Early Life Stages Of Zebrafish Research Articles

Developmental toxicity and mechanism of dibutyl phthalate and alternative diisobutyl phthalate in the early life stages of zebrafish (Danio rerio)

Diisobutyl phthalate (DiBP), is widely chemical replacement for Dibutyl phthalate (DBP). Although DBP and DiBP have been detected in surface water worldwide, few studies to date have systematically assessed the risks of DBP and its alternatives to aquatic organisms. The present study compared DBP and DiBP for their individual and joint toxicity as well as thyroid hormone levels in zebrafish embryo. Transcripts of key genes related to the hypothalamic-pituitary-thyroid (HPT) axis were investigated in developing zebrafish larvae by application of real time polymerase chain reaction. The median half-lethal concentrations of DBP and DiBP to zebrafish at 96 h were 0.545 mg L−1 and 1.149 mg L−1, respectively. The joint toxic effect of DBP–DiBP (0.25–0.53 mg L−1) with the same ratio showed a synergistic effect. Thyroid hormones levels increased with exposure to 10 μg L−1 of DBP or 50 μg L−1 of DiBP, and exposure to both compounds significantly increased thyroid gland-specific transcription of thyroglobulin gene (tg), hyronine deiodinase (dio2), and transthyretin (ttr), indicating an adverse effect associated with the HPT axis. Molecular docking results indicated that DBP (−7.10 kcal/M and −7.53 kcal/M) and DiBP (−6.63 kcal/M and −7.42 kcal/M) had the same docking energy with thyroid hormone receptors. Our data facilities an understand of potential harmful effects of DBP and its alternative (DiBP).

Exposure to acifluorfen induces developmental toxicity in the early life stage of zebrafish

Acifluorfen, a selective herbicide from the diphenyl ether family, targets broad leaf weeds. Diphenyl ether inhibits chlorophyll production in green plants by inhibiting protoporphyrinogen oxidase (PPO), causing cellular damage. Despite its known impacts on plants, the influence of acifluorfen on zebrafish embryo development remains unclear. In this study, we explored the LC50 of acifluorfen in early-stage wild-type zebrafish, determining it to be 54.99 mg/L. Subsequent examinations revealed morphological changes in zebrafish, including reduced body length. Using the cmlc2:dsRED transgenic model, we observed heart dysfunction in acifluorfen-exposed zebrafish, marked by an enlarged heart area, edema, and decreased heart rate. In response to dose-dependent acifluorfen exposure, the inhibition of angiogenesis in the brain was observed in transgenic zebrafish models (fli1a:eGFP). Organ malformations, specifically in the liver and pancreas, were noted, in lfabp:dsRED;elastase:eGFP transgenic models, indicating reduced organ size in acifluorfen-exposed zebrafish. Furthermore, acifluorfen heightened the expression of apoptosis-related genes (casp8, casp9, and tp53) in zebrafish embryos. We then determined whether acifluorfen affected the viability of zebrafish liver (ZFL) cells based on its effects on liver development in vivo. The results indicated that the proliferation of ZFL cells decreased significantly in a dose-dependent manner. Additionally, acifluorfen-treated ZFL cells exhibited a slight increase in apoptotic cells stained with annexin V and propidium iodide. In summary, these findings establish a baseline concentration for acifluorfen's effects on aquatic ecosystems and non-target organisms.

Embryotoxicity Induced by Triclopyr in Zebrafish (Danio rerio) Early Life Stage.

Triclopyr, an auxin-like herbicide that is widely employed for managing weeds in food crops and pastures, has been identified in various environmental settings, particularly aquatic ecosystems. Limited understanding of the environmental fate of this herbicide, its potential repercussions for both the environment and human health, and its insufficient monitoring in diverse environmental compartments has caused it to be recognized as an emerging contaminant of concern. In this study, we have investigated how triclopyr affects zebrafish, considering a new alternative methodology. We focused on the endpoints of developmental toxicity, neurotoxicity, and behavior of zebrafish embryos and larvae. We determined that triclopyr has a 96 h median lethal concentration of 87.46 mg/L (341.01 µM). When we exposed zebrafish embryos to sublethal triclopyr concentrations (0.5, 1, 5, 10, and 50 μM) for up to 144 h, we found that 50 µM triclopyr delayed zebrafish egg hatchability. Yolk sac malabsorption was significant at 0.5, 1, 5, and 10 µM triclopyr. In zebrafish larvae, uninflated swim bladder was significant only at 50 µM triclopyr. Furthermore, zebrafish larvae had altered swimming activity after exposure to 10 µM triclopyr for 144 h. In summary, these comprehensive results indicate that even low triclopyr concentrations can elicit adverse effects during early zebrafish development.

