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Background: The Fish Embryo Acute Toxicity (FET) test with the zebrafish (Danio rerio) embryo, the OECD test guideline (TG) 236, has been designed as an alternative for acute fish toxicity testing such as the OECD Acute Fish Toxicity Test (TG 203). To provide equivalent sensitivity to the acute fish test, the original FET test was designed to use o...
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Etridiazole (EDZ), a thiadiazole-containing toxic chemical, is widely used as a fungicide. Regular usage of EDZ may reach and contaminate water bodies, but its adverse effects on aquatic vertebrates have not been well studied. Therefore, the present study aimed to evaluate the harmful effects of EDZ using zebrafish (ZF) (Danio rerio) embryos. ZF em...
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... The 1-hpf zebrafish larvae were cultured for 48 and 96 hpf at 28.5 °C using Ru(II) complex (5 μM) and Ru@ DNA (5 μM). In the early development of zebrafish (48 hpf), the primary organs, including the heart, liver, brain, and pancreas, have fully developed and could undergo natural food intake, metabolism, and excretion [36,37] (Fig. 5A). ...
Identifying tumor cells can be challenging due to cancer’s complex and heterogeneous nature. Here, an efficacious phosphorescent probe that can precisely highlight tumor cells has been created. By combining the ruthenium(II) complex with oligonucleotides, we have developed a nanosized functional ruthenium(II) complex (Ru@DNA) with dimensions ranging from 300 to 500 nm. Our research demonstrates that Ru@DNA can readily traverse biomembranes via ATP-dependent endocytosis without carriers. Notably, the nanosized ruthenium(II) complex exhibits rapid and selective accumulation within tumor cells, possibly attributed to the nanoparticles’ enhanced permeation and retention (EPR) effect. Ru@DNA can also effectively discern and label the transplanted cancer cells in the zebrafish model. Moreover, Ru@DNA is efficiently absorbed into the intestine and further distributed in the pancreas. Our findings underscore the potential of Ru@DNA as a DNA-based nanodevice derived from a functional ruthenium(II) complex. This innovative nanodevice holds promise as an efficient phosphorescent probe for both in vitro and in vivo imaging of living tumor cells.
... The zebrafish eggs and larvae did not display any negative impacts from the experiment, and every embryo developed into a normal larva without any abnormal development. Similar tests have been conducted previously by Rebecca et al. [35][36][37]. ...
Mosquito-borne diseases pose a global health threat, with pathogens like Malaria, Dengue fever, and others transmitted by mosquitoes. Our study focuses on evaluating the toxicity of genetically engineered mosquito larvicidal algae (Chlamydomonas reinhardtii) to non-target organisms, specifically Zebrafish. We conducted a 90-day experiment, feeding Zebrafish different combinations of larvicidal algae and commercial fish feed. Statistical analysis revealed no significant differences in mortality, allergenicity, or moribundity among groups. Hematology, molecular analysis, and necropsy showed no physiological differences. Our findings indicate that the transgenic algae (TN72.cry11Ba) had no adverse effects on adult Zebrafish or their larvae. This study confirmed the safety of algae on non-target organisms, such as zebrafish.
... Meanwhile, the relative short period of these early life stages make them ideal for the acute testing of various pollutants on a suite of endpoints related to differentiation and development, which are vital for growth and survival during this critical developmental period . For example, the Fish Embryo Acute Toxicity (FET) (i.e., OECD test guideline (TG) 236) has been proposed as an alternative for acute fish toxicity testing such as the OECD Acute Fish Toxicity Test (TG 203) (Léonard et al. 2005;Sobanska et al. 2018), which is in accordance with the idea to reduce, refine and replace animal testing (3 R) by alternative test methods proposed by OECD (Dang et al. 2017;von Hellfeld et al. 2020). Since life cycle toxicity tests are only feasible for certain species, it is essential to use data from partial life cycle studies to estimate effects for the whole life cycle (Hutchinson et al. 1998). ...
Rapid evaluation of the toxicity of metals using fish embryo acute toxicity is facilitative to ecological risk assessment of aquatic organisms. However, this approach has seldom been utilized for the comparative study on the effects of different metals to fish. In this study, acute and sub-chronic tests were used to compare the toxicity of Se(IV) and Cd in the embryos and larvae of Japanese medaka (Oryzias latipes). The embryos with different levels of dechorionation and/or pre-exposure were also exposed to Se(IV) and Cd at various concentrations. The results showed that the LC50-144 h of Cd was 1.3–5.2 folds higher than that of Se(IV) for the embryos. In contrast, LC50-96 h of Se(IV) were 200–400 folds higher than that of Cd for the larvae. Meanwhile, dechorionated embryos were more sensitive to both Se and Cd than the intact embryos. At elevated concentrations, both Se and Cd caused mortality and deformity in the embryos and larvae. In addition, pre-exposure to Cd at the embryonic stages enhanced the resistance to Cd in the larvae. However, pre-exposure to Se(IV) at the embryonic stages did not affect the toxicity of Se(IV) to the larvae. This study has distinguished the nuance differences in effects between Se(IV) and Cd after acute and sub-chronic exposures with/without chorion. The approach might have a potential in the comparative toxicology of metals (or other pollutants) and in the assessment of their risks to aquatic ecosystems.
