Development of a Zebrafish Embryo-Based Test System for Thyroid Hormone System Disruption: 3Rs in Ecotoxicological Research.

There is increasing concern regarding pollutants disrupting the vertebrate thyroid hormone (TH) system, which is crucial for development. Thus, identification of TH system-disrupting chemicals (THSDCs) is an important requirement in the Organisation for Economic Co-operation and Development (OECD) testing framework. The current OECD approach uses different model organisms for different endocrine modalities, leading to a high number of animal tests. Alternative models compatible with the 3Rs (replacement, reduction, refinement) principle are required. Zebrafish embryos, not protected by current European Union animal welfare legislation, represent a promising model. Studies show that zebrafish swim bladder inflation and eye development are affected by THSDCs, and the respective adverse outcome pathways (AOPs) have been established. The present study compared effects of four THSDCs with distinct molecular modes of action: Propylthiouracil (PTU), potassium perchlorate, iopanoic acid, and the TH triiodothyronine (T3) were tested with a protocol based on the OECD fish embryo toxicity test (FET). Effects were analyzed according to the AOP concept from molecular over morphological to behavioral levels: Analysis of thyroid- and eye-related gene expression revealed significant effects after PTU and T3 exposure. All substances caused changes in thyroid follicle morphology of a transgenic zebrafish line expressing fluorescence in thyrocytes. Impaired eye development and swimming activity were observed in all treatments, supporting the hypothesis that THSDCs cause adverse population-relevant changes. Findings thus confirm that the FET can be amended by TH system-related endpoints into an integrated protocol comprising molecular, morphological, and behavioral endpoints for environmental risk assessment of potential endocrine disruptors, which is compatible with the 3Rs principle. Environ Toxicol Chem 2024;00:1-18. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Contribution of the eye and of opn4xa function to circadian photoentrainment in the diurnal zebrafish.

The eye is instrumental for controlling circadian rhythms in mice and human. Here, we address the conservation of this function in the zebrafish, a diurnal vertebrate. Using lakritz (lak) mutant larvae, which lack retinal ganglion cells (RGCs), we show that while a functional eye contributes to masking, it is largely dispensable for the establishment of circadian rhythms of locomotor activity. Furthermore, the eye is dispensable for the induction of a phase delay following a pulse of white light at CT 16 but contributes to the induction of a phase advance upon a pulse of white light at CT21. Melanopsin photopigments are important mediators of photoentrainment, as shown in nocturnal mammals. One of the zebrafish melanopsin genes, opn4xa, is expressed in RGCs but also in photosensitive projection neurons in the pineal gland. Pineal opn4xa+ projection neurons function in a LIGHT ON manner in contrast to other projection neurons which function in a LIGHT OFF mode. We generated an opn4xa mutant in which the pineal LIGHT ON response is impaired. This mutation has no effect on masking and circadian rhythms of locomotor activity, or for the induction of phase shifts, but slightly modifies period length when larvae are subjected to constant light. Finally, analysis of opn4xa;lak double mutant larvae did not reveal redundancy between the function of the eye and opn4xa in the pineal for the control of phase shifts after light pulses. Our results support the idea that the eye is not the sole mediator of light influences on circadian rhythms of locomotor activity and highlight differences in the circadian system and photoentrainment of behaviour between different animal models.

Embryonic exposures to chemicals acting on brain aromatase lead to different locomotor effects in zebrafish larvae.

Pathways underlying neurodevelopmental effects of endocrine disruptors (EDs) remain poorly known. Expression of brain aromatase (aroB), responsible for estrogen production in the brain of teleosts, is regulated by estrogenic EDs and could play a role in their behavioral effects. We exposed zebrafish eleutheroembryos (0-120 h post-fertilization) to various concentrations of 16 estrogenic chemicals (incl. bisphenols and contraceptives), and of 2 aroB inhibitors. Behavior was monitored using a photomotor response test procedure. Both aroB inhibitors (clotrimazole and prochloraz) and a total of 6 estrogenic EDs induced significant behavioral alterations, including DM-BPA, BPC and BPS-MPE, three bisphenol substitutes which behavioral effects were, to our knowledge, previously unknown. However, no consensus was reported on the effects among tested substances. It appears that behavioral changes could not be linked to groups of substances defined by their specificity or potency to modulate aroB expression, or by their structure. Altogether, behavioral effects of estrogenic EDs in 120 h post-fertilization larvae appear unrelated to aroB but are nonetheless not to be neglected in the context of environmental safety.

