Zebrafish Embryo Toxicity Test Research Articles

A Review on Unlocking Toxicity: How Zebrafish embryos Help Ensure Safe Chemicals

Aquatic ecotoxicity testing has benefited greatly from zebrafish embryo toxicity testing (ZFET) because of its high throughput, low cost, sensitivity to a variety of toxicants, and ethical issues. This study highlights the benefits of ZFET over conventional fish toxicity testing, such as its capacity to detect teratogenic defects and possible long-term impacts. The limitations of ZFET are also addressed, notably its emphasis on acute dosages and difficulties with interspecies extrapolation. In addition to the generation of in vitro alternatives utilizing zebrafish cell lines, recent developments in ZFET technology are also highlighted, including the use of transgenic zebrafish lines and high-throughput screening techniques. The paper's conclusion includes a discussion of potential prospects for ZFET research. These include improving interspecies extrapolation methods, optimizing chronic toxicity assessment procedures, and integrating ZFET with other ecotoxicity testing strategies. ZFET is a great instrument for environmental protection and a crucial component in avoiding chemical contamination of aquatic ecosystems, so long as it is acknowledged for what it is and is open to future improvement

Indigo dyes: Toxicity, teratogenicity, and genotoxicity studies in zebrafish embryos

Wastewater released by textile dyeing industries is a major source of pollution. Untreated wastewater released from indigo dyeing operations affects aquatic ecosystems and threatens their biodiversity. We have assessed the toxicity of natural and synthetic indigo dye in zebrafish embryos, using the endpoints of teratogenicity, genotoxicity, and histopathology. The zebrafish embryo toxicity test (ZFET) was conducted, exposing embryos to ten concentrations of natural and synthetic indigo dyes; the 96-hour LC50 values were approximately 350 and 300 mg/L, respectively. Both dyes were teratogenic, causing egg coagulation, tail detachment, yolk sac edema, pericardial edema, and tail bend, with no significant difference in effects between the natural and synthetic dyes. Both dyes were genotoxic (using comet assay for DNA damage). Real-time RT-PCR studies showed upregulation of the DNA-repair genes FEN1 and ERCC1. Severe histological changes were seen in zebrafish larvae following exposure to the dyes. Our results show that indigo dyes may be teratogenic and genotoxic to aquatic organisms, underscoring the need for development of sustainable practices and policies for mitigating the environmental impacts of textile dyeing.

Integrated mRNA- and miRNA-sequencing analyses unveil the underlying mechanism of tobacco pollutant-induced developmental toxicity in zebrafish embryos

Tobacco pollutants are prevalent in the environment, leading to inadvertent exposure of pregnant females. Studies of these pollutants' toxic effects on embryonic development have not fully elucidated the potential underlying mechanisms. Therefore, in this study, we aimed to investigate the developmental toxicity induced by cigarette smoke extract (CSE) at concentrations of 0.25, 1, and 2.5% using a zebrafish embryo toxicity test and integrated transcriptomic analysis of microRNA (miRNA) and messenger RNA (mRNA). The findings revealed that CSE caused developmental toxicity, including increased mortality and decreased incubation rate, in a dose-dependent manner. Moreover, CSE induced malformations and apoptosis, specifically in the head and heart of zebrafish larvae. We used mRNA and miRNA sequencing analyses to compare changes in the expression of genes and miRNAs in zebrafish larvae. The bioinformatics analysis indicates that the mechanism underlying CSE-induced developmental toxicity was associated with compromised genetic material damage repair, deregulated apoptosis, and disturbed lipid metabolism. The enrichment analysis and RT-qPCR show that the ctsba gene plays a crucial function in embryo developmental apoptosis, and the fads2 gene mainly regulates lipid metabolic toxicity. The results of this study improve the understanding of CSE-induced developmental toxicity in zebrafish embryos and contribute insights into the formulation of novel preventive strategies against tobacco pollutants during early embryonic development.

Synergistic induction of apoptosis in lung cancer cells through co-delivery of PLGA phytol/α-bisabolol nanoparticles.