Multilevel assessment of carbamazepine effects: An integrative approach using zebrafish early-life stages

Carbamazepine (CBZ) is the most commonly used drug in epilepsy treatment, and its metabolites are commonly detected among persistent pharmaceuticals in the aquatic environment. This study aimed to investigate CBZ effects on early-life-stage zebrafish (Danio rerio) (from 2 to 168 hpf) by employing of an integrative approach linking endpoints from molecular to individual level: (i) development; (ii) locomotor activity; (iii) biochemical markers (lactate dehydrogenase, glutathione-S-transferase, acetylcholinesterase and catalase) and (iv) transcriptome analysis using microarray. A 168 h - LC50 of 73.4 mg L−1 and a 72 h - EC50 of 66.8 mg L−1 for hatching were calculated while developmental effects (oedemas and tail deformities) were observed at CBZ concentrations above 37.3 mg L−1. At the biochemical level, AChE activity proved to be the most sensitive parameter, as evidenced by its decrease across all concentrations tested (∼25% maximum reduction, LOEC (lowest observed effect concentration) < 0.6 μg L−1). Locomotor behaviour seemed to be depressed by CBZ although this effect was only evident at the highest concentration tested (50 mg L−1). Molecular analysis revealed a dose-dependent effect of CBZ on gene expression. Although only 25 genes were deregulated in organisms exposed to CBZ when compared to controls, both 0.6 and 2812 μg L−1 treatments impaired gene expression related to development (e.g. crygmxl1, org, klf2a, otos, stx16 and tob2) and the nervous system (e.g. Rtn3, Gdf10, Rtn3), while activated genes were associated with behavioural response (e.g. prlbr and taar). Altogether, our results indicate that environmentally relevant CBZ concentrations might affect biochemical and genetic traits of fish. Thus, the environmental risk of CBZ cannot be neglected, especially in a realistic scenario of constant input of domestic effluents into aquatic systems.

'Environmental standard limit concentration' arsenic exposure is associated with anxiety, depression, and autism-like changes in early-life stage zebrafish

Arsenic is a worldwide environmental pollutant that can impair human health. Previous studies have identified mental disorders induced by arsenic, but the environmental exposure concentrations in the early life stages associated with these disorders are poorly understood. In the present study, early-life stage zebrafish were used to explore the effects on mental disorders under 'environmental standard limit concentrations' arsenic exposures of 5, 10, 50, 150, and 500 μg/L. The results showed that arsenic exposure at these concentrations changed the locomotor behavior in larval zebrafish and was further associated with anxiety, depression, and autism-like behavior in both larval and juvenile zebrafish. Changes were noted at benchmark dose limit (BMDL) concentrations as low as 0.81 μg/L. Transcriptomics showed that immediate early genes (IEGs) fosab, egr1, egr2a, ier2b, egr3, and jund were decreased after arsenic exposure in larval and juvenile zebrafish. Nervous system impairment and anxiety, depression, and autism-like behaviors in early-life stage zebrafish at 'environmental standard limit concentrations' may be attributed to the downregulation of IEGs. These findings in zebrafish provided new experimental support for an arsenic toxicity threshold for mental disorders, and they suggest that low levels of environmental chemicals may be causative developmental factors for mental disorders.

An Integrated Metabolomics-Based Model, and Identification of Potential Biomarkers, of Perfluorooctane Sulfonic Acid Toxicity in Zebrafish Embryos.

