Developmental toxicity assay using high content screening of zebrafish embryos

Typically, time‐consuming standard toxicological assays using the zebrafish (Danio rerio) embryo model evaluate mortality and teratogenicity after exposure during the first 2 days post‐fertilization. Here we describe an automated image‐based high content screening (HCS) assay to identify the teratogenic/embryotoxic potential of compounds in zebrafish embryos in vivo. Automated image acquisition was performed using a high content microscope system. Further automated analysis of embryo length, as a statistically quantifiable endpoint of toxicity, was performed on images post‐acquisition. The biological effects of ethanol, nicotine, ketamine, caffeine, dimethyl sulfoxide and temperature on zebrafish embryos were assessed. This automated developmental toxicity assay, based on a growth‐retardation endpoint should be suitable for evaluating the effects of potential teratogens and developmental toxicants in a high throughput manner. This approach can significantly expedite the screening of potential teratogens and developmental toxicants, thereby improving the current risk assessment process by decreasing analysis time and required resources. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.     

In vivo quantitative study of sized-dependent transport and toxicity of single silver nanoparticles using zebrafish embryos

Nanomaterials possess distinctive physicochemical properties (e.g., small sizes and high surface area-to-volume ratios) and promise a wide variety of applications, ranging from the design of high quality consumer products to effective disease diagnosis and therapy. These properties can lead to toxic effects, potentially hindering advances in nanotechnology. In this study, we have synthesized and characterized purified and stable (nonaggregation) silver nanoparticles (Ag NPs, 41.6 ± 9.1 nm in average diameter) and utilized early developing (cleavage-stage) zebrafish embryos (critical aquatic and eco- species) as in vivo model organisms to probe the diffusion and toxicity of Ag NPs. We found that single Ag NPs (30-72 nm diameters) passively diffused into the embryos through chorionic pores via random Brownian motion and stayed inside the embryos throughout their entire development (120 hours-post-fertilization, hpf). Dose- and size-dependent toxic effects of the NPs on embryonic development were observed, showing the possibility of tuning biocompatibility and toxicity of the NPs. At lower concentrations of the NPs (≤0.02 nM), 75-91% of embryos developed into normal zebrafish. At the higher concentrations of NPs (≥0.20 nM), 100% of embryos became dead. At the concentrations in between (0.02-0.2 nM), embryos developed into various deformed zebrafish. Number and sizes of individual Ag NPs embedded in tissues of normal and deformed zebrafish at 120 hpf were quantitatively analyzed, showing deformed zebrafish with higher number of larger NPs than normal zebrafish and size-dependent nanotoxicity. By comparing with our previous studies of smaller Ag NPs (11.6 ± 3.5 nm), we found striking size-dependent nanotoxicity that, at the same molar concentration, the larger Ag NPs (41.6 ± 9.1 nm) are more toxic than the smaller Ag NPs (11.6 ± 3.5 nm).     

Development of a quantitative morphological assessment of toxicant‐treated zebrafish larvae using brightfield imaging and high‐content analysis

One of the rate‐limiting procedures in a developmental zebrafish screen is the morphological assessment of each larva. Most researchers opt for a time‐consuming, structured visual assessment by trained human observer(s). The present studies were designed to develop a more objective, accurate and rapid method for screening zebrafish for dysmorphology. Instead of the very detailed human assessment, we have developed the computational malformation index, which combines the use of high‐content imaging with a very brief human visual assessment. Each larva was quickly assessed by a human observer (basic visual assessment), killed, fixed and assessed for dysmorphology with the Zebratox V4 BioApplication using the Cellomics® ArrayScan® V^TI high‐content image analysis platform. The basic visual assessment adds in‐life parameters, and the high‐content analysis assesses each individual larva for various features (total area, width, spine length, head–tail length, length–width ratio, perimeter–area ratio). In developing the computational malformation index, a training set of hundreds of embryos treated with hundreds of chemicals were visually assessed using the basic or detailed method. In the second phase, we assessed both the stability of these high‐content measurements and its performance using a test set of zebrafish treated with a dose range of two reference chemicals (trans‐retinoic acid or cadmium). We found the measures were stable for at least 1 week and comparison of these automated measures to detailed visual inspection of the larvae showed excellent congruence. Our computational malformation index provides an objective manner for rapid phenotypic brightfield assessment of individual larva in a developmental zebrafish assay. Copyright © 2016 John Wiley & Sons, Ltd.     