Whole-genome sequencing identifies interferon-induced protein IFI6/IFI27-like as a strong candidate gene for VNN resistance in European sea bass.

BACKGROUND: Viral nervous necrosis (VNN) is a major disease that affects European sea bass, and understanding the biological mechanisms that underlie VNN resistance is important for the welfare of farmed fish and sustainability of production systems. The aim of this study was to identify genomic regions and genes that are associated with VNN resistance in sea bass. RESULTS: We generated a dataset of 838,451 single nucleotide polymorphisms (SNPs) identified from whole-genome sequencing (WGS) in the parental generation of two commercial populations (A: 2371 individuals and B: 3428 individuals) of European sea bass with phenotypic records for binary survival in a VNN challenge. For each population, three cohorts were submitted to a red-spotted grouper nervous necrosis virus (RGNNV) challenge by immersion and genotyped on a 57K SNP chip. After imputation of WGS SNPs from their parents, quantitative trait loci (QTL) were mapped using a Bayesian sparse linear mixed model (BSLMM). We found several QTL regions that were specific to one of the populations on different linkage groups (LG), and one 127-kb QTL region on LG12 that was shared by both populations and included the genes ZDHHC14, which encodes a palmitoyltransferase, and IFI6/IFI27-like, which encodes an interferon-alpha induced protein. The most significant SNP in this QTL region was only 1.9 kb downstream of the coding sequence of the IFI6/IFI27-like gene. An unrelated population of four large families was used to validate the effect of the QTL. Survival rates of susceptible genotypes were 40.6% and 45.4% in populations A and B, respectively, while that of the resistant genotype was 66.2% in population B and 78% in population A. CONCLUSIONS: We have identified a genomic region that carries a major QTL for resistance to VNN and includes the ZDHHC14 and IFI6/IFI27-like genes. The potential involvement of the interferon pathway, a well-known anti-viral defense mechanism in several organisms (chicken, human, or fish), in survival to VNN infection is of particular interest. Our results can lead to major improvements for sea bass breeding programs through marker-assisted genomic selection to obtain more resistant fish.

Temperature and feeding frequency impact the survival, growth, and metamorphosis success of Solea solea larvae.

Human-induced climate change impacts the oceans, increasing their temperature, changing their circulation and chemical properties, and affecting marine ecosystems. Like most marine species, sole has a biphasic life cycle, where one planktonic larval stage and juvenile/adult stages occur in a different ecological niche. The year-class strength, usually quantified by the end of the larvae stage, is crucial for explaining the species' recruitment. We implemented an experimental system for rearing larvae under laboratory conditions and experimentally investigated the effects of temperature and feeding frequencies on survival, development (growth), and metamorphosis success of S. solea larvae. Specific questions addressed in this work include: what are the effects of feeding regimes on larvae development? How does temperature impact larvae development? Our results highlight that survival depends on the first feeding, that the onset of metamorphosis varies according to rearing temperature and that poorly fed larvae take significantly longer to start (if they do) metamorphosing. Moreover, larvae reared at the higher temperature (a +4°C scenario) showed a higher incidence in metamorphosis defects. We discuss the implications of our results in an ecological context, notably in terms of recruitment and settlement. Understanding the processes that regulate the abundance of wild populations is of primary importance, especially if these populations are living resources exploited by humans.

Thermal imprinting during embryogenesis modifies skin repair in juvenile European sea bass (Dicentrarchus labrax).

