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Watersheds and estuaries are impacted by multiple anthropogenic stressors that affect their biodiversity and functioning. Assessing their ecological quality has consequently remained challenging for scientists and stakeholders. In this paper, we propose a multidisciplinary approach to identify the stressors in seven small French estuaries located in agricultural watersheds. We collected data from landscape (geography, hydrobiology) to estuary (pollutant chemistry) and fish individual scales (environmental signatures, proteomics). This integrative approach focused on the whole hydrosystems, from river basins to estuaries. To characterize each watershed, we attempted to determine the land use considering geographic indicators (agricultural and urbanised surfaces) and landscape patterns (hedges density and riparian vegetation). Juveniles of European flounder (Platichthys flesus) were captured in September, after an average residence of five summer months in the estuary. Analyses of water, sediments and biota allowed to determine the concentrations of dissolved inorganic nitrogen species, pesticides and trace elements in the systems. Environmental signatures were also measured in flounder tissues. These environmental parameters were used to establish a typology of the watersheds. Furthermore, data from proteomics on fish liver were combined with environmental signatures to determine the responses of fish to stressors in their environments. Differential protein abundances highlighted a dysregulation related to the detoxification of xenobiotics (mainly pesticides) in agricultural watersheds, characterized by intensive cereal and vegetable crops and high livestock. Omics also revealed a dysregulation of proteins associated with the response to hypoxia and heat stress in some estuaries. Furthermore, we highlighted a dysregulation of proteins involved in urea cycle, immunity and metabolism of fatty acids in several systems. Finally, the combination of environmental and molecular signatures appears to be a relevant method to identify the major stressors operating within hydrosystems., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jean Laroche reports financial support was provided by the “Agence de l'eau Loire-Bretagne” (AELB, project ECOEST)., (Copyright © 2024. Published by Elsevier Ltd.)

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. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

A two months common garden experiment was carried out to explore the potential differences of energy metabolism in northern core (France, 50°N and 47°N) vs southern peripheral (Portugal, 41°N) populations of European flounder Platichthys flesus , submitted to cold condition (CC: water temperature = 10 °C) and to warm and hypoxic condition (WHC: water temperature = 22 °C, and moderate hypoxia with O 2 saturation = 40% during the last 6 days). Convergent growth rates (in length) were observed in the different populations and conditions, when the southern peripheral population of Portugal did not grow under cold conditions. A general reduction in liver lipid storage was observed in all populations subjected to WHC when compared to CC, whereas muscle lipid storage was unaffected. The thermal and hypoxia treatment induced changes in muscle phospholipids (PL) ratios: phosphatidylserine/PL, phosphatidylinositol/PL, between northern and southern populations. Fish from northern estuaries displayed marked anaerobiosis in WHC (increased liver LDH activity) vs marked aerobiosis under CC (higher muscle CS and CCO activities). Contrariwise, fish from the southern estuary displayed equilibrium between anaerobiosis and aerobiosis activities in WHC. Flounders from the southern population exhibited generally lower G6PDH activity (proxy for anabolism and for defense against oxidative damage), tissue-specific anaerobiosis response (muscle LDH activity) and lower CS and CCO muscle activities (aerobiosis markers) when compared to northern populations. Globally, these inter-population differences in bioenergetics suggest that southern peripheral vs northern core populations have developed differential capacity to cope with interacting stressors and that much of this variation is more likely due to local adaptation. [ABSTRACT FROM AUTHOR]

Abstract: Estuaries are important areas highly susceptible to anthropogenic degradations like pollution. Estuarine species have thus to cope with many types of constraints depending on the estuaries'' characteristics. The European flounder Platichthys flesus is considered as a sentinel species for the monitoring of estuarine water quality. In this study, juvenile flounders (0+ group) were sampled from three contrasted Channel estuaries, i.e. the Seine, the Canche and the Tamar, and we characterized their liver proteomes by using a two-dimensional electrophoresis based proteomic approach. We showed that 27 protein spots differentially accumulated between the 3 populations. Six of these proteins were identified by MALDI TOF–TOF mass spectrometry. Flounders from the Seine and from the Tamar, two highly polluted estuaries, displayed common differences, i.e. an increase of the energetic- and the glutathione-metabolism. The most accumulated protein in the Seine''s samples (6.7-fold) was a Vitelline Membrane Outer layer protein 1 homolog, suggesting oogenesis deregulation in these juvenile (sexually immature) flounders. Future works applying this kind of proteomic approach on flounders experimentally exposed to conditions that mimic environmental constraints will help to better understand the significance of these environmental proteomic signatures. [Copyright &y& Elsevier]