This study explored the potential of poly-(lactic-co-glycolic) acid (PLGA) nanoparticles to enhance the effectiveness of anticancer treatments through combination therapy with phytol and α-bisabolol. The encapsulation efficiency of the nanoparticles was investigated, highlighting the role of ionic interactions between the drugs and the polymer. Characterization of PLGA-Phy+Bis nanoparticles was carried out using DLS with zeta potential and HR-TEM for size determination. Spectrophotometric measurements evaluated the encapsulation efficiency, loading efficiency, and in vitro drug release. FTIR analysis assessed the chemical interactions between PLGA and the drug actives, ensuring nanoparticle stability. GC-MS was employed to analyze the chemical composition of drug-loaded PLGA nanocarriers. Cytotoxicity was evaluated via the MTT assay, while Annexin V-FITC/PI staining and western blot analysis confirmed apoptotic cell death. Additionally, toxicity tests were performed on L-132 cells and in vivo zebrafish embryos. The study demonstrates high encapsulation efficiency of PLGA-Phy+Bis nanoparticles, which exhibit monodispersity and sizes of 189.3±5nm (DLS) and 268±54 nm (HR-TEM). Spectrophotometric analysis confirmed efficient drug encapsulation and release control. FTIR analysis revealed nanoparticle structural stability without chemical interactions. MTT assay results demonstrated the promising anticancer potential of all thethree nanoparticle types (PLGA-Phy, PLGA-Bis, and PLGA-Phy+Bis) against lung cancer cells. Apoptosis was confirmed through Annexin V-FITC/PI staining and western blot analysis, which also revealed changes in Bax and Bcl-2 protein expression. Furthermore, the nanoparticles exhibited non-toxicity in L-132 cells and zebrafish embryo toxicity tests. PLGA-Phy+Bis nanoparticles exhibited efficient encapsulation, controlled release, and low toxicity. Apoptosis induction in A549 cells and non-toxicity in healthy cells highlight their clinical potential.

Unsymmetrical dimethylhydrazine (UDMH) exposure to zebrafish embryos causes lethal and severe teratogenic effects

The assessment of lethal and teratogenic effects of unsymmetrical dimethylhydrazine (UDMH) using zebrafish embryo toxicity test for 72 h was performed. Following UDMH concentrations were studied for embryotoxic effects: 0.64M, 0.13M, 0.06M, 0.04M, 5x10-3M and 1,7x10-3M after 3 h embryo exposure to UDMH at 7-10 hpf. It was shown that high UDMH concentrations (0,64М, 0,13М, and 0,06М) possess significant embryotoxic action to D.rerio embryos followed by concentration-dependent embryo lysis. 0.04M UDMH and vapors caused severe teratogenic effects and growth retardation prior to hatching resulted in embryo mortality to 48 hpf and 72 hpf, respectively. Zebrafish embryos exposure to low UDMH concentrations of 5x10-3M and 1,7x10-3M caused no significant lethal, but pronounced teratogenic effect (in latter case accumulated from 24 to 72 hpf), with observed malformations of spinal cord and tail tip, yolk sac and pericardial edemas, hypopigmentation. Thus short term 3 h. exposure of D.rerio embryos to UDMH at gastrulation stage induces alterations that further revealed themselves throughout embryonic development causing dose-dependent lethal and teratogenic effects.

Using Electroporation to Improve and Accelerate Zebrafish Embryo Toxicity Testing.