Fish skin is a multifunctional tissue that develops during embryogenesis, a developmental stage highly susceptible to epigenetic marks. In this study, the impact of egg incubation temperature on the regeneration of a cutaneous wound caused by scale removal in juvenile European sea bass was evaluated. Sea bass eggs were incubated at 11, 13.5 and 16 °C until hatching and then were reared at a common temperature until 9 months when the skin was damaged and sampled at 0, 1 and 3 days after scale removal and compared to the intact skin from the other flank. Skin damage elicited an immediate significant (p < 0.001) up-regulation of pcna in fish from eggs incubated at higher temperatures. In fish from eggs incubated at 11 °C there was a significant (p < 0.001) up-regulation of krt2 compared to fish from higher thermal backgrounds 1 day after skin damage. Damaged epidermis was regenerated after 3 days in all fish irrespective of the thermal background, but in fish from eggs incubated at 11 °C the epidermis was significantly (p < 0.01) thinner compared to other groups, had less goblet cells and less melanomacrophages. The thickness of the dermis increased during regeneration of wounded skin irrespective of the thermal background and by 3 days was significantly (p < 0.01) thicker than the dermis from the intact flank. The expression of genes for ECM remodelling (mmp9, colXα, col1α1, sparc, and angptl2b) and innate immunity (lyg1, lalba, sod1, csf-1r and pparγ) changed during regeneration but were not affected by egg thermal regime. Overall, the results indicate that thermal imprinting of eggs modifies the damage-repair response in juvenile sea bass skin.

Exposure of zebrafish to an environmental mixture of persistent organic pollutants triggers an increase in anxiety-like syndrome but does not affect boldness in unexposed offspring.

Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are persistent organic pollutants (POPs) that are present as complex mixtures in all environmental compartments, including aquatic ecosystems. However, little is known about the effects of such complex mixtures on teleost behaviour. In this study, zebrafish (Danio rerio) were chronically exposed to an environmentally relevant mixture (MIX) containing 22 PCB and 7 PBDE congeners through diet from 5 days post fertilization onwards. MIX-exposed F0 fish produced offspring (F1 and F2 generations) that were fed using plain food and grown until adulthood. In each generation, five behavioural traits (i.e. boldness, activity, sociality, exploration and anxiety) were evaluated by the mean of different experimental set-ups. Two distinct behavioural syndromes were identified: boldness, positively correlated to activity and exploration; and anxiety, associated with low sociality. F0 fish did not display any behavioural disruption resulting from POP exposure whereas F1 MIX fish were bolder than fish from other generations but did not differ significantly from F1 controls. F2 MIX fish displayed a higher anxiety syndrome than F2 controls. This is of particular importance since such behavioural changes in offspring generations may have persistent ecological consequences, may affect fitness and hence cause detrimental effects on wild fish populations exposed to POP mixtures.

Revealing the Increased Stress Response Behavior through Transcriptomic Analysis of Adult Zebrafish Brain after Chronic Low to Moderate Dose Rates of Ionizing Radiation.

High levels of ionizing radiation (IR) are known to induce neurogenesis defects with harmful consequences on brain morphogenesis and cognitive functions, but the effects of chronic low to moderate dose rates of IR remain largely unknown. In this study, we aim at defining the main molecular pathways impacted by IR and how these effects can translate to higher organizational levels such as behavior. Adult zebrafish were exposed to gamma radiation for 36 days at 0.05 mGy/h, 0.5 mGy/h and 5 mGy/h. RNA sequencing was performed on the telencephalon and completed by RNA in situ hybridization that confirmed the upregulation of oxytocin and cone rod homeobox in the parvocellular preoptic nucleus. A dose rate-dependent increase in differentially expressed genes (DEG) was observed with 27 DEG at 0.05 mGy/h, 200 DEG at 0.5 mGy/h and 530 DEG at 5 mGy/h. Genes involved in neurotransmission, neurohormones and hypothalamic-pituitary-interrenal axis functions were specifically affected, strongly suggesting their involvement in the stress response behavior observed after exposure to dose rates superior or equal to 0.5 mGy/h. At the individual scale, hypolocomotion, increased freezing and social stress were detected. Together, these data highlight the intricate interaction between neurohormones (and particularly oxytocin), neurotransmission and neurogenesis in response to chronic exposure to IR and the establishment of anxiety-like behavior.