Known for their high stability and surfactant properties, per- and polyfluoroalkyl substances (PFAS) have been widely used in a range of manufactured products. Despite being largely phased out due to concerns regarding their persistence, bioaccumulation, and toxicity, legacy PFAS such as perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid continue to persist at high levels in the environment, posing risks to aquatic organisms. We used high-resolution magic angle spinning nuclear magnetic resonance spectroscopy in intact zebrafish (Danio rerio) embryos to investigate the metabolic pathways altered by PFOS both before and after hatching (i.e., 24 and 72 h post fertilization [hpf], respectively). Assessment of embryotoxicity found embryo lethality in the parts-per-million range with no significant difference in mortality between the 24- and 72-hpf exposure groups. Metabolic profiling revealed mostly consistent changes between the two exposure groups, with altered metabolites generally associated with oxidative stress, lipid metabolism, energy production, and mitochondrial function, as well as specific targeting of the liver and central nervous system as key systems. These metabolic changes were further supported by analyses of tissue-specific production of reactive oxygen species, as well as nontargeted mass spectrometric lipid profiling. Our findings suggest that PFOS-induced metabolic changes in zebrafish embryos may be mediated through previously described interactions with regulatory and transcription factors leading to disruption of mitochondrial function and energy metabolism. The present study proposes a systems-level model of PFOS toxicity in early life stages of zebrafish, and also identifies potential biomarkers of effect and exposure for improved environmental biomonitoring. Environ Toxicol Chem 2024;43:896-914. © 2024 SETAC.

Elucidating the effects of pure glyphosate and a commercial formulation on early life stages of zebrafish using a complete biomarker approach: All-or-nothing!

The search for new methods in the toxicology field has increased the use of early life stages of zebrafish (Danio rerio) as a versatile organism model. Here, we use early stages of zebrafish to evaluate glyphosate as pure active ingredient and within a commercial formulation in terms of oxidative stress. Biomarkers involved in the oxidative status were evaluated along with other markers of neurotoxicity, genotoxicity, cytotoxicity, energy balance and motor performance, and the selected tools were evaluated by its sensitivity in determining early-warning events. Zebrafish embryos exposed to glyphosate active ingredient and glyphosate-based formulation were under oxidative stress, but only the commercial formulation delayed the embryogenesis, affected the cholinergic neurotransmission and induced DNA damage. Both altered the motor performance of larvae at very low concentrations, becoming larvae hypoactive. The energy balance was also impaired, as embryos under oxidative stress had lower lipids reserves. Although data suggest that glyphosate-based formulation has higher toxicity than the active ingredient itself, the most sensitive biomarkers detected early-warning effects at very low concentrations of the active ingredient. Biochemical biomarkers of defense system and oxidative damage were the most sensitive tools, detecting pro-oxidant responses at very low concentrations, along with markers of motor performance that showed high sensitivity and high throughput, suitable for detecting early effects linked to neurotoxicity. Alterations on morphology during embryogenesis showed the lowest sensitivity, thus morphological alterations appeared after several alterations at biochemical levels. Tools evaluating DNA damage and cell proliferation showed mid-sensitivity, but low throughput, thus they could be used as complementary markers.

Cortisol Quantification for Assessing Stress-Induced Changes in Zebrafish Larvae.

The stress response, mainly mediated by cortisol, plays a critical role in the regulation of physiological and behavioral homeostasis through a variety of mechanisms. Different aquatic animal models have been widely employed to understand the pathobiology of stress and stress-related brain disorders. The early life stress can affect the hypothalamic-pituitary-interrenal (HPI) axis and induce cellular and molecular impairments that impact the brain functioning later in life. However, these alterations have been poorly explored mainly due to the lack of suitable models. In this chapter, the vortex flow stimulation, an acute stress that causes a forced swimming and activates the HPI axis, is described and its correlations with behavioral outputs reported. To this end, the early life stages of zebrafish are used as animal models for modeling stress disorders experimentally. The behavioral despair model can be employed as an initial screening tool for assessing neural circuit activation and motor alterations. Taken together, the implementation of this strategy in this animal model allows the analysis of stress responses in a simple manner and its correlation with neural circuitries and motor alterations.

Effects of exposure to 3,6-DBCZ on neurotoxicity and AhR pathway during early life stages of zebrafish (Danio rerio)