Proteomic analysis of ametryn toxicity in zebrafish embryos

Ametrym (AMT) is the most widely used herbicide and frequently detected in the aquatic environment. AMT also represent a potential health risk to aquatic organisms and animals, including humans. However, little data are available on their toxicity to zebrafish (Danio rerio). The aim of the present study was to evaluate the toxicological effects of AMT exposure on zebrafish embryos. In the acute toxicity test, 6 hpf embryos were exposed to various concentrations of AMT for 24 or 48 h. The results indicated that AMT induced malformation in larvae. To investigate the toxicological mechanism on the protein expression level. A proteomic approach was employed to investigate the proteome alterations of zebra fish embryos exposed to 20 mg/L AMT for 48 h. Among 2925 unique proteins identified, 298 differential proteins (> or <1.3‐fold, P < 0.05) were detected in the treated embryos as compared to the corresponding proteins in the untreated embryos. Gene ontology analysis showed that these up‐regulated proteins were most involved in glycolysis, lipid transport, protein polymerization, and nucleotide binding, and the down‐regulated proteins were related to microtubule‐based process, protein polymerization, oxygen transport. Moreover, KEGG pathway analysis indicated that tight junction, ribosome, and oxidative phosphorylation were inhibited in the treated embryos. These findings provide new insight into the mechanisms of toxicity induced by AMT.     

Cadmium significantly changes major morphometrical points and cardiovascular functional parameters during early development of zebrafish

Living organisms are commonly exposed to cadmium and other toxic metals. A vast body of research has shown the significant effects of these toxic metals on developmental processes. In order to study the role of toxic metals on early developmental stages of eukaryotes, we explored the effect of cadmium (Cd²⁺) contaminant on zebrafish. Thus, zebrafish embryos were exposed to 3 mg/L (16.7 μM) Cd²⁺ for 96 h and imaged every 24 h from the exposure onwards. Hatching rates of the eggs were determined at 72 h, followed by analyses at 96 h for: survival rate, morphometrical factors, and functional parameters of the cardiovascular system. Interestingly enough, significant hatching delays along with smaller cephalic region and some morphological abnormalities were observed in the treatment group. Moreover, substantial changes were noticed in the length of notochord and embryo, absorption of yolk sac with shorter extension, area of swimming bladder, as well as pericardium sac after Cd²⁺ treatment. Cadmium also caused significant abnormalities in heart physiology which could be the leading cause of mentioned morphological deformities. Herein, our results shine light on systematic acute embryological effects of cadmium in the early development of zebrafish for the first time.     

Four-dimensional imaging and quantification of gene expression in early developing zebrafish (Danio rerio) embryos.

Four-dimensional (4D) imaging is a powerful tool for studying three-dimensional (3D) changes in an organism through time. Different imaging systems for obtaining 3D data from in vivo specimens have been developed but usually involved large and expensive machines. We successfully used a simple inverted compound microscope and a commercially available program to study and quantify in vivo changes in sonic hedgehog (shh) expression during early development in a green fluorescence protein (GFP) transgenic zebrafish (Danio rerio) line. We applied the 4D system to study the effect of 100 microM cadmium exposure on shh expression. In control zebrafish embryos, shh:GFP expression was detected at about 9 h post-fertilization (hpf) and increased steadily in the next 7 h, peaking at about 17 hpf and decreasing in the following 4 h. In the same time period, different shh expression volumes were observed in cadmium-treated and control embryos. Embryos affected by cadmium-exposure demonstrated a down-regulation in shh expression. The number of GFP-expressing cells measured by flow cytometry decreased, and expression of neurogenin-1, a downstream target of the shh signaling pathway, was down-regulated, providing additional supporting data on the effects of cadmium on shh. In summary, we demonstrated the setup of a 4D imaging system and its application to the quantification of gene expression.     