Zebrafish have emerged as a useful model for biomedical research and have been used in environmental toxicology studies. However, the presence of the chorion during the embryo stage limits cellular exposure to toxic elements and creates the possibility of a false-negative or reduced sensitivity in fish embryo toxicity testing (FET). This paper presents the use of electroporation as a technique to improve the delivery of toxic elements inside the chorion, increasing the exposure level of the toxins at an early embryo stage (<3 h post-fertilization). A custom-made electroporation device with the required electrical circuitry has been developed to position embryos between electrodes that provide electrical pulses to expedite the entry of molecules inside the chorion. The optimized parameters facilitate material entering into the chorion without affecting the survival rate of the embryos. The effectiveness of the electroporation system is demonstrated using Trypan blue dye and gold nanoparticles (AuNPs, 20-40 nm). Our results demonstrate the feasibility of controlling the concentration of dye and nanoparticles delivered inside the chorion by optimizing the electrical parameters, including pulse width, pulse number, and amplitude. Next, we tested silver nanoparticles (AgNPs, 10 nm), a commonly used toxin that can lower mortality, affect heart rate, and cause phenotypic defects. We found that electroporation of AgNPs reduces the exposure time required for toxicity testing from 4 days to hours. Electroporation for FET can provide rapid entry of potential toxins into zebrafish embryos, reducing the time required for toxicity testing and drug delivery experiments.

Evaluating the in vitro developmental toxicity potency of a series of petroleum substance extracts using new approach methodologies (NAMs)

The present study evaluates the in vitro developmental toxicity and the possible underlying mode of action of DMSO extracts of a series of highly complex petroleum substances in the mouse embryonic stem cell test (mEST), the zebrafish embryotoxicity test (ZET) and the aryl hydrocarbon receptor reporter gene assay (AhR CALUX assay). Results show that two out of sixteen samples tested, both being poorly refined products that may contain a substantial amount of 3- to 7-ring polycyclic aromatic compounds (PACs), induced sustained AhR activation in the AhR CALUX assay, and concentration-dependent developmental toxicity in both mEST and ZET. The other samples tested, representing highly refined petroleum substances and petroleum-derived waxes (containing typically a very low amount or no PACs at all), were negative in all assays applied, pointing to their inability to induce developmental toxicity in vitro. The refining processes applied during the production of highly refined petroleum products, such as solvent extraction and hydrotreatment which focus on the removal of undesired constituents, including 3- to 7-ring PACs, abolish the in vitro developmental toxicity. In conclusion, the obtained results support the hypothesis that 3- to 7-ring PACs are the primary inducers of the developmental toxicity induced by some (i.e., poorly refined) petroleum substances and that the observed effect is partially AhR-mediated.

The Biocompatibility of a Ginger-Containing Herbal Toothpaste on Developing Zebrafish Embryos

The biocompatibility of toothpaste in an oral cavity should be approved by clinical trials. Nowadays, herbal toothpaste is increasing in popularity due to its natural ingredients. Being genetically similar to humans, zebrafish are used in potential toxicity testing. The zebrafish embryotoxicity test is a fast and straightforward method to study chemical toxicity during embryogenesis. Objective: This study aimed to evaluate if there was any biocompatibility of the toothpaste on zebrafish embryos. Methods: Adult AB strain zebrafish were used according to Institutional Animal Care and Use Committee protocols. Normally dividing, spherical embryos were exposed to herbal toothpaste with ginger (Gumgumix, Turkey) (50 mg/L) and conventional toothpaste (Signal, Expert Protection, Bulgaria) (50 mg/L) in well plates containing 20 embryos, having four replicates. Developmental effects, mortality, and hatching rates were evaluated for 72h. Results: Zebrafish embryos exposed to conventional toothpaste had a higher mortality rate than those exposed to herbal toothpaste; they hatched later and delayed in development. There was no difference between herbal toothpaste and the control group regarding mortality and hatching rates (p &gt; 0.005). Conclusion: The herbal toothpaste showed higher biocompatibility on zebrafish embryos compared to the conventional toothpaste under the condition of this study.

Advancing the use of new approach methodologies for assessing teratogenicity: Building a tiered approach

