetic reserves were highly depleted in Seine vs. Douarnenez fish. The Seine fish displaying a reduced capacity to manage the oxidative stress and a higher energetic metabolism. An increase in the content of muscle membrane phospholipids (sphingomyelin, phosphatidylserine, free sterols) was detected in the Seine vs. Douarnenez fish. The data integration allowed to hypothesize relationships between membrane phospholipids, xenobiotic metabolism, bioenergetics, and antioxidant defence. The genetic diversity considering neutral markers was maintained in the heavily polluted Seine population compared with the Douarnenez population. Finally, we suggest that the high physiological cost of tolerance to toxicants in the Seine flounder population could compromise its capacity to respond in the future to an additional stressor like warming waters in shallow depth. Thus, this population could be submitted to an ecological risk.

Cellular, humoral and molecular responses in rainbow trout (Oncorhynchus mykiss) exposed to a herbicide and subsequently infected with infectious hematopoietic necrosis virus.

Aquatic ecosystems are now chronically polluted by a cocktail of many chemical substances. There is now clear evidence of associations between exposure to pollutants and greater susceptibility to pathogens. The aim of the present study was to characterize the defense capacities of rainbow trout (Oncorhynchus mykiss), chronically exposed to pendimethalin (PD), to subsequent experimental challenge with the infectious hematopoietic necrosis virus (IHNV). Immunological responses were examined at different organizational levels, from individuals to gene expression. No negative effects of PD were noted on the Fulton index nor on the liver or spleen somatic indices (LSI; SSI) before viral infection, but the infectious stress seems to generate a weak but significant decrease in Fulton and LSI values, which could be associated with consumption of energy reserves. During the viral challenges, the distribution of cumulative mortality was slightly different between infected groups. The impact of the virus on fish previously contaminated by PD started earlier and lasted longer than controls. The proportion of seropositive fish was lower in the fish group exposed to PD than in the control group, with similar quantities of anti-IHNV antibodies secreted in positive fish, regardless of the treatment. While no significant differences in C3-1 expression levels were detected throughout the experiment, TNF1&2, TLR3, Il-1ß and IFN expression levels were increased in all infected fish, but the difference was more significant in fish groups previously exposed to herbicide. On the other hand, ß-def expression was decreased in the pendimethalin-IHNV group compared to that in fish only infected by the virus (control-IHNV group).

Effects of oil spill response technologies on marine microorganisms in the high Arctic.

We studied how exposure to oil spill response technologies affect marine microorganisms during Arctic winter and spring. Microorganisms were exposed to chemically dispersed oil (DISP), in situ burnt oil (ISB), and natural attenuated oil (NATT) in mesocosms from February to May. We subsampled the mesocosms and studied the effects of oil in laboratory incubations as changes in biomass of the major functional groups: bacteria, heterotrophic-nanoflagellates, dinoflagellates, ciliates, pico- and nanophytoplankton, and diatoms over two 14-day periods. In winter, the majority of polycyclic aromatic hydrocarbons (PAHs) remained encapsulated in the ice, and the low concentrations of PAHs in water led to minute changes in biomass of the investigated groups. In spring, however, when the PAHs were partially released from the melting ice, the biomass of many functional groups in DISP and NATT decreased significantly, while the changes in ISB were less pronounced. The overall biomass reduction, as observed in this study, could lead to a disrupted transfer of energy from the primary producers to the higher trophic levels in oil affected areas.

The effect of hypoxia and hydrocarbons on the anti-predator performance of European sea bass (Dicentrarchus labrax).

Hydrocarbons contamination and hypoxia are two stressors that can coexist in coastal ecosystems. At present, few studies evaluated the combined impact of these stressors on fish physiology and behavior. Here, we tested the effect of the combination of hypoxia and petrogenic hydrocarbons on the anti-predator locomotor performance of fish. Specifically, two groups of European sea bass (Dicentrarchus labrax) were exposed to clean water (Ctrl) or oil-contaminated water (Oil). Subsequently, fish of both groups were placed in normoxic (norx) or hypoxic (hyp) experimental tanks (i.e. four groups of fish were formed: Ctrl norx, Ctrl hyp, Oil norx, Oil hyp). In these tanks, escape response was elicited by a mechano-acoustic stimulus and recorded with a high speed camera. Several variables were analyzed: escape response duration, responsiveness (percentage of fish responding to the stimulation), latency (time taken by the fish to initiate a response), directionality (defined as away or toward the stimulus), distance-time variables (such as speed and acceleration), maneuverability variables (such as turning rate), escape trajectory (angle of flight) and distancing of the fish from the stimulus. Results revealed (i) effects of stressors (Ctrl hyp, Oil norx and Oil hyp) on the directionality; (ii) effects of Oil norx and Oil hyp on maneuverability and (iii) effects of Oil hyp on distancing. These results suggest that individual stressors could alter the escape response of fish and that their combination could strengthen these effects. Such an impact could decrease the probability of prey escape success. By investigating the effects of hydrocarbons (and the interaction with hypoxia) on the anti-predator behavior of fish, this work increases our understanding of the biological impact of oil spill. Additionally, the results of this study are of interest for oil spill impact evaluation and also for developing new ecotoxicological tools of ecological significance.

Underwater hyperspectral classification of deep sea corals exposed to 2-methylnaphthalene.

Coral reefs around the world are under threat due to anthropogenic impacts on the environment. It is therefore important to develop methods to monitor the status of the reefs and detect changes in the health condition of the corals at an early stage before severe damage occur. In this work, we evaluate underwater hyperspectral imaging as a method to detect changes in health status of both orange and white color morphs of the coral species Lophelia pertusa. Differing health status was achieved by exposing 60 coral samples to the toxic compound 2-methylnaphthalene in concentrations of 0 mg L-1 to 3.5 mg L-1. A machine learning model was utilized to classify corals according to lethal concentration (LC) levels LC5 (5% mortality) and LC25 (25% mortality), solely based on their reflectance spectra. All coral samples were classified to correct concentration group. This is a first step towards developing a remote sensing technique able to assess environmental impact on deep-water coral habitats over larger areas.

Food deprivation reduces social interest in the European sea bass Dicentrarchus labrax.

Periods of food deprivation of several months are common events for fishes and in such conditions, fitness will be determined by their capacity to maximize food encounters while minimizing predation risk. In this context, the propensity to take risks and the willingness to associate with conspecifics are particularly important as they contribute to alleviating the trade-off between predation avoidance and foraging efficiency. This study examined to what extent food deprivation modulates fish risk-taking and social behaviours, as well as the relationship between them. To address these issues, juvenile European sea bass were either fed daily with a maintenance ration or food deprived for a period of 3 weeks. Risk taking and sociability were assessed through measurements of fish willingness to explore a novel environment, and to interact with a novel object or a conspecific. Multivariate analysis allowed the identification of three behaviours: risk taking, exploratory activity and solitariness. Food-deprived fish interacted less with conspecifics than control fish; however, no difference in terms of risk taking and exploratory patterns was observed. Finally, the relationship between risk taking and solitariness was influenced by feeding status. When food-deprived, fish with a higher propensity to take risk displayed increased solitariness, while when fed normally, they interacted more with conspecifics.