Dichloroacetate-induced developmental toxicity and production of reactive oxygen species in zebrafish embryos

Dichloroacetate (DCA) is one of the toxic by products that are formed during the chlorine disinfection process of drinking water. In this study, the developmental toxicity of DCA has been determined in zebrafish (Danio rerio) embryos. Embryos were exposed to different concentrations (4, 8, 16, and 32 mM) of the compound at the 4 h postfertilization (hpf) stage of development, and were observed for different developmental toxic effects at 8, 24, 32, 55, 80, and 144 hpf. Exposure of embryos to 8-32 mM of DCA resulted in significant increases in the heart rate and blood flow of the 55 and 80 hpf embryos that turned into significant decreases at the 144 hpf time point. At 144 hpf, malformations of mouth structure, notochord bending, yolk sac edema and behavioral effects including perturbed swimming and feeding behaviors were also observed. DCA was also found to produce time- and concentration-dependent increases in embryonic levels of superoxide anion (O2*-) and nitric oxide (NO), at various stages of development. The results of the study suggest that DCA-induced developmental toxic effects in zebrafish embryos are associated with production of reactive oxygen species in those embryos.     

Modulation by ellagic acid of DCA-induced developmental toxicity in the zebrafish (Danio rerio)

The ability of ellagic acid (EA) to modulate dichloroacetic acid (DCA)-induced developmental toxicity and oxidative damage was examined in zebrafish embryos. Embryos were exposed to 20 mM EA administered concomitantly with 32 mM DCA at 4 hours postfertilization (hpf) and 20 h later. Embryos were observed through 144 hpf for developmental malformations, and production of superoxide anion (SA) and nitric oxide (NO) was determined in embryonic homogenates. DCA was shown to produce developmental abnormalities and significant levels of SA and NO in zebrafish embryos. EA exposure alleviated the developmental malformations observed in treated embryos and decreased the levels of SA and NO in those same embryos. Less than 10% of DCA + EA exposed embryos showed developmental malformations compared to 100% of embryos treated with DCA alone. Animals in this group that developed malformations were shown to have fewer defects than those treated with DCA only. Taken together, the results confirm the involvement of oxidative stress in the developmental toxicity of DCA in zebrafish embryos, and suggest possible protection against those effects with the use of antioxidants.     

Disruption of circulation by ethanol promotes fetal alcohol spectrum disorder (FASD) in medaka (Oryzias latipes) embryogenesis

Japanese medaka (Oryzias latipes) embryos exposed to ethanol have developed craniofacial, cardiovascular and skeletal defects which can be compared with the phenotypic features of fetal alcohol spectrum disorder (FASD) observed in human. The present experiment was designed to show that the disruption in circulation by ethanol during embryogenesis is a potential cause of FASD. Fertilized eggs were exposed to ethanol (0, 100 and/or 400 mM) for 24 or 48 h at various developmental stages (Iwamatsu stages 4-30) and were analyzed at 6 day post fertilization (dpf). It was observed that controls and the embryos exposed to 100 mM ethanol were in circulating state; however, a significant number of embryos of stages 4-24 exposed to 400 mM ethanol had disrupted circulation. Compared to controls, protein and RNA contents were significantly reduced in non-circulating embryos. Lipid peroxidation (LPO) analysis was made at 3, 6, 24, 48, 96 and 144 hour post fertilization (hpf). LPO was increased with the advancement of morphogenesis; however, ethanol or the circulation status had no effect. We further analyzed alcohol dehydrogenase (Adh 5 and adh8) and aldehyde dehydrogenase (Aldh9A and Aldh1A2) enzyme mRNAs in the embryos exposed to 400 mM ethanol for 24 h. A developmental stage-specific reduction in these enzyme mRNAs by ethanol was observed. We conclude that ethanol-induced disruption in circulation during embryogenesis is a potential cause of the development of FASD features in medaka.     