DHF-BAHPC molecule exerts ameliorative antioxidant status and reduced cadmium-induced toxicity in zebrafish (Danio rerio) embryos

In this study, we report the antioxidant and antitoxic potential of chemically synthesized 4-oxo-2-phenyl-4H-chromene-7,8-diyl bis((1-amino-2-hydroxypropyl)carbamate) (DHF-BAHPC) compound using in vitro and in vivo assays. The DHF-BAHPC was synthesized by linking 7,8-Dihydroxyflavone (DHF) with two molecules of Fmoc-threonine and characterized by Ultraviolet-visible spectroscopy (UV–vis), Fourier-transform infrared spectroscopy (FT-IR), ¹H NMR, ¹³C NMR and Mass spectrometry (MS). In vitro, antioxidant assay results revealed that DHF-BAHPC has a dose-dependent radical scavenging potential towards DPPH, ABTS, FRAP and H₂O₂ radicals with an IC₅₀ range of 15.45, 66.27, 25.71, 4.375 μg/mL, respectively. Furthermore DHF-BAHPC treatment significantly altered cadmium (Cd) intoxicated zebrafish embryos by rescuing the developmental changes associated with severe histological and reduced the level of defensive antioxidant activities (SOD, CAT, GPx and GST). The overall results of the present study represented that DHF-BAHPC may be used as a potential drug in redox-based therapeutics.     

Development of a screening assay to identify teratogenic and embryotoxic chemicals using the zebrafish embryo

We developed and optimized a screening procedure, in which zebrafish embryos were explored as a model for the evaluation of the specific embryotoxic and teratogenic potential of chemicals. A selection of known positive (retinoic acid, valproic acid, caffeine, lithium chloride) and negative (glucose, saccharin) compounds for developmental toxicity were used to evaluate this method. We exposed embryos and evaluated embryotoxicity and morphological characteristics of the embryos at 24, 48, 72 and 144 h post fertilization. After evaluation of the induced effects, concentration–response curves were created for both embryotoxicity and teratogenic effects. Values for teratogenic indices (TI) were calculated as the ratio LC₅₀/EC₅₀. The results obtained were compared to existing data from studies with laboratory animals and humans. We demonstrated that our classification of the compounds, based on TI values, allows to distinguish teratogens from non-teratogens and supports the application of zebrafish embryos as an alternative method for developmental toxicity studies to predict effects in mammals.     

菲并咪唑衍生物的体外抗肿瘤活性及其对斑马鱼胚胎发育的毒性效应研究

目的 设计合成菲并咪唑衍生物L271,并研究其外抗肿瘤活性,及其对斑马鱼胚胎发育的毒性效应.方法 以1,10-邻菲啰啉-5,6-二酮和2-甲基苯甲醛为原料,运用微波辅助合成技术制备了菲并咪唑衍生L271.采用噻唑蓝(MTT)比色法研究菲并咪唑衍生物L271对人宫颈癌细胞Hela和人肺腺癌细胞A549在体外生长的抑制作用.以斑马鱼为模型,研究L271对斑马鱼胚胎发育的形态、孵化率、死亡率和畸形率的影响,评价L271对斑马鱼胚胎发育的毒性效应.结果 经电喷雾质谱技术(ESI-MS)表征确证成功合成了目标化合物L271.新合成的菲并咪唑衍生物L271对人宫颈癌细胞Hela和人肺腺癌细胞A549均有增殖抑制作用,尤其对人宫颈癌细胞Hela的IC50值达到(8.38±0.09)μmol/L,与同等条件下吡柔比星的抗肿瘤活性相当[IC50=(6.97±0.07)μmol/L].与对照组相比,L271浓度≥15μmol/L暴露组能够引起斑马鱼出现尾鳍萎缩、脊柱弯曲、卵黄囊水肿和心包囊水肿等畸形现象;L271浓度≥30μmol/L可显著降低斑马鱼的孵化率和提高死亡率(P＜0.05);在48、72、96 hpf时,L271对斑马鱼胚胎半致死浓度LC5o分别是37.331μmol/L(95％CI:35.535-39.301)、34.911μmol/L(95％CI:33.213-36.729)、30.283μmol/L(95％CI:29.590-30.980);在L271浓度≥15μmol/L时,随着L271浓度的逐渐增加,各暴露组正常胚胎百分率渐下降,胚胎死亡率逐渐上升,而胚胎畸形率先升后降.结论 L271对人宫颈癌细胞Hela具有良好的抗肿瘤活性,且与吡柔比星的相当;L271浓度≤10μmol/L对斑马鱼胚胎发育无明显毒性效应.     