Many New Approach Methodologies (NAMs) have been developed for the safety assessment of new ingredients. Research into reproductive toxicity and teratogenicity is a particularly high priority, especially given their mechanistic complexity. Forty-six non-teratogenic and 39 teratogenic chemicals were screened for teratogenic potential using the in silico DART model from the OECD QSAR Toolbox; the devTox quickPredict™ (devTox assay) test and the Zebrafish Embryotoxicity Test (ZET). The sensitivity and specificity were 94.7% and 84.1%, respectively, for the DART tree (83 chemicals), 86.1% and 35.6% for the devTox (81 chemicals) and 77.8% and 76.7% for the ZET (57 chemicals). Fifty-three chemicals were tested in all three assays and when results were combined and based on a “2 out of 3 rule”, the sensitivity and specificity were 96.0% and 71.4%, respectively. The specificity of the devTox assay for a sub-set of 43 chemicals was increased from 26.1% to 82.6% by incorporating human plasma concentrations into the assay interpretation. When all 85 chemicals were assessed in a decision tree approach, there was an excellent predictivity and assay robustness of 90%. In conclusion, all three models exhibited a good sensitivity and specificity, especially when outcomes from all three were combined or used in “2 out of 3” or a tiered decision tree approach. The latter is an interesting predictive approach for evaluating the teratogenic potential of new chemicals. Future investigations will extend the number of chemicals tested, as well as explore ways to refine the results and obtain a robust Integrated Testing Strategy to evaluate teratogenic potential.

In Silico and In Vitro Methods in the Characterization of Beta-Carotene as Pharmaceutical Material via Acetylcholine Esterase Inhibitory Actions.

Molecular docking is widely used in the assessment of the therapeutic potential of pharmaceutical agents. The binding properties of beta-carotene (BC) to acetylcholine esterase (AChE) proteins were characterized using the molecular docking method. The mechanism of AChE inhibition was assessed by an experimental in vitro kinetic study. In addition, the role of BC action was tested by the zebrafish embryo toxicity test (ZFET). The results of the docking ability of BC to AChE showed significant ligand binding mode. The kinetic parameter, i.e., the low AICc value shown as the compound was the competitive type of inhibition of AChE. Further, BC also showed mild toxicity at a higher dose (2200 mg/L) in ZFET assessment with changes in biomarkers. The LC50 value of BC is 1811.94 mg/L. Acetylcholine esterase (AChE) plays a pivotal role in the hydrolysis of acetylcholine, which leads to the development of cognitive dysfunction. BC possesses the regulation of acetylcholine esterase (AChE) and acid phosphatase (AP) activity to prevent neurovascular dysfunction. Therefore, the characterization of BC could be used as a pharmaceutical agent for the treatment of cholinergic neurotoxicity-associated neurovascular disorders such as developmental toxicity, vascular dementia, and Alzheimer's disease due to its AChE and AP inhibitory actions.

A metabolomics approach to reveal the mechanism of developmental toxicity in zebrafish embryos exposed to 6-propyl-2-thiouracil

A crucial component of a substance registration and regulation is the evaluation of human prenatal developmental toxicity. Current toxicological tests are based on mammalian models, but these are costly, time consuming and may pose ethical concerns. The zebrafish embryo has evolved as a promising alternative model to study developmental toxicity. However, the implementation of the zebrafish embryotoxicity test is challenged by lacking information on the relevance of observed morphological alterations in fish for human developmental toxicity. Elucidating the mechanism of toxicity could help to overcome this limitation. Through LC-MS/MS and GC-MS metabolomics, we investigated whether changes to the endogenous metabolites can indicate pathways associated with developmental toxicity. To this aim, zebrafish embryos were exposed to different concentrations of 6-propyl-2-thiouracil (PTU), a compound known to induce developmental toxicity. The reproducibility and the concentration-dependence of the metabolome response and its association with morphological alterations were studied. Major morphological findings were reduced eye size, and other craniofacial anomalies; major metabolic changes included increased tyrosine, pipecolic acid and lysophosphatidylcholine levels, decreased methionine levels, and disturbance of the ‘Phenylalanine, tyrosine and tryptophan biosynthesis’ pathway. This pathway, and the changes in tyrosine and pipecolic acid levels could be linked to the mode of action of PTU, i.e., inhibition of thyroid peroxidase (TPO). The other findings suggested neurodevelopmental impairments. This proof-of-concept study demonstrated that metabolite changes in zebrafish embryos are robust and provide mechanistic information associated with the mode of action of PTU.