Environmental microplastics disrupt swimming activity in acute exposure in Danio rerio larvae and reduce growth and reproduction success in chronic exposure in D. rerio and Oryzias melastigma.

Microplastics (MPs), widely present in aquatic ecosystems, can be ingested by numerous organisms, but their toxicity remains poorly understood. Toxicity of environmental MPs from 2 beaches located on the Guadeloupe archipelago, Marie Galante (MG) and Petit-Bourg (PB) located near the North Atlantic gyre, was evaluated. A first experiment consisted in exposing early life stages of zebrafish (Danio rerio) to MPs at 1 or 10 mg/L. The exposure of early life stages to particles in water induced no toxic effects except a decrease in larval swimming activity for both MPs exposures (MG or PB). Then, a second experiment was performed as a chronic feeding exposure over 4 months, using a freshwater fish species, zebrafish, and a marine fish species, marine medaka (Oryzias melastigma). Fish were fed with food supplemented with environmentally relevant concentrations (1% wet weight of MPs in food) of environmental MPs from both sites. Chronic feeding exposure led to growth alterations in both species exposed to either MG or PB MPs but were more pronounced in marine medaka. Ethoxyresorufin-O-deethylase (EROD) and acetylcholinesterase (AChE) activities were only altered for marine medaka. Reproductive outputs were modified following PB exposure with a 70 and 42% decrease for zebrafish and marine medaka, respectively. Offspring of both species (F1 generation) were reared to evaluate toxicity following parental exposure on unexposed larvae. For zebrafish offspring, it revealed premature mortality after parental MG exposure and parental PB exposure produced behavioural disruptions with hyperactivity of F1 unexposed larvae. This was not observed in marine medaka offspring. This study highlights the ecotoxicological consequences of short and long-term exposures to environmental microplastics relevant to coastal marine areas, which represent essential habitats for a wide range of aquatic organisms.

The insecticide permethrin induces transgenerational behavioral changes linked to transcriptomic and epigenetic alterations in zebrafish (Danio rerio).

The pyrethroid insecticide permethrin is widely used for agricultural and domestic purposes. Previous data indicated that it acts as a developmental neurotoxicant and can induce transgenerational effects in non-target organisms. However, associated underlying mechanisms remain unclear. The aim of this study was to investigate permethrin-related transgenerational effects in the zebrafish model, and to identify possible molecular mechanisms underlying inheritance. Zebrafish (F0) were exposed to permethrin during early-life (2 h post-fertilization up to 28 days). The F1 and F2 offspring generations were obtained by pairing exposed F0 males and females, and were bred unexposed. Locomotor and anxiety behavior were investigated, together with transcriptomic and epigenomic (DNA methylation) changes in brains. Permethrin exposed F0 fish were hypoactive at adulthood, while males from the F1 and F2 generations showed a specific decrease in anxiety-like behavior. In F0, transcriptomic data showed enrichment in pathways related to glutamatergic synapse activity, which may partly underlie the behavioral effects. In F1 and F2 males, dysregulation of similar pathways was observed, including a subset of differentially methylated regions that were inherited from the F0 to the F2 generation and indicated stable dysregulation of glutamatergic signaling. Altogether, the present results provide novel evidence on the transgenerational neurotoxic effects of permethrin, as well as mechanistic insight: a transient exposure induces persistent transcriptional and DNA methylation changes that may translate into transgenerational alteration of glutamatergic signaling and, thus, into behavioral alterations.

Chronic feeding exposure to virgin and spiked microplastics disrupts essential biological functions in teleost fish.