The Nicotine-Evoked Locomotor Response: A Behavioral Paradigm for Toxicity Screening in Zebrafish (Danio rerio) Embryos and Eleutheroembryos Exposed to Methylmercury

This study is an adaptation of the nicotine-evoked locomotor response (NLR) assay, which was originally utilized for phenotype-based neurotoxicity screening in zebrafish embryos. Zebrafish embryos do not exhibit spontaneous swimming until roughly 4 days post-fertilization (dpf), however, a robust swimming response can be induced as early as 36 hours post-fertilization (hpf) by means of acute nicotine exposure (30–240μM). Here, the NLR was tested as a tool for early detection of locomotor phenotypes in 36, 48 and 72 hpf mutant zebrafish embryos of the non-touch-responsive maco strain; this assay successfully discriminated mutant embryos from their non-mutant siblings. Then, methylmercury (MeHg) was used as a proof-of-concept neurotoxicant to test the effectiveness of the NLR assay as a screening tool in toxicology. The locomotor effects of MeHg were evaluated in 6 dpf wild type eleutheroembryos exposed to waterborne MeHg (0, 0.01, 0.03 and 0.1μM). Afterwards, the NLR assay was tested in 48 hpf embryos subjected to the same MeHg exposure regimes. Embryos exposed to 0.01 and 0.03μM of MeHg exhibited significant increases in locomotion in both scenarios. These findings suggest that similar locomotor phenotypes observed in free swimming fish can be detected as early as 48 hpf, when locomotion is induced with nicotine.     

Japanese medaka (Oryzias latipes): developmental model for the study of alcohol teratology

BACKGROUND: Animal models are necessary to investigate the mechanism of alcohol-induced birth defects. We have used Japanese medaka (Oryzias latipes) as a non-mammalian model to elucidate the molecular mechanism(s) of ethanol teratogenesis. METHODS: Medaka eggs, within 1 hr post-fertilization (hpf) were exposed to waterborne ethanol (0-1000 mM) in hatching solution for 48 hr. Embryo development was observed daily until 10 days post-fertilization (dpf). The concentration of embryonic ethanol was determined enzymatically. Cartilage and bones were stained by Alcian blue and calcein, respectively and skeletal and cardiovascular defects were assessed microscopically. Genetic gender of the embryos was determined by PCR. Levels of two isoenzymes of alcohol dehydrogenase (Adh) mRNAs were determined by semi-quantitative and real-time RT-PCR. RESULTS: The concentration of ethanol required to cause 50% mortality (LC50) in 10 dpf embryos was 568 mM, however, the embryo absorbed only 15-20% of the waterborne ethanol at all ethanol concentrations. The length of the lower jaw and calcification in tail fin cartilaginous structures were reduced by ethanol exposure. Active blood circulation was exhibited at 50+ hpf in embryos treated with 0-100 mM ethanol; active circulation was delayed and blood clots developed in embryos treated with 200-400 mM ethanol. The deleterious effects of ethanol were not gender-specific. Moreover, ethanol treatment was unable to alter the constitutive expression of either Adh5 or Adh8 mRNA in the medaka embryo. CONCLUSIONS: Preliminary results suggested that embryogenesis in medaka was significantly affected by ethanol exposure. Phenotypic features normally associated with ethanol exposure were similar to that observed in mammalian models of fetal alcohol syndrome. The results further indicated that medaka embryogenesis might be used as an alternative non-mammalian model for investigating specific alterations in gene expression as a means to understand the molecular mechanism(s) of ethanol-induced birth defects.     

Nephrotoxicity assessments of acetaminophen during zebrafish embryogenesis

We used a green fluorescent kidney line, Tg(wt1b:GFP), as a model to access the acetaminophen (AAP)-induced nephrotoxicity dynamically. Zebrafish (Danio rerio) embryos at different developmental stages (12–60 hpf) were treated with different dosages of AAP (0–45 mM) for different time courses (12–60 h). Results showed that zebrafish embryos exhibited no evident differences in survival rates and morphological changes between the mock-treated control (0 mM) and 2.25 mM AAP-exposure (12–72 hpf) groups. In contrast, after higher doses (22.5 and 45 mM) of exposure, embryos displayed malformed kidney phenotypes, such as curved, cystic pronephric tube, pronephric duct, and a cystic and atrophic glomerulus. The percentages of embryos with malformed kidney phenotypes increased as the exposure dosages of AAP increased. Interestingly, under the same exposure time course (12 h) and dose (22.5 mM), embryos displayed higher percentages of severe defects at earlier developmental stage of exposure (12–24 hpf), whereas embryos displayed higher percentages of mild defects at later exposure (60–72 hpf). With an exposure time course less than 24 h of 45 mM AAP, no embryo survived by the developmental stage of 72 hpf. These results indicated that AAP-induced nephrotoxicity depended on the exposure dose, time course and developmental stages. Immunohistochemical experiments showed that the cells' morphologies of the pronephric tube, pronephric duct and glomerulus were disrupted by AAP, and consequently caused cell death. Real-time RT-PCR revealed embryos after AAP treatment decreased the expression of cox2 and bcl2, but increased p53 expression. In conclusion, AAP-induced defects on glomerulus, pronephric tube and pronephric duct could be easily and dynamically observed in vivo during kidney development in this present model.     

