Intra-specific variability of root biomass production (RP) of six rooted macrophytes, i.e. Juncus effusus, Phragmites australis, Schoenoplectus lacustris, Typha latifolia, Phalaris arundinacea, and Iris pseudacorus grown from clones, in response to Cu exposure was investigated. Root biomass production varied widely for all these macrophytes in control conditions (0.08 μM) according to the sampling site. Root biomass production of T. latifolia and I. pseudacorus in the 2.5–25 μM Cu range depended on the sampling location but not on the Cu dose in the growth medium. For P. australis, J. effusus, S. lacustris, and P. arundinacea, an intra-specific variability of RP depending on both the sampling location and the Cu-dose was evidenced. This intra-specific variability of RP depending on the sampling location and of Cu-tolerance for these last four species suggests that Cu constitutive tolerance for all rooted macrophytes is not a species-wide trait but it exhibits variability for some species.

A new gravel-contact assay using rainbow trout, Oncorhynchus mykiss, embryos was developed to assess the toxicity of polycyclic aromatic hydrocarbons (PAHs) and other hydrophobic compounds. Environmentally realistic exposure conditions were mimicked with a direct exposure of eyed rainbow trout embryos incubated onto chemical-spiked gravels until hatching at 10 °C. Several endpoints were recorded including survival, hatching delay, hatching success, biometry, developmental abnormalities, and DNA damage (comet and micronucleus assays). This bioassay was firstly tested with two model PAHs, fluoranthene and benzo[a]pyrene. Then, the method was applied to compare the toxicity of three PAH complex mixtures characterized by different PAH compositions: a pyrolytic extract from a PAH-contaminated sediment (Seine estuary, France) and two petrogenic extracts from Arabian Light and Erika oils, at two environmental concentrations, 3 and 10 μg g−1 sum of PAHs. The degree and spectrum of toxicity were different according to the extract considered. Acute effects including embryo mortality and decreased hatching success were observed only for Erika oil extract. Arabian Light and pyrolytic extracts induced mainly sublethal effects including reduced larvae size and hemorrhages. Arabian Light and Erika extracts both induced repairable DNA damage as revealed by the comet assay versus the micronucleus assay. The concentration and proportion of methylphenanthrenes and methylanthracenes appeared to drive the toxicity of the three PAH fractions tested, featuring a toxic gradient as follows: pyrolytic < Arabian Light < Erika. The minimal concentration causing developmental defects was as low as 0.7 μg g−1 sum of PAHs, indicating the high sensitivity of the assay and validating its use for toxicity assessment of particle-bound pollutants.

The growing need to evaluate the quality of aquatic ecosystems led to the development of numerous monitoring tools. Among them, the development of biomarker-based procedures, that combine precocity and relevance, is recommended. However, multi-biomarker approaches are often hard to interpret, and produce results that are not easy to integrate in the environmental policies framework. Integrative index have been developed, and one of the most used is the integrated biomarker response (IBR). However, an analysis of available literature demonstrated that the IBR suffers from a frequent misuse and a bias in its calculation. Then, we propose here a new calculation method based on both a more simple formula and a permutation procedure. Together, these improvements should rightly avoid the misuse and bias that were recorded. Additionally, a case study illustrates how the new procedure enabled to perform a reliable classification of site along a pollution gradient based on biomarker responses used in the IBR calculations.

Most persistent organic pollutants, due to their hydrophobic properties, accumulate in aquatic sediments and represent a high risk for sediment quality. To assess the toxicity of hydrophobic pollutants, a novel approach was recently proposed as an alternative to replace, refine and reduce animal experimentation: the medaka embryo–larval sediment contact assay (MELAc). This assay is performed with Japanese medaka embryos incubated on a natural sediment spiked with the compound being tested. With the aim of improving this assay, our study developed a reference exposure protocol with an artificial sediment specifically designed to limit natural sediment composition uncertainties and preparation variability. The optimum composition of the new artificial sediment was tested using a model polycyclic aromatic hydrocarbon (PAH), fluoranthene. The sediment was then validated with two other model PAHs, benz[a]anthracene and benzo[a]pyrene. Various developmental end points were recorded, including survival, embryonic heartbeat, hatching delay, hatching success, larval biometry and abnormalities. The final artificial sediment composition was set at 2.5 % dry weight (dw) Sphagnum peat, 5 % dw kaolin clay and 92.5 % dw silica of 0.2- to 0.5-mm grain size. In contrast with natural sediments, the chemical components of this artificial matrix are fully defined and readily identifiable. It is totally safe for fish embryos and presents relatively high sorption capacities for hydrophobic compounds. Studies with other hydrophobic and metallic contaminants and mixtures should be performed to further validate this artificial sedimen

In the last 10 years, behavior assessment has been developed as an indicator of neurotoxicity and an integrated indicator of physiological disruption. Polycyclic aromatic hydrocarbon (PAH) release into the environment has increased in recent decades resulting in high concentrations of these compounds in the sediment of contaminated areas. We evaluated the behavioral consequences of long-term chronic exposure to PAHs, by exposing zebrafish to diets spiked with three PAH fractions at environmentally relevant concentrations. Fish were exposed to these chemicals from their first meal (5 days postfertilization) until they became reproducing adults (at 6 months old). The fractions used were representative of PAHs of pyrolytic (PY) origin and of two oils differing in composition (a heavy fuel oil (HO) and a light crude oil (LO)). Several tests were carried out to evaluate circadian spontaneous swimming activity, responses to a challenge (photomotor response), exploratory tendencies, and anxiety levels. We found that dietary PAH exposure was associated with greater mobility, lower levels of exploratory activity, and higher levels of anxiety, particularly in fish exposed to the HO fraction and, to a lesser extent, the LO fraction. Finally, our results indicate that PAH mixtures of different compositions, representative of situations encountered in the wild, can induce behavioral disruptions resulting in poorer fish performance.