Toxicity of polyethylene (PE) and polyvinyl chloride (PVC) microplastics (MPs), either virgin or spiked with chemicals, was evaluated in two short-lived fish using a freshwater species, zebrafish, and a marine species, marine medaka. Exposures were performed through diet using environmentally relevant concentrations of MPs over 4 months. No modification of classical biomarkers, lipid peroxidation, genotoxicity or F0 behaviour was observed. A significant decrease in growth was reported after at least two months of exposure. This decrease was similar between species, independent from the type of MPs polymer and the presence or not of spiked chemicals, but was much stronger in females. The reproduction was evaluated and it revealed a significant decrease in the reproductive output for both species and in far more serious numbers in medaka. PVC appeared more reprotoxic than PE as were MPs spiked with PFOS and benzophenone-3 compared to MPs spiked with benzo[a]pyrene. Further, PVC-benzophenone-3 produced behavioural disruption in offspring larvae. These results obtained with two species representing different aquatic environments suggest that microplastics exert toxic effects, slightly different according to polymers and the presence or not of sorbed chemicals, which may lead in all cases to serious ecological disruptions.

Genetic Parameters and Genome-Wide Association Studies of Quality Traits Characterised Using Imaging Technologies in Rainbow Trout, Oncorhynchus mykiss.

One of the top priorities of the aquaculture industry is the genetic improvement of economically important traits in fish, such as those related to processing and quality. However, the accuracy of genetic evaluations has been hindered by a lack of data on such traits from a sufficiently large population of animals. The objectives of this study were thus threefold: (i) to estimate genetic parameters of growth-, yield-, and quality-related traits in rainbow trout (Oncorhynchus mykiss) using three different phenotyping technologies [invasive and non-invasive: microwave-based, digital image analysis, and magnetic resonance imaging (MRI)], (ii) to detect quantitative trait loci (QTLs) associated with these traits, and (iii) to identify candidate genes present within these QTL regions. Our study collected data from 1,379 fish on growth, yield-related traits (body weight, condition coefficient, head yield, carcass yield, headless gutted carcass yield), and quality-related traits (total fat, percentage of fat in subcutaneous adipose tissue, percentage of fat in flesh, flesh colour); genotypic data were then obtained for all fish using the 57K SNP Axiom® Trout Genotyping array. Heritability estimates for most of the 14 traits examined were moderate to strong, varying from 0.12 to 0.67. Most traits were clearly polygenic, but our genome-wide association studies (GWASs) identified two genomic regions on chromosome 8 that explained up to 10% of the genetic variance (cumulative effects of two QTLs) for several traits (weight, condition coefficient, subcutaneous and total fat content, carcass and headless gutted carcass yields). For flesh colour traits, six QTLs explained 1-4% of the genetic variance. Within these regions, we identified several genes (htr1, gnpat, ephx1, bcmo1, and cyp2x) that have been implicated in adipogenesis or carotenoid metabolism, and thus represent good candidates for further functional validation. Finally, of the three techniques used for phenotyping, MRI demonstrated particular promise for measurements of fat content and distribution, while the digital image analysis-based approach was very useful in quantifying colour-related traits. This work provides new insights that may aid the development of commercial breeding programmes in rainbow trout, specifically with regard to the genetic improvement of yield and flesh-quality traits as well as the use of invasive and/or non-invasive technologies to predict such traits.

Chemicals sorbed to environmental microplastics are toxic to early life stages of aquatic organisms.

Microplastics are ubiquitous in aquatic ecosystems, but little information is currently available on the dangers and risks to living organisms. In order to assess the ecotoxicity of environmental microplastics (MPs), samples were collected from the beaches of two islands in the Guadeloupe archipelago, Petit-Bourg (PB) located on the main island of Guadeloupe and Marie-Galante (MG) on the second island of the archipelago. These samples have a similar polymer composition with mainly polyethylene (PE) and polypropylene (PP). However, these two samples are very dissimilar with regard to their contamination profile and their toxicity. MPs from MG contain more lead, cadmium and organochlorine compounds while those from PB have higher levels of copper, zinc and hydrocarbons. The leachates of these two samples of MPs induced sublethal effects on the growth of sea urchins and on the pulsation frequency of jellyfish ephyrae but not on the development of zebrafish embryos. The toxic effects are much more marked for samples from the PB site than those from the MG site. This work demonstrates that MPs can contain high levels of potentially bioavailable toxic substances that may represent a significant ecotoxicological risk, particularly for the early life stages of aquatic animals.