Green synthesis of photocatalytic TiO2/Ag nanoparticles for an efficient water remediation

This work reports a green method to produce efficient photocatalytic nanopowders. Titanium dioxide nanoparticles were easily synthetized by an extract of Thymus vulgaris, and then decorated with commercial silver nanoparticles. A detailed morphological, structural, and chemical characterization was performed by scanning electron microscopy (SEM), Brunauer-Emmett-Teller (BET) adsorption–desorption of N2, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction analysis (XRD), Raman spectroscopy, transmission electron microscopy (TEM), and UV–Vis spectroscopy. The synthetized TiO2 nanoparticles showed a mean size of 10–15 nm, and had the anatase crystallographic phase. The photocatalytic efficiency of the TiO2 nanoparticles was demonstrated by the photo-degradation under UV-light irradiation of different pollutants in aqueous solution (methylene blue, diclofenac, and sodium dodecyl sulfate). TiO2 was coupled to Ag nanoparticles with different percentage in weight (from 0.25 to 3%), to investigate an eventual improvement of its photocatalytic efficiency. The best photocatalytic composite among the tested compositions was the one with the lowest Ag percentage (0.25%). Zebrafish embryo toxicity tests ruled out the toxicity of the synthetized nanoparticles. The present results offer a new, green, and easy method to prepare TiO2/Ag nanoparticles with high photocatalytic efficiency. The proposed materials are therefore promising for applications in photocatalysis, especially for wastewater remediation and reuse.

Intergrading Water Quality Parameters, Benthic Fauna and Acute Toxicity Test for Risk Assessment on an Urban-Rural River

Climate change, river pollution and loss of biodiversity are increasing and becoming global environmental concerns. The Yellow River is China’s mother river, providing water for about 114 million residents in towns and cities along its route. Yet in 2012, the Yellow River received 4.474 × 109 tons of sewage containing a large number of exogenous pollutants, posing a huge ecological and public health threat. Water quality safety is not only a matter of ecosystem health but also of human survival and social development. Therefore, the effects of pollutants on water quality safety should be carefully studied, which is important to ensure the sustainability of the Yellow River and the surrounding cities and towns. In this study, water and sediment samples from the Jishan River of the Juancheng, a typical city in the lower reaches of the Yellow River, were collected and evaluated by integrating the traditional physicochemical water indicators, benthic Index of Biological Integrity (B-IBI) and zebrafish embryotoxicity test. The results showed that water dissolved oxygen, pH, total nitrogen and total phosphorus were strongly correlated with zebrafish embryonic teratogenicity, lethality, abundance index and Shannon Winner index. A total of 21 benthic species were collected, including mollusks, arthropods and annelids, with the upper reaches having the largest biomass and B-IBI values indicating the urban reaches have better biological integrity than rural reaches. The teratogenic rate of zebrafish embryos in raw water was greatest in rural rivers and was significantly different from the negative control (p &lt; 0.05). When exposed to 100 mg/mL sediment samples, embryo hatching rates were inversely correlated with teratogenic rates, with lethal rates reaching over 96% in all rural reaches. The results showed that the water quality safety at the rural farms in the Yellow River transfer-type towns is poor, and they especially recommended that the river near the farms in rural areas should be monitored with emphasis.

The role of receptor-mediated activities of 4- and 5-ring unsubstituted and methylated polycyclic aromatic hydrocarbons (PAHs) in developmental toxicity.