Polyhalogenated carbazoles (PHCZs) are emerging environmental pollutants, yet limited information is available on their embryotoxicity and neurotoxicity. Therefore, the current work was performed to investigate the adverse effects of 3,6-dibromocarbazole (3,6-DBCZ), a typical PHCZs homolog, on the early life stages of zebrafish larvae. It revealed that the 96-hour post-fertilization (hpf) median lethal concentration (LC50) value of 3,6-DBCZ in zebrafish larvae was determined to be 0.7988 mg/L. Besides, 3,6-DBCZ reduced survival rates at concentrations ≥ 1 mg/L and decreased hatching rates at ≥ 0.25 mg/L at 48 hpf. In behavior tests, it inhibited locomotor activities and reduced the frequency of recorded acceleration states in response to optesthesia (a sudden bright light stimulus) at concentrations ≥ 160 μg/L. Meanwhile, 3,6-DBCZ exposure decreased the frequency of recorded acceleration states in the startle response (tapping mode) at concentrations ≥ 6.4 μg/L. Pathologically, with the transgenic zebrafish model (hb9-eGFP), we observed a strikingly decreased axon length and number in motor neurons after 3,6-DBCZ treatment, which may be ascribed to the activation of the AhR signaling pathway, as evidenced by the molecular docking analysis and Microscale thermophoresis (MST) assay suggested that 3,6-DBCZ binding to AhR-ARNT2 compound proteins. Through interaction with AhR-ARNT, a striking reduction of the anti-oxidative stress (sod1/2, nqo1, nrf2) and neurodevelopment-related genes (elavl3, gfap, mbp, syn2a) were observed after 3,6-DBCZ challenge, accompanied by a marked increased inflammatory genes (TNFβ, IL1β, IL6). Collectively, our findings reveal a previously unrecognized adverse effect of 3,6-DBCZ on zebrafish neurodevelopment and locomotor behaviors, potentially mediated through the activation of the AhR pathway. Furthermore, it provides direct evidence for the toxic concentrations of 3,6-DBCZ and the potential target signaling in zebrafish larvae, which may be beneficial for the risk assessment of the aquatic ecosystems.

Combined inhibitory effects of microcystin-LR and microcystin-RR on growth and development in zebrafish larvae

Microcystins (MCs) are the most widespread, frequently found, and seriously toxic cyanobacterial toxins in aquatic environments. Microcystin-leucine-arginine (MCLR) and microcystin-arginine-arginine (MCRR) are the most studied MCs. Normally, their levels are low and they coexist in the environment; however, they may also interact with each other. The developmental toxicity of MCLR in the presence of MCRR in the early life stage of zebrafish (from 2 to 120 h post fertilization) was investigated for the first time in this study. Our findings revealed that MCRR treatment marginally elevated thyroxine (T4) and 3,5,3′-triiodothyronine (T3) levels, whereas MCLR treatment alone resulted in a significant increase in T3 and T4 levels, indicating a cooperative effect. Furthermore, clear changes in the expression levels of genes involved in growth and development, accompanied by growth inhibition, were observed after co-treatment with MCRR and MCLR. In addition, zebrafish larvae subjected to MCRR and/or MCLR treatment showed increased levels of superoxide dismutase, glutathione, and malondialdehyde, and decreased levels of catalase in the MCRR + MCLR group, indicating oxidative stress and lipid peroxidation. Thus, we investigated the synergistic developmental toxicity of MCRR and MCLR during the early life stages of zebrafish development.

Analysis of vascular disruption in zebrafish embryos as an endpoint to predict developmental toxicity

Inhibition of angiogenesis is an important mode of action for the teratogenic effect of chemicals and drugs. There is a gap in the availability of simple, experimental screening models for the detection of angiogenesis inhibition. The zebrafish embryo represents an alternative test system which offers the complexity of developmental differentiation of an entire organism while allowing for small-scale and high-throughput screening. Here we present a novel automated imaging-based method to detect the inhibition of angiogenesis in early life stage zebrafish. Video subtraction was used to identify the location and number of functional intersegmental vessels according to the detection of moving blood cells. By exposing embryos to multiple tyrosine kinase inhibitors including SU4312, SU5416, Sorafenib, or PTK787, we confirmed that this method can detect concentration-dependent inhibition of angiogenesis. Parallel assessment of arterial and venal aorta ruled out a potential bias by impaired heart or blood cell development. In contrast, the histone deacetylase inhibitor valproic acid did not affect ISV formation supporting the specificity of the angiogenic effects. The new test method showed higher sensitivity, i.e. lower effect concentrations, relative to a fluorescent reporter gene strain (Tg(KDR:EGFP)) exposed to the same tyrosine kinase inhibitors indicating that functional effects due to altered tubulogenesis or blood transport can be detected before structural changes of the endothelium are visible by fluorescence imaging. Comparison of exposure windows indicated higher specificity for angiogenesis when exposure started at later embryonic stages (24 h post-fertilization). One of the test compounds was showing particularly high specificity for angiogenesis effects (SU4312) and was, therefore, suggested as a model compound for the identification of molecular markers of angiogenic disruption. Our findings establish video imaging in wild-type strains as viable, non-invasive, high-throughput method for the detection of chemical-induced angiogenic disruption in zebrafish embryos.