... In further experiments designed to further investigate larvae behavior after DTG exposure, FA was added as above at the 10 hpf stage and then DTG was added at 72 hpf (see experimental exposure and analysis timelines in Figure 1). As negative control, for all experiments, embryos were exposed to 0.1% DMSO in fish water (expected mortality rate < 10%) [81,82]. ...
Dolutegravir (DTG) is one of the most prescribed antiretroviral drugs for treating people with HIV infection, including women of child-bearing potential or pregnant. Nonetheless, neuropsychiatric symptoms are frequently reported. Early reports suggested that, probably in relation to folic acid (FA) shortage, DTG may induce neural tube defects in infants born to women taking the drug during pregnancy. Subsequent reports did not definitively confirm these findings. Recent studies in animal models have highlighted the association between DTG exposure in utero and congenital anomalies, and an increased risk of neurologic abnormalities in children exposed during in utero life has been reported. Underlying mechanisms for DTG-related neurologic symptoms and congenital anomalies are not fully understood. We aimed to deepen our knowledge on the neurodevelopmental effects of DTG exposure and further explore the protective role of FA by the use of zebrafish embryos. We treated embryos at 4 and up to 144 h post fertilization (hpf) with a subtherapeutic DTG concentration (1 μM) and observed the disruption of the anterior–posterior axis and several morphological malformations in the developing brain that were both prevented by pre-exposure (2 hpf) and rescued by post-exposure (10 hpf) with FA. By whole-mount in situ hybridization with riboprobes for genes that are crucial during the early phases of neurodevelopment (ntl, pax2a, ngn1, neurod1) and by in vivo visualization of the transgenic Tg(ngn1:EGFP) zebrafish line, we found that DTG induced severe neurodevelopmental defects over time in most regions of the nervous system (notochord, midbrain–hindbrain boundary, eye, forebrain, midbrain, hindbrain, spinal cord) that were mostly but not completely rescued by FA supplementation. Of note, we observed the disruption of ngn1 expression in the dopaminergic regions of the developing forebrain, spinal cord neurons and spinal motor neuron projections, with the depletion of the tyrosine hydroxylase (TH)+ dopaminergic neurons of the dorsal diencephalon and the strong reduction in larvae locomotion. Our study further supports previous evidence that DTG can interfere with FA pathways in the developing brain but also provides new insights regarding the mechanisms involved in the increased risk of DTG-associated fetal neurodevelopmental defects and adverse neurologic outcomes in in utero exposed children, suggesting the impairment of dopaminergic pathways.
... All data presented the validity criteria. The effects were defined according to the study of von Hellfeld et al. (2020). ...
The increasing pressure on freshwater systems due to intensive anthropogenic use is a big challenge in central-northern Namibia and its catchment areas, the Kunene and the Kavango Rivers, and the Cuvelai-Etosha Basin, that provide water for more than 1 million people. So far, there is no comprehensive knowledge about the ecological status and only few knowledge about the water quality. Therefore, it is crucial to learn about the state of the ecosystem and the ecological effects of pollutants to ensure the safe use of these resources. The surface waters of the three systems were sampled, and three bioassays were applied on three trophic levels: algae, daphnia, and zebrafish embryos. Additionally, in vitro assays were performed to analyze mutagenicity (Ames fluctuation), dioxin-like potential (micro-EROD), and estrogenicity (YES) by mechanism-specific effects. The results show that acute toxicity to fish embryos and daphnia has mainly been detected at all sites in the three catchment areas. The systems differ significantly from each other, with the sites in the Iishana system showing the highest acute toxicity. At the cellular level, only weak effects were identified, although these were stronger in the Iishana system than in the two perennial systems. Algae growth was not inhibited, and no cytotoxic effects could be detected in any of the samples. Mutagenic effects and an estrogenic potential were detected at three sites in the Iishana system. These findings are critical in water resource management as the effects can adversely impact the health of aquatic ecosystems and the organisms within them.
... 19 The embryotoxicity test evaluates lethality, sublethality, and teratogenic parameters within the first 120 h post fertilization (hpf). 20 Fertilized embryos (n = 20) at 2 hpf were transferred to Petri dishes (60 mm × 15 mm) and incubated with different extract concentrations (31.25, 62.5, 125, 250, and 750 μg/ mL). Embryos were exposed to the extract solutions (8 mL) since 2 hpf and continued until 5 days post fertilization (dpf), with daily replacement of the solutions (Supporting Figure S1). ...