The present study evaluated the aryl hydrocarbon receptor (AhR), estrogen receptor-α (ER-α), and retinoic acid receptor (RAR) mediated activities of nine 4- and 5-ring unsubstituted and monomethylated polycyclic aromatic hydrocarbons (PAHs) using a series of Chemical-Activated LUciferase gene eXpression (CALUX) assays. The potential role of these aforementioned receptors in relation to the developmental toxicity of these PAHs was further assessed in the zebrafish embryotoxicity test (ZET). The results show that all nine tested PAHs were AhR agonists, benz[a]anthracene (BaA) and 8-methyl-benz[a]anthracene (8-MeBaA) were ER-α agonists, and none of the tested PAHs induced ER-α antagonistic or RAR (ant)agonistic activities. In the AhR CALUX assay, all the methylated PAHs showed higher potency (lower EC50) in activating the AhR than their respective unsubstituted PAHs, implying that the addition of a methyl substituent on the aromatic ring of PAHs could enhance their AhR-mediated activities. Co-exposure of zebrafish embryos with each individual PAH and an AhR antagonist (CH223191) counteracted the observed developmental retardations and embryo lethality to a certain extent, except for 8-methyl-benzo[a]pyrene (8-MeBaP). Co-exposure of zebrafish embryos with either of the two estrogenic PAHs (i.e., BaA and 8-MeBaA) and an ER-α antagonist (fulvestrant) neutralized embryo lethality induced by 50 μM BaA and the developmental retardations induced by 15 μM 8-MeBaA. Altogether, our findings suggest that the observed developmental retardations in zebrafish embryos by the PAH tested may partially be AhR- and/or ER-α-mediated, whereas the RAR seems not to be relevant for the PAH-induced developmental toxicity in the ZET.

Estimating risk of cardiovascular pharmaceuticals in freshwaters using zebrafish embryotoxicity test - statins threat revealed

Cardiovascular pharmaceuticals (CVPs) are globally present in inland waters and have also been found in the sediment and plasma of fish from the Sava River, Croatia. Based on the previous research, CVPs amiodarone (AMI), ramipril (RAM), simvastatin (SIM), and verapamil (VER) have been selected for this study. Their effect has been investigated, individually and in a mixture, on the development of the zebrafish embryo Danio rerio (Hamilton, 1822) within the first 96 h of development. Upon exposure to environmentally relevant concentrations of tested CVPs (0.1, 1, and 10 μg/L) zebrafish survival and development as apparent from observed morphological abnormalities, heartbeat rates and changes in behavior, hatching success, larval length and oxidative stress level were monitored. The CVP causing the highest mortality and pathological changes was SIM (1 and 10 μg/L), which corresponds well with the observed effects during zebrafish exposure to CVPs' mixtures (4 and 40 μg/L). All pharmaceuticals affected cardiac function and decreased heart rate. SIM (1 μg/L), VER and RAM (10 μg/L) decreased larval length, while induced oxidative stress was recorded in the SIM- and VER-exposed specimens. Behavioral alterations of zebrafish were observed only in AMI-treated group (10 μg/L). Our amino acid sequence comparison and structural and docking analysis showed a highly conserved binding site between human and zebrafish HMG-CoA reductase for SIM and its main metabolite simvastatin acid. Using these ecotoxicological bioassays on a zebrafish model with particular emphasis on sublethal endpoints, the risk of CVPs, especially statins, for fish in inland waters has been identified.

Dynamic regulation of gene expression and morphogenesis in the zebrafish embryo test after exposure to all-trans retinoic acid

The zebrafish embryotoxicity test (ZET) is widely used in developmental toxicology. The analysis of gene expression regulation in ZET after chemical exposure provides mechanistic information about the effects of chemicals on morphogenesis in the test. The gene expression response magnitude has been shown to change with exposure duration. The objective of this work is to study the effect of the exposure duration on the magnitude of gene expression changes in the all-trans retinoic acid (ATRA) signaling pathway in the ZET. Retinoic acid regulation is a key driver of morphogenesis and is therefore employed here as an indicator for the regulation of developmental genes. A teratogenic concentration of 7.5 nM of ATRA was given at 3 hrs post fertilization (hpf) for a range of exposure durations until 120 hrs of development. The expression of a selection of genes related to ATRA signaling and downstream developmental genes was determined. The highest magnitudes of gene expression regulation were observed after 2–24 hrs exposure with an optimal response after 4 hrs. Longer exposures showed a decrease in the gene expression response, although continued exposure to 120 hpf caused malformations and lethality. This study shows that assessment of gene expression regulation at early time points after the onset of exposure in the ZET may be optimal for the prediction of developmental toxicity. We believe these results could help optimize sensitivity in future studies with ZET.