Effects of gadolinium (Gd) and a Gd-based contrast agent (GBCA) on early life stages of zebrafish (Danio rerio)

Gadolinium (Gd) is one of the rare earth elements (REY) and is widely used in magnetic resonance imaging (MRI) contrast agents. Anthropogenic Gd enrichment has frequently been found in wastewater treatment plant effluents in industrialised countries, rising concerns regarding effects on aquatic biota. This study investigates the acute toxicity and sublethal effects of Gd in two forms, as inorganic salt (GdCl3) and as Gd-based contrast agent (GBCA), on early life stages of zebrafish (Danio rerio). Nominal exposure concentrations ranged from 3 to 3000 μg L−1, with an exposure duration of 96 h. None of the two tested compounds were acutely toxic to embryos and larvae. Similarly, we did not observe any effects on larval development and locomotive behaviour. However, we found significant changes in the brain activity of larvae exposed to the highest concentrations of GdCl3 and the GBCA. Our findings show that Gd can have sublethal effects on developing fish at lower concentrations than reported previously, highlighting the necessity of investigating the long-term fate and effects of GBCAs released into the aquatic environment.

Uptake and toxicity of micro-/nanoplastics derived from naturally weathered disposable face masks in developing zebrafish: Impact of COVID-19 pandemic on aquatic life

The unprecedented proliferation of disposable face masks during the COVID-19 pandemic, coupled with their improper disposal, threatens to exacerbate the already concerning issue of plastic pollution. This study evaluates the role of environmentally weathered masks as potential sources of microplastics (MPs) and nanoplastics (NPs) and assesses their adverse impact on the early life stages of zebrafish. Experimental findings revealed that a single disposable mask could release approximately 1.79 × 109 particles, with nearly 70% measuring less than 1 μm, following 60 days of sunlight exposure and subsequent sand-induced physical abrasion. Remarkably, the MPs/NPs (MNPs) emanating from face masks have the potential to permeate the outer layer (chorion) of zebrafish embryos. Furthermore, due to their minute size, these particles can be consumed by the larvae's digestive system and subsequently circulated to other tissues, including the brain. Exposure to mask-derived MNPs at concentrations of 1 and 10 μg/L led to significant cases of developmental toxicity, incited oxidative stress, and prompted cell apoptosis. A subsequent metabolomics analysis indicated that the accumulation of these plastic particles perturbed metabolic functions in zebrafish larvae, primarily disrupting amino acid and lipid metabolism. The outcomes of this research underscore the accelerating possibility of environmental aging processes and physical abrasion in the release of MNPs from disposable face masks. Most importantly, these results shed light on the possible ecotoxicological risk posed by improperly disposed of face masks.

Newly synthesized derivatives with a thiosemicarbazide group reduce the viability of cancer cell lines. Acute toxicity assessment in Zebrafish (Danio rerio) early life stages

Due to the variability and ability of tumor to mutate, as well as the heterogeneity of tumor tissue, such drugs are sought that would act selectively and multidirectionally on the cancer cell. Therefore, two newly synthesized semicarbazide structured substances were evaluated for anticancer properties in our study: 1a and 1b. In order to evaluate the cytotoxicity and selectivity of the tested compounds, MTT and Neutral Red uptake assay on cell lines (HEK293, LN229, 769-P, HepG2 and NCI-H1563) and cell cycle analysis were performed. Acute toxicity and cardiotoxicity were also evaluated in the zebrafish model. The tested compounds (1a, 1b) showed cytotoxic activity, with the greatest selectivity noted against the glioblastoma multiforme cell line (LN229). However, compound 1b showed stronger selective activity than 1a. Both of compounds were shown to significantly affect the M phase of the cell cycle. Whereas, the conducted toxicological examination of newly synthesized thiosemicarbazide derivates showed, that direct exposition of Danio rerio embryos to compound 1a, but not 1b, causes a concentration-dependent increase in developmental malformations, indicating possible teratogenic effects.

Testing biological actions of medicinal plants from northern Vietnam on zebrafish embryos and larvae: Developmental, behavioral, and putative therapeutical effects.