Taxifolin mitigates oxidative DNA damage in vitro and protects zebrafish (Danio rerio) embryos against cadmium toxicity

Taxifolin (TAX) is a natural source of bioflavonoid found in various conifers. In this study, initially we investigated the antioxidant potential of TAX under in vitro assays such as 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), ferric-ion reducing power (FRAP) and hydroxyl radical (OH). The activities of DPPH, ABTS, FRAP and OH radical levels were significantly inhibited by TAX with an IC₅₀ values of 16.48, 66.34, 18.17 and 11.42 μg/ml, respectively. Secondly, TAX exhibited a strong protection against OH mediated DNA damage on pUC19 plasmid DNA at 1.0 μg/ml. Finally, we evaluated the protective mechanism of TAX against cadmium intoxicated zebrafish embryos (Danio rerio). We found that embryos exposed to 100 μM Cd exhibited significantly reduced survival, delayed hatching and phenotypic abnormalities at 24, 48, 72 and 96 hours post fertilization (hpf). Similarly, Cd intoxicated embryos showed significantly increased cardiac function (131 beats/min) at 60 hpf. Conversely, treatment with TAX (0.1, 1.0 and 10 μM) significantly enhanced the antioxidant enzyme levels (SOD, CAT, GPx and GR) by reducing the lipid peroxidation (MDA) in zebrafish embryos. Collectively, our results concluded that TAX could act as a potent redox scavenger against oxidative DNA damage and also functions as a crucial suppressor of Cd toxicity in zebrafish embryos.     

Induced toxicity in early-life stage zebrafish (Danio rerio) and its behavioral analysis after exposure to non-doped,nitrogen-doped and nitrogen,sulfur-co doped carbon quantum dots

In this study, the effects of doping of CQDs with alternative functional groups (dopants) were evaluated through embryonic development of zebrafish (Danio rerio). The CQDs were synthesized using simple and low-cost sources: Non-doped (citric acid was used as the carbon source), nitrogen-doped (N-doped) and nitrogen, sulfur-co-doped (N,S-doped). The CQDs induced significant toxicity to zebrafish (>150 μg/mL) and the toxic effects were dose-dependent. The N,S-doped CQDs were the most toxic (LD50 = 149.92 μg/mL), followed by the N-doped CQDs (LD50 = 399.95 μg/mL) while the non-doped CQDs were the least toxic (LD50 = 548.48 μg/mL) of the three. The growth rate (GR) was affected following the toxicity pattern (GR_NS-doped<GR_N-doped<GR_non-doped <GR_blanc), which, in turn, greatly depends on the type of dopant. Morphological malformations, such as pericardial edema, yolk sac edema, tail and spinal curvature were observed to zebrafish embryos as the toxicity, concentration and exposure time to the nanomaterial increased. Behavioral analysis showed that locomotor activity increases as the toxicity of the nanomaterial rises. The differences in toxicity, growth rate and malfunctions of CQDs were attributed to their doping with different heteroatoms. The N,S-doped CQDs, unequivocally, exhibited the most pronounced effects.     

In vivo bioluminescent monitoring of chemical toxicity using heme oxygenase-luciferase transgenic mice

Transgenic mice expressing the luciferase (luc) gene under the control of the heme oxygenase-1 promoter (Ho1) were used to measure the induction of heme oxygenase in response to known toxicants. Transgenic Ho1-luc expression was visualized in vivo using a low-light imaging system (IVIS). Ho1-luc activation was compared to Ho1-luc expression, HO1 protein levels, standard markers of toxicity, and histology. Male and female Ho1-luc transgenic mice were exposed to acute doses of cadmium chloride (CdCl2, 3.7 mg/kg), doxorubicin (15 mg/kg), and thioacetamide (300 mg/kg). These agents induced the expression of Ho1-luc in the liver and other tissues to varying degrees. The greatest increase in Ho1-luc activity was observed in the liver in response to CdCl2; intermediate responses were observed for doxorubicin and thioacetamide. Induction of the Ho1-luc transgene by these agents was similar to endogenous protein levels of heme oxygenase as assessed by Western blotting, and generally correlated with plasma levels of circulating enzymes reflecting hepatic or general tissue damage. Histopathology confirmed the toxic effects of CdCl2 on liver and kidney; doxorubicin on kidney, liver, and intestine; and thioacetamide on the liver. Tissue damage was much more pronounced than the luciferase expression following thioacetamide treatment when compared with tissue damage and bioluminescence of the other toxicants. Nevertheless, the induction of Ho1-luc expression following exposure to these agents suggests that the Ho1-luc transgenic mouse may prove useful as a model for in vivo screening of compounds that induce luciferase expression as a marker of toxicity.     