Evaluation of Anaesthetic Effect of Commercial Basil Ocimum basilicum on Zebrafish (Danio rerio) Embryos

Essential oils (EOs) are natural compounds used in various fields such as traditional medicine, gastronomy, and agriculture, often used against plant and animal parasites. In the present study, the toxic and pathological effects of basil (Ocimum basilicum) essential oil on the development of zebrafish embryos was investigated. The manufacturer datasheet reported linalool and eugenol as major compounds. The effects of the essential oil were evaluated through a zebrafish embryo toxicity test (ZFET) following the OECD n°236 guidelines. Embryos were exposed to different essential oil concentrations (50, 100, and 200 μL/L). Results showed mortality effects in the experimental groups in a dose-dependent manner. Moreover, zebrafish exposed to the higher concentration showed several pathological alterations; particularly, delayed hatching, pericardial edema, and a significant reduction of heart rate after 96 h post-fertilization (hpf) compared to the control group. Results reported here suggest an anaesthetic effect of the O. basilicum at low concentrations, due to the possible synergist effect of the main components such as linalool and eugenol which act on the GABAergic system evoking bradycardia.

Data-Driven Endpoint Selection in Data-Poor Scenarios: Bioassay Design for Shale Gas Flowback and Produced Waters

Big data approaches have greatly improved scientific decision making, but they are highly dependent on the availability of data, impeding their use in data-poor scenarios. In addition to data abundance, enhancing data diversity is likewise a way to access knowledge. Herein, we propose a data-driven method for toxicity endpoint selection when directly relevant data are deficient, and shale gas exploitation sites were used as an example scenario. From the 1173 substances in the U.S. Environmental Protection Agency’s HFList, the most concerning endpoints in zebrafish embryo toxicity tests (FET) were inferred using a newly developed relational database (RDB) strategy that integrated chemical, high-throughput screening (HTS) bioactivity, genome, and FET endpoint information. This RDB strategy based on text mining and data fusion approaches enabled the integration of 255 bioactive contaminants, 955 HTS bioassays with known modes of action (MoAs), 214 gene ontologies, 65 pathways, and 27 phenotypic data and predicted measurement endpoints within 10 MoAs for shale gas pollution. This data-driven approach was further validated using zebrafish FET and transcriptomic sequencing with field-collected samples and achieved 89% and 97% accuracy for the predictive ontologies and pathways, respectively. This highlighted the applicability of RDB-based data-driven strategies for predicting toxicity endpoints from a priori knowledge of contaminants by improving data diversity.

Toxicity of single and combined 4-epianhydrotetracycline and cadmium at environmentally relevant concentrations on the zebrafish embryos (Danio rerio)

The combined pollution of antibiotics and heavy metals has attracted a worldwide attention in the recent years. 4-epianhydrotetracycline (EATC) is the major degradation product of tetracycline (TC), which has been detected frequently in environment and its concentration is even higher than TC under some circumstances. Cadmium (Cd) is a common heavy metal contaminant and has highly toxic to organisms, plants and humans even at low doses. In the present study, zebrafish (Danio rerio) embryo toxicity test was performed to investigate the single and combined effects of EATC and Cd on aquatic organisms. Exposure to EATC and Cd at environmentally relevant concentrations had a series of hazardous impacts on the embryonic development, including lethality, hatching rate, heart rate and teratogenic effects. Compared to the contaminant existed alone, combined pollution produced stronger toxicity, which appeared as the decreasing of heart rate and hatching rate, and the increasing of malformation of zebrafish embryos. After 96 h exposure, the reactive oxygen species (ROS) levels in zebrafish embryos were increased significantly, revealing that EATC-Cd co-exposure resulted in potential oxidative stress-induced damage. Acridine orange (AO) staining showed that combined exposure resulted in stronger cell apoptosis. The potential health risks of the combined pollution of EATC and Cd should be paid more attention to higher level vertebrates and humans.

P06-11 Validation of zebrafish embryotoxicity test (ZET) as a qualified alternative assay for its regulatory use

P06-11 Validation of zebrafish embryotoxicity test (ZET) as a qualified alternative assay for its regulatory use