Evaluating the risks and benefits of using traditional medicinal plants is of utmost importance for a huge fraction of the human population, in particular in Northern Vietnam. Zebrafish are increasingly used as a simple vertebrate model for testing toxic and physiological effects of compounds, especially on development. Here, we tested 12 ethanolic extracts from popular medicinal plants collected in northern Vietnam for their effects on zebrafish survival and development during the first 4 days after fertilization. We characterized more in detail their effects on epiboly, hatching, growth, necrosis, body curvature, angiogenesis, skeletal development and mostly increased movement behavior. Finally, we confirm the effect on epiboly caused by the Mahonia bealei extract by staining the actin filaments and performing whole genome gene expression analysis. Further, we show that this extract also inhibits cell migration of mouse embryo fibroblasts. Finally, we analyzed the chemical composition of the Mahonia bealei extract and test the effects of its major components. In conclusion, we show that traditional medicinal plant extracts are able to affect zebrafish early life stage development to various degrees. In addition, we show that an extract causing delay in epiboly also inhibits mammalian cell migration, suggesting that this effect may serve as a preliminary test for identifying extracts that inhibit cancer metastasis.

Study of metabolism and potential toxicity of nine synthetic opioid analogs using the zebrafish larvae model.

The use of novel psychoactive substances (NPSs) has dramatically increased worldwide, and among them, synthetic opioids are one of the fastest growing groups, where cinnamylpiperazines and 2-benzylbenzimidazoles represent two of the most relevant subclasses. However, the data on their toxicity and metabolism are still limited. The aim of the present study was to evaluate the toxicity and metabolic pathways of some compounds belonging to these families, namely, AP-237, 2-methyl AP-237, isotonitazene, flunitazene, etodesnitazene, metonitazene, metodesnitazene, N-pyrrolidino etonitazene, and butonitazene. The study was performed using a zebrafish early life stages model. In fact, zebrafish (Danio rerio) embryos and larvae have recently been recognized as a suitable animal model in alternative to mammals, because they require less time and resources and do not need complex procedures for ethics approval. The cellular toxicity after a single administration was assessed at the fourth day post-fertilization with acridine orange staining. Possible morphological defects were evaluated with a light microscope after 24 h of exposure to 1μmol/L concentration of each drug. Subsequently, the larvae were euthanized and underwent analysis of drug metabolites using UPLC coupled to an Orbitrap high-resolution mass spectrometer. High rates of morphological defects, as well as of cellular death, were detected, but no significant difference in mortality between treatment and control groups was observed. In addition, several metabolites, mainly produced through monohydroxylation, N-dealkylation, and O-dealkylation, were identified in the larvae extracts.

Impact of environmentally relevant concentrations of fluoxetine on zebrafish larvae: From gene to behavior

Fluoxetine is widely prescribed for the treatment of depressive states, acting at the level of the central nervous system, consequently affecting non-target organisms. This study aimed to investigate the influence of environmentally relevant fluoxetine concentrations (1–1000 ng/L) on Danio rerio development, assessing both embryotoxicity and behavior, antioxidant defense, gene expression and neurotransmitter levels at larval stage. Exposure to fluoxetine during early development was found to be able to accelerate embryo hatching in embryos exposed to 1, 10 and 100 ng/L, reduce larval size in 1000 ng/L, and increase heart rate in 10, 100 and 1000 ng/L exposed larvae. Behavioral impairments (decreased startle response and increased larvae locomotor activity) were associated with effects on monoaminergic systems, detected through the downregulation of key genes (vmat2, mao, tph1a and th2). In addition, altered levels of neurochemicals belonging to the serotonergic and dopaminergic systems (increased levels of tryptophan and norepinephrine) highlighted the sensitivity of early life stages of zebrafish to low concentrations of fluoxetine, inducing effects that may compromise larval survival. The obtained data support the necessity to test low concentrations of SSRIs in environmental risk assessment and the use of biomarkers at different levels of biological organization for a better understanding of modes of action.

Behavioural, developmental and biochemical effects in zebrafish caused by ibuprofen, irgarol and terbuthylazine