Environmental conditions in estuarine habitats can vary greatly and influence the composition of fish assemblages and their trophic interrelationships. We investigated feeding habits and patterns of diet overlap in juvenile plaice ( Pleuronectes platessa) and flounder ( Platichthys flesus) from two estuarine habitats in the Irish Sea. Plaice was found to vary its diet significantly across environments, whereas flounder exhibited a more consistent and homogeneous feeding pattern. Importantly, sympatric fish sampled at the same station were shown to reduce diet overlap. The results support the view that environmental heterogeneity in estuaries maintains a wide range of selective forces, the net outcome of which can produce a diverse array of feeding adaptations among interacting species. [ABSTRACT FROM AUTHOR]

Abstract: One of the present concerns of fish biologists involves defining and identifying nursery habitats in the context of conservation and resource management strategies. Fish nursery studies usually report upon nursery occupation during the latter juvenile stages, despite the fact that recruitment to nurseries can start early in life, during the larval phase. Here we investigated the use of a temperate estuarine nursery area, the Lima estuary (NW Portugal), by initial development stages of flatfish species before and after metamorphosis, integrating the larval and juvenile phases. The Lima estuarine flatfish community comprised twelve taxa, seven of which were present as pelagic larvae, six as juveniles and three as adults. There was a general trend of increasing spring–summer abundance of both larvae and juveniles, followed by a sharp winter decrease, mainly of larval flatfishes. The Lima estuary was used by Solea senegalensis, Platichthys flesus and Solea solea as a nursery area, with direct settlement for the two first species. In contrast, indirect settlement was suggested for S. solea, with metamorphosis occurring outside the estuarine area. Estuarine recruitment of S. senegalensis varied between years, with young larvae occurring in the estuary throughout a prolonged period that lasted 6–9months, corroborating the protracted spawning season. P. flesus, the second most abundant species, exhibited a typical spring estuarine recruitment, without inter-annual variations. Developed larvae arrived in the estuary during spring, whereas the 0-group juveniles emerged in the following summer period. The present study contributes new insight to our understanding of the economically important S. senegalensis, and highlights the importance of integrating the planktonic larval phase into traditional flatfish nursery studies. [ABSTRACT FROM AUTHOR]

Abstract: Several flatfishes spawn in oceanic waters and pelagic larvae are transported inshore to settle in the nursery areas, usually estuaries, where they remain during their juvenile life. Nursery areas appear as extremely important habitats, not only for juveniles but also for the earlier planktonic larval fish. Yet, the majority of nursery studies tend to focus only on one development stage, missing an integrative approach of the entire early life that fishes spent within a nursery ground. Thus, the present study assessed the influence of environmental parameters on the dynamics of the larval and juvenile flatfishes, throughout their nursery life in the Lima Estuary. Between April 2002 and April 2004, fortnightly subsurface ichthyoplankton samples were collected and juveniles were collected from October 2003 until September 2005. Larval assemblages comprised nine flatfish species, while only six were observed among the juvenile assemblages. Solea senegalensis and Platichthys flesus were the most abundant species of both fractions of the Lima Estuary flatfishes. Larval flatfish assemblages varied seasonally, without relevant differences between lower and middle estuary. Platichthys flesus dominated the spring samples and summer and autumn periods were characterized by an increase of overall abundance and diversity of larval flatfishes, mainly S. senegalensis, associated with temperature increase and reduced river flow. On the contrary, during the winter abundance sharply decreased, as a consequence of higher river run-off that might compromised the immigration of incompetent marine larvae. Juvenile flatfishes were more abundant in the middle and upper areas of the estuary, but the species richness was higher near the river mouth. Sediment type, distance from the river mouth, salinity, temperature and dissolved oxygen were identified as the main environmental factors structuring the juvenile flatfish assemblages. Juveniles were spatially discrete, with the most abundant species S. senegalensis and P. flesus associated with the middle and upper estuary, while the remaining species were associated with the lower estuarine areas. The larval fraction exhibited distinct dynamics from the juvenile estuarine flatfish community. Larval flatfishes showed a strong seasonal structure mainly regulated by biological features as the spawning season and also by seasonal variations of water characteristics. On the other hand, juvenile flatfishes were markedly controlled by site specific characteristics such as sediments structure, distance from the river mouth and salinity regime. The present study emphasized the idea that the environmental control varies throughout the ontogenetic development, stressing the importance of integrating all the early life of a species in flatfish nursery studies. [Copyright &y& Elsevier]