Morphometric analysis of developing zebrafish embryos allows predicting teratogenicity modes of action in higher vertebrates

The early identification of teratogens in humans and animals is mandatory for drug discovery and development. Zebrafish has emerged as an alternative model to traditional preclinical models for predicting teratogenicity and other potential chemical-induced toxicity hazards. To prove its predictivity, we exposed zebrafish embryos from 0 to 96 h post fertilization to a battery of 31 compounds classified as teratogens or non-teratogens in mammals. The teratogenicity score was based on the measurement of 16 phenotypical parameters, namely heart edema, pigmentation, body length, eye size, yolk size, yolk sac edema, otic vesicle defects, otoliths defects, body axis defects, developmental delay, tail bending, scoliosis, lateral fins absence, hatching ratio, lower jaw malformations and tissue necrosis. Among the 31 compounds, 20 were detected as teratogens and 11 as non-teratogens, resulting in 94.44 % sensitivity, 90.91 % specificity and 87.10 % accuracy compared to rodents. These percentages decreased slightly when referred to humans, with 87.50 % sensitivity, 81.82 % specificity and 74.19 % accuracy, but allowed an increase in the prediction levels reported by rodents for the same compounds. Positive compounds showed a high correlation among teratogenic parameters, pointing out at general developmental delay as major cause to explain the physiological/morphological malformations. A more detailed analysis based on deviations from main trends revealed potential specific modes of action for some compounds such as retinoic acid, DEAB, ochratoxin A, haloperidol, warfarin, valproic acid, acetaminophen, dasatinib, imatinib, dexamethasone, 6-aminonicotinamide and bisphenol A. The high degree of predictivity and the possibility of applying mechanistic approaches makes zebrafish a powerful model for screening teratogenicity.     

Developmental toxicity of albendazole and its three main metabolites in zebrafish embryos

Albendazole (ABZ) is used as an anthelmintic drug in humans and animals. ABZ has been shown to cause developmental toxicity in experimental animals, however it is not clear if this is caused by the parent compound or a metabolite. Zebrafish embryos were exposed from 1 to 144 hpf (hours post fertilization) to investigate the developmental toxicity of ABZ, the first metabolite albendazole sulphoxide and the subsequent metabolites albendazole sulphone (ABZSO₂) and albendazole-2-aminosulphone (ABZSO₂NH₂). The results showed that ABZ caused malformations of head and tail and embryonic lethality from 0.3 μM. In contrast, the metabolites did not display developmental toxicity at any tested concentration. Dechorionation did not influence the developmental toxic potential of ABZ and ABZSO, indicating that bioavailability was not a limiting factor. Chemical analysis showed that at sublethal concentrations, most of ABZ was metabolized to ABZSO. The results demonstrate that in zebrafish embryos ABZ rather than ABZSO displays developmental toxicity.     

Identification of environmental chemicals that induce yolk malabsorption in zebrafish using automated image segmentation

Environmental factors affecting nutrient availability during development can cause predisposition to diseases later in life. To identify chemicals in the environment capable of altering nutrient mobilization, we analyzed yolk malabsorption in the zebrafish embryo, which relies on maternally-derived yolk for nutrition during its first week of life. Embryos of the transgenic zebrafish line HGn50D, which fluoresce in the yolk syncytial layer, were exposed from two to five days post fertilization to different chemicals. We developed a software package to automatically and accurately segment and quantify the area of the fluorescing yolk in images captured at the end of the treatment period. Based on this quantification, we found that prochloraz decreased yolk absorption, while butralin, tetrabromobisphenol A, tetrachlorobisphenol A and tributyltin increased yolk absorption. Given the number and variety of industrial chemicals in commerce today, development of automated image processing to perform high-speed quantitative analysis of biological effects is an important step for enabling high throughput screening to identify chemicals altering nutrient absorption.     