A comparison of behavioral and reproductive parameters between wild-type, transgenic and mutant zebrafish: Could they all be considered the same "zebrafish" for reglementary assays on endocrine disruption?

Transgenic zebrafish models are efficiently used to study the effects of endocrine disrupting chemicals (EDC); thereby informing on their mechanisms of action. However, given the reported differences between zebrafish strains at the genetical, physiological and behavioral levels; care should be taken before using these transgenic models for EDC testing. In the present study, we undertook a set of experiments in different transgenic and/or mutant zebrafish strains of interest for EDC testing: casper, cyp19a1a-eGFP, cyp19a1a-eGFP-casper, cyp11c1-eGFP, cyp11c1-eGFP-casper. Some behavioral traits, and some biochemical and reproductive physiological endpoints commonly used in EDC testing were assessed and compared to those obtained in WT AB zebrafish to ensure that transgene insertion and/or mutations do not negatively modify basal reproductive physiology or behavior of the fish. Behavioral traits considered as anxiety and sociality have been monitored. Sociality was evaluated by monitoring the time spent near congeners in a shuttle box while anxiety was evaluated using the Novel tank diving test. No critical difference was observed between strains for either sociality or anxiety level. Concerning reproduction, no significant difference in the number of eggs laid per female, in the viability of eggs or in the female circulating VTG concentrations was noted between the 5 transgenic/mutants and the WT AB zebrafish studied. In summary, the transgene insertion and the mutations had no influence on the endpoints measured in basal conditions. These results were a prerequisite to the use of these transgenic/mutant models for EDC testing. Next step will be to determine the sensitivity of these biological models to chemical exposure to accurately validate their use in existing fish assays for EDC testing.

An environmentally relevant mixture of polychlorinated biphenyls (PCBs) and polybrominated diphenylethers (PBDEs) disrupts mitochondrial function, lipid metabolism and neurotransmission in the brain of exposed zebrafish and their unexposed F2 offspring.

Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are persistent organic pollutants still present in aquatic environments despite their total or partial ban. Previously, we observed that an environmentally realistic mixture of these compounds affects energy balance, growth, and reproduction in exposed zebrafish (F0), and behavior in their unexposed offspring (F1-F4). In the present work, we performed lipidomic and transcriptomic analyses on brains of zebrafish (F0-F2) from exposed and control lineages to identify molecular changes that could explain the observed phenotypes. The use of both technologies highlighted that F0 zebrafish displayed impaired mitochondrial function and lipid metabolism regulation (depletion in triacylglycerols and phospholipids) which can explain disruption of energy homeostasis. A subset of the regulated biological pathways related to energetic metabolism and neurotransmission were inherited in F2. In addition, there were increasing effects on epigenetic pathways from the F0 to the F2 generation. Altogether, we show that the effects of an environmental exposure to PCBs and PBDEs on energetic metabolism as well as neurotransmission extend over 2 generations of zebrafish, possibly due to transgenerational epigenetic inheritance.

Sources of variation of DNA methylation in rainbow trout: combined effects of temperature and genetic background.

Phenotypic plasticity is a key component of the ability of organisms to respond to changing environmental conditions. In this study, we aimed to study the establishment of DNA methylation marks in response to an environmental stress in rainbow trout and to assess whether these marks depend on the genetic background. The environmental stress chosen here was temperature, a known induction factor of epigenetic marks in fish. To disentangle the role of epigenetic mechanisms such as DNA methylation in generating phenotypic variations, nine rainbow trout isogenic lines with no genetic variability within a line were used. For each line, half of the eggs were incubated at standard temperature (11°C) and the other half at high temperature (16°C), from eyed-stage to hatching. In order to gain a first insight into the establishment of DNA methylation marks in response to an early temperature regime (control 11°C vs. heated 16°C), we have studied the expression of 8 dnmt3 (DNA methyltransferase) genes, potentially involved in de novo methylation, and analysed global DNA methylation in the different rainbow trout isogenic lines using LUMA (LUminometric Methylation Assay). Finally, finer investigation of genome-wide methylation patterns was performed using EpiRADseq, a reduced-representation library approach based on the ddRADseq (Double Digest Restriction Associated DNA) protocol, for six rainbow trout isogenic lines. We have demonstrated that thermal history during embryonic development alters patterns of DNA methylation, but to a greater or lesser extent depending on the genetic background.