In aquatic environments, polycyclic aromatic hydrocarbons (PAHs) mostly occur as complex mixtures, for which risk assessment remains problematic. To better understand the effects of PAH mixture toxicity on fish early life stages, this study compared the developmental toxicity of three PAH complex mixtures. These mixtures were extracted from a PAH-contaminated sediment (Seine estuary, France) and two oils (Arabian Light and Erika). For each fraction, artificial sediment was spiked at three different environmental concentrations roughly equivalent to 0.5, 4, and 10 μg total PAH g−1 dw. Japanese medaka embryos were incubated on these PAH-spiked sediments throughout their development, right up until hatching. Several endpoints were recorded at different developmental stages, including acute endpoints, morphological abnormalities, larvae locomotion, and genotoxicity (comet and micronucleus assays). The three PAH fractions delayed hatching, induced developmental abnormalities, disrupted larvae swimming activity, and damaged DNA at environmental concentrations. Differences in toxicity levels, likely related to differences in PAH proportions, were highlighted between fractions. The Arabian Light and Erika petrogenic fractions, containing a high proportion of alkylated PAHs and low molecular weight PAHs, were more toxic to Japanese medaka early life stages than the pyrolytic fraction. This was not supported by the toxic equivalency approach, which appeared unsuitable for assessing the toxicity of the three PAH fractions to fish early life stages. This study highlights the potential risks posed by environmental mixtures of alkylated and low molecular weight PAHs to early stages of fish development.

Alexandrium catenella (group IV) and Alexandrium tamarense (group III) (Dinophyceae) are two cryptic invasive phytoplankton species belonging to the A. tamarense species complex. Their worldwide spread is favored by the human activities, transportation and climate change. In order to describe their diversity in the Mediterranean Sea and understand their settlements and maintenances in this area, new microsatellite markers were developed based on Thau lagoon (France) samples of A. catenella and A. tamarense strains. In this study twelve new microsatellite markers are proposed. Five of these microsatellite markers show amplifications on A. tamarense and ten on A. catenella. Three of these 12 microsatellite markers allowed amplifications on both cryptic species. Finally, the haplotypic diversity ranged from 0.000 to 0.791 and 0.000 to 0.942 for A. catenella and A. tamarense respectively.

Due to hydrophobic and persistent properties, polycyclic aromatic hydrocarbons (PAHs) have a high capacity to accumulate in sediment. Sediment quality criteria, for the assessment of habitat quality and risk for aquatic life, include understanding the fate and effects of PAHs. In the context of European regulation (REACH and Water Framework Directive), the first objective was to assess the influence of sediment composition on the toxicity of two model PAHs, benzo[a]pyrene and fluoranthene using 10-day zebrafish embryo-larval assay. This procedure was undertaken with an artificial sediment in order to limit natural sediment variability. A suitable sediment composition might be then validated for zebrafish and proposed in a new OECD guideline for chemicals testing. Second, a comparative study of toxicity responses from this exposure protocol was then performed using another OECD species, the Japanese medaka. The potential toxicity of both PAHs was assessed through lethal (e.g., survival, hatching success) and sublethal endpoints (e.g., abnormalities, PMR, and EROD) measured at different developmental stages, adapted to the embryonic development time of both species. Regarding effects observed for both species, a suitable artificial sediment composition for PAH toxicity testing was set at 92.5 % dry weight (dw) silica of 0.2-0.5-mm grain size, 5 % dw kaolin clay without organic matter for zebrafish, and 2.5 % dw blond peat in more only for Japanese medaka. PAH bioavailability and toxicity were highly dependent on the fraction of organic matter in sediment and of the K (ow) coefficients of the tested compounds. The biological responses observed were also dependent of the species under consideration. Japanese medaka embryos appeared more robust than zebrafish embryos for understanding the toxicity of PAHs following a sediment contact test, due to the longer exposure duration and lower sensitivity of sediment physical properties.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants that can be present at high levels as mixtures in polluted aquatic environments. Many PAHs are potent mutagens and several are well-known carcinogens. Despite numerous studies on individual compounds, little is known about the toxicity of PAHs mixtures that are encountered in environmental situations. In the present work, zebrafish were continuously fed from 5 days post-fertilisation to 14 months post-fertilisation (mpf) with a diet spiked with fractions of either pyrolytic (PY), petrogenic light oil (LO), or petrogenic heavy oil (HO) origin at three concentrations. A decrease in survival was identified after 3 mpf in fish fed with the highest concentration of HO or LO, but not for PY. All PAH fractions caused preneoplastic and neoplastic disorders in long-term-exposed animals. Target tissues were almost exclusively of epithelial origin, with the bile duct epithelium being the most susceptible to chronic exposure to all PAH fractions, and with germ cells being the second most responsive cells. Significantly higher incidences of neoplasms were observed with increasing PAH concentration and exposure duration. The most severe carcinogenic effects were induced by dietary exposure to HO compared to exposure to LO or PY (45, 30 and 7 %, respectively, after 9 to 10 months of exposure to an intermediate concentration of PAHs). In contrast, earliest carcinogenic effects were detected as soon as 3 mpf after exposure to LO, including the lowest concentration, or to PY. PAH bioactivation and genotoxicity in blood was assessed by ethoxyresorufin-O-deethylase activity quantification and comet and micronuclei assays, respectively, but none of these were positive. Chronic dietary exposure of zebrafish to PAH mixtures results in carcinogenotoxic events that impair survival and physiology of exposed fish.