The increasing use of chemicals and their release into aquatic ecosystems are harming aquatic biota. Despite extensive ecotoxicological research, many environmental pollutants' ecological effects are still unknown. This study examined the spatial avoidance, behavioural and biochemical impacts of ibuprofen, irgarol, and terbuthylazine on the early life stages of zebrafish (Danio rerio) under a range of ecologically relevant concentrations (0–500 μg/L). Embryos were exposed following the OECD guideline “fish embryo toxicity test” complemented with biochemical assessment of AChE activity and behavioural analyses (swimming activity) using the video tracking system Zebrabox. Moreover, spatial avoidance was assessed by exposing 120 hpf-old larvae of D. rerio to a gradient of each chemical, by using the heterogeneous multi-habitat assay system (HeMHAS). The results obtained revealed that the 3 compounds delayed hatching at concentrations of 50 and 500 μg/L for both ibuprofen and irgarol and 500 μg/L for terbuthylazine. Moreover, all chemicals elicited a dose-dependent depression of movement (swimming distance) with LOEC values of 5, 500 and 50 μg/L for ibuprofen, irgarol and terbuthylazine, respectively. Zebrafish larvae avoided the three chemicals studied, with 4 h-AC50 values for ibuprofen, irgarol, and terbuthylazine of 64.32, 79.86, and 131.04 μg/L, respectively. The results of the HeMHAS assay suggest that larvae may early on avoid (just after 4 h of exposure) concentrations of the three chemicals that may later induce, apical and biochemical effects. Findings from this study make clear some advantages of using HeMHAS in ecotoxicology as it is: ecologically relevant (by simulating a chemically heterogeneous environmental scenario), sensitive (the perception of chemicals and the avoidance can occur at concentrations lower than those producing lethal or sublethal effects) and more humane and refined approach (organisms are not mandatorily exposed to concentrations that can produce individual toxicity).

Hepatotoxic response of perfluorooctane sulfonamide (PFOSA) in early life stage zebrafish (Danio rerio) is greater than perfluorooctane sulfonate (PFOS)

Perfluorooctane sulfonamide (PFOSA), a typical perfluorooctane sulfonate precursor (PreFOS), has been detected in the aquatic environment globally. However, the effects of PFOSA at levels measured in the environment have not been well characterized in aquatic organisms. In this study, we evaluated the transcriptional, biochemical, histopathological, and morphological effects of PFOSA to characterize the underlying mechanisms of toxicity by using a universal model in aquatic ecotoxicology, zebrafish (Danio rerio). Transcriptional changes in PFOSA-exposed zebrafish predicted hepatic fibrosis and associated immune function. Subsequent, sublethal impacts were observed, which included significant alterations in liver-specific protein levels, increased immune cell numbers, and liver pathological structural damage. In addition, we compared the effects caused by PFOSA and perfluorooctane sulfonate (PFOS) at the same exposure concentration and found a greater hepatotoxic effect of PFOSA relative to PFOS, indicating that the adverse impacts of PFOSA may be more severe. This was the first study to comparatively explore the hepatotoxic response of PFOSA and PFOS in aquatic organisms, which can be used for ecological risk assessments of PreFOS compounds.

Comparative analysis between zebrafish and an automated live-cell assay to classify developmental neurotoxicant chemicals

Modern toxicology's throughput has dramatically increased due to alternative models, laboratory automation, and machine learning. This has enabled comparative studies across species and assays to prioritize chemical hazard potential and to understand how different model systems might complement one another. However, such comparative studies of high-throughput data are still in their infancy, with more groundwork needed to firmly establish the approach. Therefore, this study aimed to compare the bioactivity of the NIEHS Division of Translational Toxicology's (DTT) 87-compound developmental neurotoxicant (DNT) library in zebrafish and an in vitro high-throughput cell culture system. The early life-stage zebrafish provided a whole animal approach to developmental toxicity assessment. Chemical hits for abnormalities in embryonic zebrafish morphology, mortality, and behavior (ZBEscreen™) were compared with chemicals classified as high-risk by the Cell Health Index (CHI™), which is an outcome class probability from a machine learning classifier using 12 parameters from the SYSTEMETRIC® Cell Health Screen (CHS). The CHS was developed to assess human toxicity risk using supervised machine learning to classify acute cell stress phenotypes in a human leukemia cell line (HL60 cells) following a 4-h exposure to a chemical of interest. Due to the design of the screen, the zebrafish assays were more exhaustive, yielding 86 total bioactive hits, whereas the SYSTEMETRIC® CHS focusing on acute toxicity identified 20 chemicals as potentially toxic. The zebrafish embryonic and larval photomotor response assays (EPR and LPR, respectively) detected 40 of the 47 chemicals not found by the zebrafish morphological screen and CHS. Collectively, these results illustrate the advantages of using two alternative models in tandem for rapid hazard assessment and chemical prioritization and the effectiveness of CHI™ in identifying toxicity within a single multiparametric assay.