Reaping the benefits of liquid handlers for high-throughput gene expression profiling in a marine model invertebrate.

BACKGROUND: Modern high-throughput technologies enable the processing of a large number of samples simultaneously, while also providing rapid and accurate procedures. In recent years, automated liquid handling workstations have emerged as an established technology for reproducible sample preparation. They offer flexibility, making them suitable for an expanding range of applications. Commonly, such approaches are well-developed for experimental procedures primarily designed for cell-line processing and xenobiotics testing. Conversely, little attention is focused on the application of automated liquid handlers in the analysis of whole organisms, which often involves time-consuming laboratory procedures. RESULTS: Here, we present a fully automated workflow for all steps, from RNA extraction to real-time PCR processing, for gene expression quantification in the ascidian marine model Ciona robusta. For procedure validation, we compared the results obtained with the liquid handler with those of the classical manual procedure. The outcome revealed comparable results, demonstrating a remarkable time saving particularly in the initial steps of sample processing. CONCLUSIONS: This work expands the possible application fields of this technology to whole-body organisms, mitigating issues that can arise from manual procedures. By minimizing errors, avoiding cross-contamination, decreasing hands-on time and streamlining the procedure, it could be employed for large-scale screening investigations.

Transcriptional regulation of the Ciona Gsx gene in the neural plate.

The ascidian neural plate consists of a defined number of identifiable cells organized in a grid of rows and columns, representing a useful model to investigate the molecular mechanisms controlling neural patterning in chordates. Distinct anterior brain lineages are specified via unique combinatorial inputs of signalling pathways with Nodal and Delta-Notch signals patterning along the medial-lateral axis and FGF/MEK/ERK signals patterning along the anterior-posterior axis of the neural plate. The Ciona Gsx gene is specifically expressed in the a9.33 cells in the row III/column 2 position of anterior brain lineages, characterised by a combinatorial input of Nodal-OFF, Notch-ON and FGF-ON. Here, we identify the minimal cis-regulatory element (CRE) of 376 bp, which can recapitulate the early activation of Gsx. We show that this minimal CRE responds in the same way as the endogenous Gsx gene to manipulation of FGF- and Notch-signalling pathways and to overexpression of Snail, a mediator of Nodal signals, and Six3/6, which is required to demarcate the anterior boundary of Gsx expression at the late neurula stage. We reveal that sequences proximal to the transcription start site include a temporal regulatory element required for the precise transcriptional onset of gene expression. We conclude that sufficient spatial and temporal information for Gsx expression is integrated in 376 bp of non-coding cis-regulatory sequences.

Patterning of brain precursors in ascidian embryos.

In terms of their embryonic origins, the anterior and posterior parts of the ascidian central nervous system (CNS) are associated with distinct germ layers. The anterior part of the sensory vesicle, or brain, originates from ectoderm lineages following a neuro-epidermal binary fate decision. In contrast, a large part of the remaining posterior CNS is generated following neuro-mesodermal binary fate decisions. Here, we address the mechanisms that pattern the anterior brain precursors along the medial-lateral axis (future ventral-dorsal) at neural plate stages. Our functional studies show that Nodal signals are required for induction of lateral genes, including Delta-like, Snail, Msxb and Trp Delta-like/Notch signalling induces intermediate (Gsx) over medial (Meis) gene expression in intermediate cells, whereas the combinatorial action of Snail and Msxb prevents the expression of Gsx in lateral cells. We conclude that despite the distinct embryonic lineage origins within the larval CNS, the mechanisms that pattern neural precursors are remarkably similar.

Detection of long non-coding RNA homology, a comparative study on alignment and alignment-free metrics.

BACKGROUND: Long non-coding RNAs (lncRNAs) represent a novel class of non-coding RNAs having a crucial role in many biological processes. The identification of long non-coding homologs among different species is essential to investigate such roles in model organisms as homologous genes tend to retain similar molecular and biological functions. Alignment-based metrics are able to effectively capture the conservation of transcribed coding sequences and then the homology of protein coding genes. However, unlike protein coding genes the poor sequence conservation of long non-coding genes makes the identification of their homologs a challenging task. RESULTS: In this study we compare alignment-based and alignment-free string similarity metrics and look at promoter regions as a possible source of conserved information. We show that promoter regions encode relevant information for the conservation of long non-coding genes across species and that such information is better captured by alignment-free metrics. We perform a genome wide test of this hypothesis in human, mouse, and zebrafish. CONCLUSIONS: The obtained results persuaded us to postulate the new hypothesis that, unlike protein coding genes, long non-coding genes tend to preserve their regulatory machinery rather than their transcribed sequence. All datasets, scripts, and the prediction tools adopted in this study are available at https://github.com/bioinformatics-sannio/lncrna-homologs .