Chipilin (Crotalaria longirostrata) is consumed as a vegetable in the preparation of traditional dishes. As a folk medicine, Chipilin extracts are used as a hypnotic and sedative agent; however, there are few reports that support these uses. This study aimed to characterize the compounds present in Chipilin leaf extracts and to investigate their sedative effect using zebrafish as an in vivo model. Extracts were obtained by maceration with water (H2O), ethanol (EtOH), and EtOH-H2O, while oleoresin was obtained by supercritical fluid extraction (SFE). Total phenolic and flavonoid contents were quantified by colorimetric methods. Phytochemical constituents were identified by gas chromatography–mass spectrometry (GC-MS) analysis. The chronic and acute toxicities of Chipilin extracts were tested in zebrafish embryos and larvae, respectively. Chipilin sedative effect was tested by the larvae response to dark–light–dark transitions. EtOH-H2O extracts had the highest value of total phenolics (5345 ± 5.1 μg GAE/g), followed by water and oleoresin (1815 ± 5.1 and 394 ± 5.1 μg GAE/g, respectively). In water extracts were identified the alkaloid trachelanthamidine, 1,2β-epoxy- and the alkyl ketone 7,9-di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione, while oleamide, α-monostearin, and erucamide were detected in all samples except in water extracts. Oleoresin extract had the lowest embryotoxicity (LC50 = 4.99 μg/mL) and the highest sedative effects. SFE is a green alternative to obtain Chipilin extracts rich in erucamide, an endocannabinoid analogue, which plays an important role in the development of the central nervous system and in modulating neurotransmitter release.
... In the present study, the timing at which developmental markers were assessed (i.e., every 24 h) did not provide sufficient resolution to distinguish between the onset of these events. A previous study aimed at documenting common abnormalities observed in the zebrafish FET test found that alterations in heartbeat and blood flow cooccurred at similar frequencies for 34 of the 48 chemicals tested (von Hellfeld et al., 2020). However, for nine of the chemicals tested, impaired or absent blood flow was found to occur with a greater frequency than impaired heartbeat. ...
... In both the sheepshead minnow and inland silversides, eye formation began by 48 hpf, pigmentation of the head and tail were noted by 72 hpf, burst movements were shown to occur by 72 hpf, and fully formed eyes were visible by 96 hpf. The timing of these developmental events is similar to that in zebrafish (Easter & Malicki, 2002;Subkhankulova et al., 2023), and alterations in these endpoints have been noted in response to a wide range of chemicals (Von Hellfeld et al., 2020). In fact, Von Hellfeld et al. (2020) found that, for 28 of the 48 chemicals used in zebrafish FET tests, at least one of these three metrics was altered. ...
... The timing of these developmental events is similar to that in zebrafish (Easter & Malicki, 2002;Subkhankulova et al., 2023), and alterations in these endpoints have been noted in response to a wide range of chemicals (Von Hellfeld et al., 2020). In fact, Von Hellfeld et al. (2020) found that, for 28 of the 48 chemicals used in zebrafish FET tests, at least one of these three metrics was altered. ...
Current regulations require that toxicity assessments be performed using standardized toxicity testing methods, often using fish. Recent legislation in both the European Union and United States has mandated that toxicity testing alternatives implement the 3Rs of animal research (replacement, reduction, and refinement) whenever possible. There have been advances in the development of alternatives for freshwater assessments, but there is a lack of analogous developments for marine assessments. One potential alternative testing method is the fish embryo toxicity (FET) test, which uses fish embryos rather than older fish. In the present study, FET methods were applied to two marine model organisms, the sheepshead minnow and the inland silverside. Another potential alternative is the mysid shrimp survival and growth test, which uses an invertebrate model. The primary objective of the present study was to compare the sensitivity of these three potential alternative testing methods to two standardized fish‐based tests using 3,4‐dichloroaniline (DCA), a common reference toxicant. A secondary objective was to characterize the ontogeny of sheepshead minnows and inland silversides. This provided a temporal and visual guide that can be used to identify appropriately staged embryos for inclusion in FET tests and delineate key developmental events (e.g., somite development, eyespot formation, etc.). Comparison of the testing strategies for assessing DCA indicated that: (1) the standardized fish tests possessed comparable sensitivity to each other; (2) the mysid shrimp tests possessed comparable sensitivity to the standardized fish tests; (3) the sheepshead minnow and inland silverside FET tests were the least sensitive testing strategies employed; and (4) inclusion of sublethal endpoints (i.e., hatchability and pericardial edema) in the marine FETs increased their sensitivity. Environ Toxicol Chem 2024;00:1–15. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.