Multi- and transgenerational effects following early-life exposure of zebrafish to permethrin and coumarin 47: Impact on growth, fertility, behavior and lipid metabolism.

Transgenerational effects induced by environmental stressors are a threat to ecosystems and human health. However, there is still limited observation and understanding of the potential of chemicals to influence life outcomes over several generations. In the present study, we investigated the effects of two environmental contaminants, coumarin 47 and permethrin, on exposed zebrafish (F0) and their progeny (F1-F3). Coumarin 47 is commonly found in personal care products and dyes, whereas permethrin is used as a domestic and agricultural pyrethroid insecticide/insect repellent. Zebrafish (F0) were exposed during early development until 28 days post-fertilization and their progeny (F1-F3) were bred unexposed. On one hand, the effects induced by coumarin 47 suggest no multigenerational toxicity. On the other hand, we found that behavior of zebrafish larvae was significantly affected by exposure to permethrin in F1 to F3 generations with some differences depending on the concentration. This suggests persistent alteration of the neural or neuromuscular function. In addition, lipidomic analyses showed that permethrin treatment was partially correlated with lysophosphatidylcholine levels in zebrafish, an important lipid for neurodevelopment. Overall, these results stress out one of the most widely used pyrethroids can trigger long-term, multi- and possibly transgenerational changes in the nervous system of zebrafish. These neurobehavioral changes echo the effects observed under direct exposure to high concentrations of permethrin and therefore call for more research on mechanisms underlying effect inheritance.

Microplastics and sorbed contaminants - Trophic exposure in fish sensitive early life stages.

The present study evaluated very small microplastic particle (MPs) transfer to zebrafish and marine medaka larvae via prey experimentally exposed to MPs from the onset of feeding. Larvae were fed Paramecium or Artemia nauplii loaded with fluorescent 1-5 or 10-20 µm MP. Pollutant accumulation was analyzed by optically tracking of benzo[a]pyrene (BaP) and recording cyp1a transcription. Paramecium transferred 1-5 µm particles only, whereas Artemia efficiently transferred both MPs. Although zebrafish and medaka larvae fed from the onset of active food intake (2-3 dph, respectively) on Paramecium and from days 6-7 post-hatch on Artemia nauplii, neither MP accumulation nor translocation to tissues was detected. MP egestion started within few hours after ingestion. Cyp1a induction and fluorescent analyses proved BaP bioavailability after transfer via Paramecium and Artemia. Unicellular or plankton organisms ingest contaminants via MPS and transfer effectively these to sensitive early life-stages of vertebrates, giving rise to whole-life exposure.

Organic contaminants sorbed to microplastics affect marine medaka fish early life stages development.

The role of polyethylene microplastics 4-6 µm size (MPs) in the toxicity of environmental compounds to fish early life stages (ELS) was investigated. Marine medaka Oryzias melastigma embryos and larvae were exposed to suspended MPs spiked with three model contaminants: benzo(a)pyrene (MP-BaP), perfluorooctanesulfonic acid (MP-PFOS) and benzophenone-3 (MP-BP3) for 12 days. There was no evidence of MPs ingestion but MPs agglomerated on the surface of the chorion. Fish ELS exposed to virgin MPs did not show toxic effects. Exposure to MP-PFOS decreased embryonic survival and prevented hatching. Larvae exposed to MP-BaP or MP-BP3 exhibited reduced growth, increased developmental anomalies and abnormal behavior. Compared to equivalent waterborne concentrations, BaP and PFOS appeared to be more embryotoxic when spiked on MPs than when alone in seawater. These results suggest a relevant pollutant transfer by direct contact of MPs to fish ELS that should be included in the ecotoxicological risk assessment of MPs.