Identification of long non-coding transcripts with feature selection: a comparative study.

BACKGROUND: The unveiling of long non-coding RNAs as important gene regulators in many biological contexts has increased the demand for efficient and robust computational methods to identify novel long non-coding RNAs from transcripts assembled with high throughput RNA-seq data. Several classes of sequence-based features have been proposed to distinguish between coding and non-coding transcripts. Among them, open reading frame, conservation scores, nucleotide arrangements, and RNA secondary structure have been used with success in literature to recognize intergenic long non-coding RNAs, a particular subclass of non-coding RNAs. RESULTS: In this paper we perform a systematic assessment of a wide collection of features extracted from sequence data. We use most of the features proposed in the literature, and we include, as a novel set of features, the occurrence of repeats contained in transposable elements. The aim is to detect signatures (groups of features) able to distinguish long non-coding transcripts from other classes, both protein-coding and non-coding. We evaluate different feature selection algorithms, test for signature stability, and evaluate the prediction ability of a signature with a machine learning algorithm. The study reveals different signatures in human, mouse, and zebrafish, highlighting that some features are shared among species, while others tend to be species-specific. Compared to coding potential tools and similar supervised approaches, including novel signatures, such as those identified here, in a machine learning algorithm improves the prediction performance, in terms of area under precision and recall curve, by 1 to 24%, depending on the species and on the signature. CONCLUSIONS: Understanding which features are best suited for the prediction of long non-coding RNAs allows for the development of more effective automatic annotation pipelines especially relevant for poorly annotated genomes, such as zebrafish. We provide a web tool that recognizes novel long non-coding RNAs with the obtained signatures from fasta and gtf formats. The tool is available at the following url: http://www.bioinformatics-sannio.org/software/ .

Mutation studies in ascidians: a review.

Historically, mutations have had a significant impact on the study of developmental processes and phenotypic evolution. Lesions in DNA are created by artificial methods or detected by natural genetic variation. Random mutations are then ascribed to genetic change by direct sequencing or positional cloning. Tunicate species of the ascidian genus Ciona represent nearly fully realized model systems in which gene function can be investigated in depth. Additionally, tunicates are valuable organisms for the study of naturally occurring mutations due to the capability to exploit genetic variation down to the molecular level. Here, we summarize the available information about how mutations are studied in ascidians with examples of insights that have resulted from these applications. We also describe notions and methodologies that might be useful for the implementation of easy and tight procedures for mutations studies in Ciona.

Ciona intestinalis as a marine model system to study some key developmental genes targeted by the diatom-derived aldehyde decadienal.

The anti-proliferative effects of diatoms, described for the first time in copepods, have also been demonstrated in benthic invertebrates such as polychaetes, sea urchins and tunicates. In these organisms PUAs (polyunsaturated aldehydes) induce the disruption of gametogenesis, gamete functionality, fertilization, embryonic mitosis, and larval fitness and competence. These inhibitory effects are due to the PUAs, produced by diatoms in response to physical damage as occurs during copepod grazing. The cell targets of these compounds remain largely unknown. Here we identify some of the genes targeted by the diatom PUA 2-trans-4-trans-decadienal (DD) using the tunicate Ciona intestinalis. The tools, techniques and genomic resources available for Ciona, as well as the suitability of Ciona embryos for medium-to high-throughput strategies, are key to their employment as model organisms in different fields, including the investigation of toxic agents that could interfere with developmental processes. We demonstrate that DD can induce developmental aberrations in Ciona larvae in a dose-dependent manner. Moreover, through a preliminary analysis, DD is shown to affect the expression level of genes involved in stress response and developmental processes.

FGF/MAPK/Ets signaling renders pigment cell precursors competent to respond to Wnt signal by directly controlling Ci-Tcf transcription.