... Embryos were assessed daily from 8 until 96 hpf under stereomicroscope (Stemi 508, Zeiss, Germany) with coupled camera (Axiocam ICc 5, Zeiss, Germany). Observations included lethal endpoints and various sublethal changes on embryos morphology, as described in previous studies (Nagel, 2002;von Hellfeld et al., 2020) (Table S2). The normal embryonic development of zebrafish was established according to the description of Kimmel et al. (1995). ...
... We also observed edemas formation in the yolk sac and pericardium, compromising the maintenance of an optimal osmotic gradient in relation to the surrounding aqueous environment, a very common toxic effect of several other toxicants, such as polycyclic aromatic hydrocarbons, polychlorinated biphenyls, brominated flame-retardants and nanoparticles (Sant and Timme-Laragy, 2018;Abe et al., 2020), suggesting that edema formation is a sensitive toxicological outcome for evaluation. Finally, Roundup Orig-inal® also increased the rate of spinal curvature of embryos, depicted by a sideway curving of the spine, which has limited recovery potential, unlike the edema effects (von Hellfeld et al., 2020). Although spinal curvature is an endpoint quite frequently observed, there is a lack of publications discussing its onset. ...
... Zebrafish (Danio rerio) is a widely used model organism for studying vertebrate toxicology, and modifications of the original OECD Fish Embryo Acute Toxicity (FET) Tests have been utilized extensively as a high-throughput screening method that can be applied to other vertebrates, including humans [16][17][18][19][20][21]. The effects of PFAS exposure on zebrafish embryos have been studied in previous research with some common non-lethal effects Toxics 2024, 12,192 2 of 23 including altered spontaneous tail bends, altered heart rate, un-or underinflated swim bladder, hatching rate, spinal/tail curvature, pericardial edema, cranial malformations, and tissue necrosis [22][23][24][25][26][27][28][29][30][31][32][33][34][35]. ...
... Curvature of the spine/upper tail similar to the scoliosis phenotype described in von Hellfeld et al. (2020) [21]. Documented on live and dead embryos. ...
The presence of per- and polyfluoroalkyl substances (PFASs) in aquatic environments is often persistent and widespread. Understanding the potential adverse effects from this group of chemicals on aquatic communities allows for better hazard characterization. This study examines impacts on zebrafish (Danio rerio) embryo physiology, behavior, and lipid levels from exposure to perfluorooctanoic acid (PFOA), perfluorohexane sulfonate (PFHxS), and heptadecafluorooctanesulfonic acid (PFOS). Embryos were exposed to lethal and sublethal levels of each chemical and monitored for alterations in physiological malformations, mortality, lipid levels, and behavior (only PFOA and PFHxS). The predicted 50% lethal concentrations for 120 hpf embryos were 528.6 ppm PFOA, 14.28 ppm PFHxS, and 2.14 ppm PFOS. Spine curvature and the inability of the 120 hpf embryos to maintain a dorsal-up orientation was significantly increased at 10.2 ppm PFHxS and 1.9 ppm PFOS exposure. All measured 120 hpf embryo behaviors were significantly altered starting at the lowest levels tested, 188 ppm PFOA and 6.4 ppm PFHxS. Lipid levels decreased at the highest PFAS levels tested (375 PFOA ppm, 14.4 PFHxS ppm, 2.42 ppm PFOS). In general, the PFAS chemicals, at the levels examined in this study, increased morphological deformities, embryo activity, and startle response time, as well as decreased lipid levels in 120 hpf zebrafish embryos.
... Studies were conducted on zebrafish (Danio rerio) in accordance with OECD guideline Protocol 236 "Fish Embryo Acute Toxicity (FET) Test" (OECD/OCDE, 2013). Morphological effects were assessed according to [50]. ...
Curcumin attracts huge attention because of its biological properties: it is antiproliferative, antioxidant, anti-inflammatory, immunomodulatory and so on. However, its usage has been limited by poor water solubility and low bioavailability. Herein, to solve these problems, we developed curcumin-loaded nanoparticles based on end-capped amphiphilic poly(N-vinylpyrrolidone). Nanoparticles were obtained using the solvent evaporation method and were characterized by dynamic and electrophoretic light scattering, transmission electron (TEM) and atomic force (AFM) microscopy. The average particle size was 200 nm, and the ζ-potential was −4 mV. Curcumin-release studies showed that nanoparticles are stable in aqueous solutions. An in vitro release study showed prolonged action in gastric, intestinal and colonic fluids, consistently, and in PBS. In vitro studies on epidermoid carcinoma and human embryonic kidney cells showed that the cells absorbed more curcumin in nanoparticles compared to free curcumin. Nanoparticles are safe for healthy cells and show high cytotoxicity for glioblastoma cells in cytotoxicity studies in vitro. The median lethal dose was determined in an acute toxicity assay on zebrafish and was 23 μM. Overall, the curcumin-loaded nanoparticles seem promising for cancer treatment.