Abstract: The relative importance of nursery areas and their relationships with several environmental variables were evaluated in nine estuarine systems along the Portuguese coast based on trawl surveys. Historical data were used to outline changes and trends in the nursery function of some of these estuaries over the past decades. The dominant flatfish species in Portuguese estuaries were Platichthys flesus (Linnaeus, 1758), Solea solea (Linnaeus, 1758), Solea senegalensis Kaup, 1858 and Monochirus hispidus Rafinesque, 1814, but their occurrence differed among the estuaries. P. flesus only occurred in estuaries north of the Tejo estuary (39°N), S. solea was quite rare along the southern Portuguese coast (south of 37°30′N), S. senegalensis occurred in estuaries throughout the coast, but its abundance varied considerably, and the occurrence of M. hispidus was limited to the Sado estuary and Ria Formosa. A Correspondence Analysis was performed to evaluate the relationships between flatfish species abundance and geomorphologic and hydrologic characteristics of estuaries (latitude, freshwater flow, estuarine area, intertidal area, mean depth and residence time). Abiotic characteristics (depth, temperature, salinity, sediment type) of nursery grounds of each flatfish species were also evaluated. Results showed that some estuaries along the Portuguese coast have nursery grounds used by several flatfish species (e.g. Ria de Aveiro, Sado estuary), while in other systems a segregation was noticed, with juveniles of different species occurring in distinct estuarine areas (e.g. Minho and Mondego estuaries). This emphasizes the relevance of niche overlap, but the potential for competition may be considerably minimized by differences in resource use patterns and by an extremely high abundance of resources. Peak densities of flatfishes recorded in nurseries areas along the Portuguese coast were within the range of values reported for other geographical areas. Inter-annual abundance variability of all the species in the Tejo and Sado estuaries was extremely high, with a drastic decrease in P. flesus in the Tejo estuary, probably related to higher water temperature in recent years due to global climate change. [Copyright &y& Elsevier]

Effects of pesticide exposure on the European flounder, Platichthys flesus, were investigated using a suppression subtractive hybridization method (SSH) to identify up- and down-regulated genes after a 30-day exposure to herbicides (a cocktail of atrazine, diuron, and isoproturon, and a single herbicide, glyphosate). A total of 256 expressed gene sequences were identified as having the potential for being differentially expressed, of which 116 could be identified by homology with databased sequences. The metabolic functions with which they are associated include energy production, general metabolism, signaling, transport, immune system, and structure. Expression of 14 of these genes was analyzed in liver, muscle, and gills by reverse transcriptase-polymerase chain reaction (RT-PCR) under experimental conditions (0, 15, and 30 days of exposure) and under field conditions (sampling in two estuaries displaying different levels of pesticide contamination). This study provides a first basis for studying the response of fish to pesticide exposure and allows the characterization of new potential genetic markers of pesticide contamination in the field. [ABSTRACT FROM AUTHOR]

Abstract: Physiological and genetic responses of flounder Platichthys flesus populations were investigated along the French Atlantic Coast in one moderately contaminated estuary (Ster) and three contaminated estuaries (Seine, Loire and Gironde). The focus of this study was to explore the relationship between stress resistance and energetic trade-offs, in order to detect possible differential physiological capacities or performances between individuals carrying particular alleles or genotypes (allozyme data) characterised as “tolerant” or “sensitive”. A general reduction of the relative fecundity, the growth rate and the condition factor was highlighted in contaminated fish populations, suggesting that survival in such polluted systems implies energetic costs for fish thus reducing the energy available for particular functions. A lower observed heterozygosity was also detected in contaminated populations with respect to the Ster, suggesting a general decrease in genetic variability in response to chemical stress (with an exception for the Seine estuary). This study confirmed the previously detected relationship between PGM 85, AAT1 95 alleles and reduced DNA damage in contaminated fish [Marchand, J., Tanguy, A., Laroche, J., Quiniou, L., Moraga, D., 2003. Responses of European flounder Platichthys flesus populations to contamination in different estuaries along the Atlantic coast of France. Mar. Ecol. Prog. Ser. 260, 273–284] and furthermore suggested that, reduced fecundity and condition factor associated to the individuals carrying the previous alleles, were also reflecting the cost of resistance to stress in polluted populations. The cost of tolerance to stress as well as the high gene flow from neighbouring populations less exposed to contamination may explain the apparently moderate increase of the suspected “tolerant” alleles in contaminated flounder populations. [Copyright &y& Elsevier]