FGF and Wnt pathways constitute two fundamental signaling cascades, which appear to crosstalk in cooperative or antagonistic fashions in several developmental processes. In vertebrates, both cascades are involved in pigment cell development, but the possible interplay between FGF and Wnt remains to be elucidated. In this study, we have investigated the role of FGF and Wnt signaling in development of the pigment cells in the sensory organs of C. intestinalis. This species possesses the basic features of an ancestral chordate, thus sharing conserved molecular developmental mechanisms with vertebrates. Chemical and targeted perturbation approaches revealed that a FGF signal, spreading in time from early gastrulation to neural tube closure, is responsible for pigment cell precursor induction. This signal is transmitted via the MAPK pathway, which activates the Ci-Ets1/2 transcription factor. Targeted perturbation of Ci-TCF, a downstream factor of the canonical Wnt pathway, indicated its contribution to pigment cell differentiation Furthermore, analyses of the Ci-Tcf regulatory region revealed the involvement of the FGF effector, Ci-Ets1/2, in Ci-Tcf transcriptional regulation in pigment cell precursors. Our results indicate that both FGF and the canonical Wnt pathways are involved in C. intestinalis pigment cell induction and differentiation. Moreover, we present a case of direct transcriptional regulation exerted by the FGF signaling cascade, via the MAPK-ERK-Ets1/2, on the Wnt downstream gene Ci-Tcf. Several examples of FGF/Wnt signaling crosstalk have been described in different developmental processes; however, to our knowledge, FGF-Wnt cross-interaction at the transcriptional level has never been previously reported. These findings further contribute to clarifying the multitude of FGF-Wnt pathway interactions.

A broad-taxa approach as an important concept in ecotoxicological studies and pollution monitoring.

Aquatic invertebrates play a pivotal role in (eco)toxicological assessments because they offer ethical, cost-effective and repeatable testing options. Additionally, their significance in the food chain and their ability to represent diverse aquatic ecosystems make them valuable subjects for (eco)toxicological studies. To ensure consistency and comparability across studies, international (eco)toxicology guidelines have been used to establish standardised methods and protocols for data collection, analysis and interpretation. However, the current standardised protocols primarily focus on a limited number of aquatic invertebrate species, mainly from Arthropoda, Mollusca and Annelida. These protocols are suitable for basic toxicity screening, effectively assessing the immediate and severe effects of toxic substances on organisms. For more comprehensive and ecologically relevant assessments, particularly those addressing long-term effects and ecosystem-wide impacts, we recommended the use of a broader diversity of species, since the present choice of taxa exacerbates the limited scope of basic ecotoxicological studies. This review provides a comprehensive overview of (eco)toxicological studies, focusing on major aquatic invertebrate taxa and how they are used to assess the impact of chemicals in diverse aquatic environments. The present work supports the use of a broad-taxa approach in basic environmental assessments, as it better represents the natural populations inhabiting various ecosystems. Advances in omics and other biochemical and computational techniques make the broad-taxa approach more feasible, enabling mechanistic studies on non-model organisms. By combining these approaches with in vitro techniques together with the broad-taxa approach, researchers can gain insights into less-explored impacts of pollution, such as changes in population diversity, the development of tolerance and transgenerational inheritance of pollution responses, the impact on organism phenotypic plasticity, biological invasion outcomes, social behaviour changes, metabolome changes, regeneration phenomena, disease susceptibility and tissue pathologies. This review also emphasises the need for harmonised data-reporting standards and minimum annotation checklists to ensure that research results are findable, accessible, interoperable and reusable (FAIR), maximising the use and reusability of data. The ultimate goal is to encourage integrated and holistic problem-focused collaboration between diverse scientific disciplines, international standardisation organisations and decision-making bodies, with a focus on transdisciplinary knowledge co-production for the One-Health approach.

Developmental toxicity of pre-production plastic pellets affects a large swathe of invertebrate taxa.

Microplastics pose risks to marine organisms through ingestion, entanglement, and as carriers of toxic additives and environmental pollutants. Plastic pre-production pellet leachates have been shown to affect the development of sea urchins and, to some extent, mussels. The extent of those developmental effects on other animal phyla remains unknown. Here, we test the toxicity of environmental mixed nurdle samples and new PVC pellets for the embryonic development or asexual reproduction by regeneration of animals from all the major animal superphyla (Lophotrochozoa, Ecdysozoa, Deuterostomia and Cnidaria). Our results show diverse, concentration-dependent impacts in all the species sampled for new pellets, and for molluscs and deuterostomes for environmental samples. Embryo axial formation, cell specification and, specially, morphogenesis seem to be the main processes affected by plastic leachate exposure. Our study serves as a proof of principle for the potentially catastrophic effects that increasing plastic concentrations in the oceans and other ecosystems can have across animal populations from all major animal superphyla.