吡嗪酰胺对斑马鱼胚胎发育和氧化应激效应的影响

目的 本文研究吡嗪酰胺(PZA)对斑马鱼胚胎的发育毒性及其产生机制。方法 采用受精后4h的斑马鱼胚胎进行吡嗪酰胺不同浓度的暴露,分别于暴露后24、48、72和96h进行死亡率、孵化率的统计。给药96h后进行斑马鱼幼鱼形态评分、行为学分析、活性氧簇(ROS)和氧化应激指标检测。结果 表明吡嗪酰胺显著抑制斑马鱼胚胎孵化,导致斑马鱼幼鱼发育畸形(如卵黄囊吸收迟滞、鱼鳔缺失、心包水肿和体节模糊等),同时吡嗪酰胺的暴露造成斑马鱼幼鱼运动行为能力的降低。吡嗪酰胺的暴露能够引起斑马鱼体内ROS、总超氧化物歧化酶(T-SOD)、过氧化氢酶(CAT)、谷胱甘肽(GSH)和丙二醛(MDA)水平的变化。结论 吡嗪酰胺对斑马鱼胚胎有明显的发育毒性,并呈剂量和时间依赖性,氧化应激在吡嗪酰胺发育毒性中起着重要的作用。     

High-throughput fluorescence imaging approaches for drug discovery using in vitro and in vivo three-dimensional models

Introduction: High-resolution microscopy using fluorescent probes is a powerful tool to investigate individual cell structure and function, cell subpopulations and mechanisms underlying cellular responses to drugs. Additionally, responses to drugs more closely resemble those seen in vivo when cells are physically connected in three-dimensional (3D) systems (either 3D cell cultures or whole organisms), as opposed to traditional monolayer cultures. Combined, the use of imaging-based 3D models in the early stages of drug development has the potential to generate biologically relevant data that will increase the likelihood of success for drug candidates in human studies.

Areas covered: The authors discuss current methods for the culturing of cells in 3D as well as approaches for the imaging of whole-animal models and 3D cultures that are amenable to high-throughput settings and could be implemented to support drug discovery campaigns. Furthermore, they provide critical considerations when discussing imaging these 3D systems for high-throughput chemical screenings.

Expert opinion: Despite widespread understanding of the limitations imposed by the two-dimensional versus the 3D cellular paradigm, imaging-based drug screening of 3D cellular models is still limited, with only a few screens found in the literature. Image acquisition in high throughput, accurate interpretation of fluorescent signal, and uptake of staining reagents can be challenging, as the samples are in essence large aggregates of cells. The authors recognize these shortcomings that need to be overcome before the field can accelerate the utilization of these technologies in large-scale chemical screens.     

Toxicity and cardiac effects of carbaryl in early developing zebrafish (Danio rerio) embryos

Carbaryl, an acetylcholinesterase inhibitor, is known to be moderately toxic to adult zebrafish and has been reported to cause heart malformations and irregular heartbeat in medaka. We performed experiments to study the toxicity of carbaryl, specifically its effects on the heart, in early developing zebrafish embryos. LC50 and EC50 values for carbaryl at 28 h post-fertilization were 44.66 microg/ml and 7.52 microg/ml, respectively, and 10 microg/ml carbaryl was used in subsequent experiments. After confirming acetylcholinesterase inhibition by carbaryl using an enzymatic method, we observed red blood cell accumulation, delayed hatching and pericardial edema, but not heart malformation as described in some previous reports. Our chronic exposure data also demonstrated carbaryl-induced bradycardia, which is a common effect of acetylcholinesterase inhibitors due to the accumulation of acetylcholine, in embryos from 1 day post-fertilization (dpf) to 5 dpf. The distance between the sinus venosus, the point where blood enters the atrium, and the bulbus arteriosus, the point where blood leaves the ventricle, indicated normal looping of the heart tube. Immunostaining of myosin heavy chains with the ventricle-specific antibody MF20 and the atrium-specific antibody S46 showed normal development of heart chambers. At the same time, acute exposure resulted in carbaryl-induced bradycardia. Heart rate dropped significantly after a 10-min exposure to 100 microg/ml carbaryl but recovered when carbaryl was removed. The novel observation of carbaryl-induced bradycardia in 1- and 2-dpf embryos suggested that carbaryl affected cardiac function possibly through an alternative mechanism other than acetylcholinesterase inhibition such as inhibition of calcium ion channels, since acetylcholine receptors in zebrafish are not functional until 3 dpf. However, the exact nature of this mechanism is currently unknown, and thus further studies are required.