Plasma vitellogenin (VTG) concentrations and the presence of the ovo-testis (intersex) condition have been recorded in male flounder (Platichthys flesus) captured from several United Kingdom (UK) estuaries since 1996 as part of the endocrine disruption in the Marine Environment (EDMAR) project and earlier programs. It has been confirmed that plasma VTG concentrations in male flounder have remained elevated in several UK estuaries (e.g., Tees, Mersey, and Tyne) throughout the period covered by this study. However, the time-series data indicate that plasma VTG, a measure of environmental estrogen contamination, has decreased in fish captured from several estuaries, especially those of the Tyne and Mersey. Shorter time-series data sets from the Forth and Clyde estuaries also suggest a decrease in estrogen contamination at these sites. Trends associated with specific point sources of estrogenic contamination show site-specific patterns. For instance, plasma VTG levels in male flounder captured near the Howdon sewage treatment outfall (Tyne) have shown a steady decline to near baseline levels in 2001, while the plasma of male fish captured at a site adjacent to the Dabholm Gut discharge in the Tees estuary have shown little evidence of a sustained decline. The occurrence of the intersex condition was detected at a low but consistent prevalence through the study period, with the majority of cases recorded in fish captured from the Tyne and Mersey estuaries. The data set does not allow conclusions to be drawn about any temporal trends associated with this condition. The significance of the findings and possible mitigating influences are discussed in terms of the impacts on wild fish and the role of effluent treatment in reducing these. [ABSTRACT FROM AUTHOR]

Estrogenic discharges are known or suspected to be present in many United Kingdom estuaries and are also occurring from offshore oil and gas installations. The aim of this study was to establish whether estrogens and their mimics are present in marine waters at concentrations that can produce biological responses in fish and, if so, to discover whether the effects are likely to be harmful to populations and communities through changes in reproductive and other physiological processes. Laboratory studies and extensive field surveys were carried out with the euryhaline flounder (Platichthys flesus), using the amount of yolk protein vitellogenin in the blood of free-living male fish as an indicator of exposure to estrogens. Exposure to the synthetic estrogen ethynylestradiol for 3 weeks confirmed that the vitellogenin response was made and showed that this species is about one order of magnitude less sensitive than the freshwater species rainbow trout. The fish did not respond to the estrogen mimic nonylphenol at concentrations of 30 g/L. Wild male flounder were sampled from four contaminated United Kingdom estuaries: the Tyne, Crouch, Thames, and Mersey. Fish from one or more sites in each estuary had significantly elevatedconcentrations of vitellogenin in their blood when compared to concentrations in fish from the clean control estuary (Alde River). Vitellogenin concentrations in the Tyne and Mersey fish were four and six orders of magnitude greater, respectively, than control concentrations. Elevated vitellogenin concentrations were also seen in some male flounder caught in coastal areas. Approximately 20% of male fish from the Mersey estuary contained oocytes in their testes, but this intersex condition was not seen elsewhere. The implications of these findings for fish populations are discussed. [ABSTRACT FROM AUTHOR]

To investigate the presence of sub-lethal contaminant effects in flounder (Platichthys flesus) populations in English estuaries, 16 sitesfrom 7 estuaries were sampled in September-December 1997. Determination of hepatic ethoxyresorufin O-deethylase (EROD) activity revealed that significant (p<0.05) induction of the mixed function oxygenase (MFO) system was apparent in flounder from several industrialised estuaries compared to the relatively unpolluted reference site - the Aldeestuary. The estuaries in increasing order of observed mean EROD activity were: Alde, Southampton Water, Humber, Tyne, Wear, Tees, Mersey. No significant gender differences in EROD induction or other measured variables were discovered for any site. Limited analytical data suggest a close link with EROD induction and hepatic polycyclic aromatic hydrocarbons (PAH)/polychlorinated biphenyls (PCB) concentrations but no strong correlations were discovered between EROD activity induction with any other measured variable across all estuaries. However, in the Tyne estuary strong negative correlations were discovered between mean female EROD and female gonado-somatic index (GSI) and femalecondition factor (CF) of r=-0.84 and -0.80, respectively. It is suggested that the `out of season' correlation of reproductive parameterswith EROD could be linked to contaminant interference, perhaps by xeno-oestrogens. It is clear that flounder populations in English industrialised estuaries are exhibiting widespread sub-lethal contaminant effects and more research is essential to understand the potential implications of this for reproduction and disease that could be affecting the future viability of these populations. [ABSTRACT FROM AUTHOR]