Transcriptional and proteomic analysis of the innate immune response to microbial stimuli in a model invertebrate chordate.

Inflammatory response triggered by innate immunity can act to protect against microorganisms that behave as pathogens, with the aim to restore the homeostatic state between host and beneficial microbes. As a filter-feeder organism, the ascidian Ciona robusta is continuously exposed to external microbes that may be harmful under some conditions. In this work, we used transcriptional and proteomic approaches to investigate the inflammatory response induced by stimuli of bacterial (lipopolysaccharide -LPS- and diacylated lipopeptide - Pam2CSK4) and fungal (zymosan) origin, in Ciona juveniles at stage 4 of metamorphosis. We focused on receptors, co-interactors, transcription factors and cytokines belonging to the TLR and Dectin-1 pathways and on immune factors identified by homology approach (i.e. immunoglobulin (Ig) or C-type lectin domain containing molecules). While LPS did not induce a significant response in juvenile ascidians, Pam2CSK4 and zymosan exposure triggered the activation of specific inflammatory mechanisms. In particular, Pam2CSK4-induced inflammation was characterized by modulation of TLR and Dectin-1 pathway molecules, including receptors, transcription factors, and cytokines, while immune response to zymosan primarily involved C-type lectin receptors, co-interactors, Ig-containing molecules, and cytokines. A targeted proteomic analysis enabled to confirm transcriptional data, also highlighting a temporal delay between transcriptional induction and protein level changes. Finally, a protein-protein interaction network of Ciona immune molecules was rendered to provide a wide visualization and analysis platform of innate immunity. The in vivo inflammatory model described here reveals interconnections of innate immune pathways in specific responses to selected microbial stimuli. It also represents the starting point for studying ontogeny and regulation of inflammatory disorders in different physiological conditions.

Application of transcriptome profiling to inquire into the mechanism of nanoplastics toxicity during Ciona robusta embryogenesis.

The growing concern on nanoplastics (<1 µm) impact on marine life has stimulated a significant amount of studies aiming to address ecotoxicity and disclose their mechanisms of action. Here, we applied an integrative approach to develop an Adverse Outcome Pathway (AOP) upon acute exposure to amino-modified polystyrene nanoparticles (PS-NH2 NPs, 50 nm), as proxy for nanoplastics, during the embryogenesis of the chordate Ciona robusta. Genes related to glutathione metabolism, immune defense, nervous system, transport by aquaporins and energy metabolism were affected by either concentration tested of 10 or 15 µg mL-1 of PS-NH2. Transcriptomic data and in vivo experiments were assembled into two putative AOPs, identifying as key events the adhesion of PS-NH2 as (molecular) initiating event, followed by oxidative stress, changes in transcription of specific genes, morphological defects, increase in reactive oxygen species level, impaired swimming behavior. As final adverse outcomes, altered larval development, reduced metamorphosis and inhibition of hatching were identified. Our study attempts to define AOPs for PS-NH2 without excluding that chemicals leaching from them might also have a potential role in the observed outcome. Overall data provide new insights into the mechanism of action of PS-NH2 NPs during chordate embryogenesis and offer further keys for a better knowledge of nanoplastics impact on early stages of marine life.

Short-Term Exposure to Nanoplastics Does Not Affect Bisphenol A Embryotoxicity to Marine Ascidian Ciona robusta.

Plastic pollution is recognized as a global environmental threat and concern is increasing regarding the potential interactions of the smallest fragments, nanoplastics (1 µm), with either physical and chemical entities encountered in the natural environment, including toxic pollutants. The smallest size of nanoplastics (<100nm) rebounds to their safety associated with remarkable biological, chemical and physical reactivity that allow them to interact with cellular machinery by crossing biological barriers and causing damage to living beings. Recent findings on nanoplastic occurrence in marine coastal waters, including the Mediterranean Sea, leave open the question on their ability to act as a vector of other contaminants of emerging concerns (CECs) concomitantly released by wastewater treatment plants and reaching marine coastal waters. Here, we assess for the first time the role of non-functionalized polystyrene nanoparticles (PS NPs, 20 nm) as a proxy for nanoplastics (1 and 10 µg/mL) alone and in combination with bisphenol A (BPA) (4.5 and 10 µM) on Ciona robusta embryos (22 h post fertilization, hpf) by looking at embryotoxicity through phenotypic alterations. We confirmed the ability of BPA to impact ascidian C. robusta embryo development, by affecting sensory organs pigmentation, either alone and in combination with PS NPs. Our findings suggest that no interactions are taking place between PS NPs and BPA in filtered sea water (FSW) probably due to the high ionic strength of seawater able to trigger the sorption surface properties of PS NPs. Further studies are needed to elucidate such peculiarities and define the risk posed by combined exposure to BPA and PS NPs in marine coastal waters.

A pan-metazoan concept for adult stem cells: the wobbling Penrose landscape.

Adult stem cells (ASCs) in vertebrates and model invertebrates (e.g. Drosophila melanogaster) are typically long-lived, lineage-restricted, clonogenic and quiescent cells with somatic descendants and tissue/organ-restricted activities. Such ASCs are mostly rare, morphologically undifferentiated, and undergo asymmetric cell division. Characterized by 'stemness' gene expression, they can regulate tissue/organ homeostasis, repair and regeneration. By contrast, analysis of other animal phyla shows that ASCs emerge at different life stages, present both differentiated and undifferentiated phenotypes, and may possess amoeboid movement. Usually pluri/totipotent, they may express germ-cell markers, but often lack germ-line sequestering, and typically do not reside in discrete niches. ASCs may constitute up to 40% of animal cells, and participate in a range of biological phenomena, from whole-body regeneration, dormancy, and agametic asexual reproduction, to indeterminate growth. They are considered legitimate units of selection. Conceptualizing this divergence, we present an alternative stemness metaphor to the Waddington landscape: the 'wobbling Penrose' landscape. Here, totipotent ASCs adopt ascending/descending courses of an 'Escherian stairwell', in a lifelong totipotency pathway. ASCs may also travel along lower stemness echelons to reach fully differentiated states. However, from any starting state, cells can change their stemness status, underscoring their dynamic cellular potencies. Thus, vertebrate ASCs may reflect just one metazoan ASC archetype.

Brain Sensory Organs of the Ascidian Ciona robusta: Structure, Function and Developmental Mechanisms.

During evolution, new characters are designed by modifying pre-existing structures already present in ancient organisms. In this perspective, the Central Nervous System (CNS) of ascidian larva offers a good opportunity to analyze a complex phenomenon with a simplified approach. As sister group of vertebrates, ascidian tadpole larva exhibits a dorsal CNS, made up of only about 330 cells distributed into the anterior sensory brain vesicle (BV), connected to the motor ganglion (MG) and a caudal nerve cord (CNC) in the tail. Low number of cells does not mean, however, low complexity. The larval brain contains 177 neurons, for which a documented synaptic connectome is now available, and two pigmented organs, the otolith and the ocellus, controlling larval swimming behavior. The otolith is involved in gravity perception and the ocellus in light perception. Here, we specifically review the studies focused on the development of the building blocks of ascidians pigmented sensory organs, namely pigment cells and photoreceptor cells. We focus on what it is known, up to now, on the molecular bases of specification and differentiation of both lineages, on the function of these organs after larval hatching during pre-settlement period, and on the most cutting-edge technologies, like single cell RNAseq and genome editing CRISPR/CAS9, that, adapted and applied to Ciona embryos, are increasingly enhancing the tractability of Ciona for developmental studies, including pigmented organs formation.

Stem cells of aquatic invertebrates as an advanced tool for assessing ecotoxicological impacts.

Environmental stressors are assessed through methods that quantify their impacts on a wide range of metrics including species density, growth rates, reproduction, behaviour and physiology, as on host-pathogen interactions and immunocompetence. Environmental stress may induce additional sublethal effects, like mutations and epigenetic signatures affecting offspring via germline mediated transgenerational inheritance, shaping phenotypic plasticity, increasing disease susceptibility, tissue pathologies, changes in social behaviour and biological invasions. The growing diversity of pollutants released into aquatic environments requires the development of a reliable, standardised and 3R (replacement, reduction and refinement of animals in research) compliant in vitro toolbox. The tools have to be in line with REACH regulation 1907/2006/EC, aiming to improve strategies for potential ecotoxicological risks assessment and monitoring of chemicals threatening human health and aquatic environments. Aquatic invertebrates' adult stem cells (ASCs) are numerous and can be pluripotent, as illustrated by high regeneration ability documented in many of these taxa. This is of further importance as in many aquatic invertebrate taxa, ASCs are able to differentiate into germ cells. Here we propose that ASCs from key aquatic invertebrates may be harnessed for applicable and standardised new tests in ecotoxicology. As part of this approach, a battery of modern techniques and endpoints are proposed to be tested for their ability to correctly identify environmental stresses posed by emerging contaminants in aquatic environments. Consequently, we briefly describe the current status of the available toxicity testing and biota-based monitoring strategies in aquatic environmental ecotoxicology and highlight some of the associated open issues such as replicability, consistency and reliability in the outcomes, for understanding and assessing the impacts of various chemicals on organisms and on the entire aquatic environment. Following this, we describe the benefits of aquatic invertebrate ASC-based tools for better addressing ecotoxicological questions, along with the current obstacles and possible overhaul approaches.

Toxicity of nanoplastics during the embryogenesis of the ascidian Ciona robusta (Phylum Chordata).

Nanoplastics are considered contaminants of emerging concern at the global scale. The recent evidence of their occurrence in seawater from the Mediterranean Sea calls for a thorough evaluation of their impact on marine life and in particular on vulnerable life stages such as planktonic embryos. Here, we investigated the impact of increasing nominal concentrations of 50 nm amino-modified (PS-NH2) and 60 nm carboxy-modified (PS-COOH) polystyrene nanoparticles (PS NPs) on the embryonic development of the ascidian Ciona robusta (phylum Chordata), a common benthic invertebrate living in Mediterranean coastal areas with the peculiarity of being an early chordate developmental model. A strong agglomeration of PS-COOH (approx. 1 µm) was observed in natural sea water (NSW) already at time 0, while PS-NH2 resulted still monodispersed (approx. 130 nm) but largely aggregated after 22 h with a microscale dimension similar to those negatively charged. However, their effect on C. robusta embryos development largely differed at 22 h: PS-COOH did not affect larvae phenotypes nor their development, while PS-NH2 caused a dose-dependent effect (EC50 (22 h) of 7.52 µg mL-1) with various degrees of phenotype malformations (from mild to severe) and impairment of larval swimming. Embryos (up to 30%) exposed to 15 µg mL-1 PS-NH2 resulted not developed and the majority was unable to hatch. Calculated PS-NH2 EC50 resulted higher than those available for other marine invertebrate species, suggesting a protective role of the egg envelopes surrounding C. robusta embryos toward nanoplastics exposure.

New approaches on the use of tunicates (Ciona robusta) for toxicity assessments.

After the accidental release of crude oil in marine environment, dispersants are applied on sea surface transferring oil into the water column where it can be broken down by biodegradation, thereby reducing potential pollution to coastal areas. Before they can be used in the wild, the ecotoxicity of such dispersants is usually evaluated with toxicity assays using algae, crustacean and fishes. Nowadays, there is a need to find alternative species to reduce the use of vertebrates both for ethical considerations and for reducing the cost of bioassays. Ciona robusta is a solitary ascidian that inhabits shallow waters and marine coastal areas. This species has been recently adopted as valuable biological model for ecotoxicity studies, thanks to its rapid embryonic and larval development, resemblance to vertebrates, and low risk of ethical issues. On this ground, the lethal and sublethal toxicity of two dispersants has been evaluated on Ciona juveniles. At this stage, the organisms become filter-feeders and the morphological alterations of the organs can be easily observed. The median lethal concentrations at 96 h (96hLC50) for Dispersant 1 (non-ionic surfactant) and for Dispersant 2 (mixture of non-ionic surfactants and anionic surfactants) are 41.6 mg/L (38.6-44.9) and 92.5 mg/L (87.7-97.5), respectively. The Ciona juvenile model was compared to Dicentrarchus labrax fish juveniles test, and it showed increased sensitivity for Ciona to these compounds. These results suggest that 96 h mortality test bioassay could be a good alternative method to 96 h mortality assay with D. labrax, limiting the use of vertebrates for dispersant toxicity.