五氯酚对斑马鱼胚胎的毒性效应研究

采用斑马鱼胚胎发育技术,对环境激素类物质五氯酚的毒性进行测定,结果表明,五氯酚(PCP)对胚胎的特定作用时间段是卵产出至发育6 h之内;PCP对胚胎发育有明显的抑制作用,会造成胚胎发育的畸形或死亡,不同时间染毒产生的可观察毒理学终点各异;随着PCP对发育48 h斑马鱼胚胎作用时间的减短,其致死效应敏感性降低,其中0 hpf组的LC0值最小,为70.8μg·L^-1,24hpf组LC0值最大,为831.8μg·L^-1;斑马鱼胚胎对孵化后0时染毒的PCP最为敏感,PCP对胚胎产生急性毒性效应的敏感指标:心胞囊肿、血液循环障碍、无心律＞孵化率降低＞停滞发育作用;斑马鱼胚胎最敏感的指标为48 h血液循环障碍和48 h半致死效应.     

Hypoxia-inducible factor-1 mediates adaptive developmental plasticity of hypoxia tolerance in zebrafish,Danio rerio

In recent years, natural and anthropogenic factors have increased aquatic hypoxia the world over. In most organisms, the cellular response to hypoxia is mediated by the master regulator hypoxia-inducible factor-1 (HIF-1). HIF-1 also plays a critical role in the normal development of the cardiovascular system of vertebrates. We tested the hypothesis that hypoxia exposures which resulted in HIF-1 induction during embryogenesis would be associated with enhanced hypoxia tolerance in subsequent developmental stages. We exposed zebrafish (Danio rerio) embryos to just 4 h of severe hypoxia or total anoxia at 18, 24 and 36 h post-fertilization (hpf). Of these, exposure to hypoxia at 24 and 36 hpf as well as anoxia at 36 hpf activated the HIF-1 cellular pathway. Zebrafish embryos that acutely upregulated the HIF-1 pathway had an increased hypoxia tolerance as larvae. The critical window for hypoxia sensitivity and HIF-1 signalling was 24 hpf. Adult male fish had a lower critical oxygen tension (P_crit) compared with females. Early induction of HIF-1 correlated directly with an increased proportion of males in the population. We conclude that mounting a HIF-1 response during embryogenesis is associated with long-term impacts on the phenotype of later stages which could influence both individual hypoxia tolerance and population dynamics.     

The induction of tumor necrosis factor‐alpha,superoxide anion,myeloperoxidase, and superoxide dismutase in the peritoneal lavage cells of mice after prolonged exposure to dichloroacetate and trichloroacetate

The induction of phagocytic activation in response to prolonged treatment with different doses of dichloroacetate (DCA) and trichloroacetate (TCA) has been investigated in mice. Groups of B6C3F1 male mice were administered 7.7, 77, 154, and 410 mg of DCA or TCA/kg/day, postorally, for 4‐ and 13‐weeks. Peritoneal lavage cells (PLCs) were isolated and assayed for the different biomarkers of phagocytyic activation, including superoxide anion (SA), tumor necrosis factor‐alpha (TNF‐α), and myeloperoxidase (MPO). In addition, the role of superoxide dismutase (SOD) in the SA production was also assessed. DCA and TCA produced significant and dose‐dependent increases in SA and TNF‐α production and in MPO activity, but the increases in response to the high doses of the compounds (>77 mg/kg/day) in the 13‐week treatment period were less significant than those produced in the 4‐week treatment period. Also, dose‐dependent increases in SOD activity were observed in both periods of treatments. In general, the results demonstrate significant induction of the biomarkers of phagocytic activation by doses of DCA and TCA that were previously shown to be noncarcinogenic, with significantly greater increases observed at the earlier period of exposure, as compared with later period. These findings may argue against the contribution of those mechanisms to the hepatotoxicity/hepatocarcinogenicity of the compounds and suggest them to be early adaptive/ protective mechanisms against their long‐term effects. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:136–144, 2010; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20322     

Dose-dependent effects of developmental mercury exposure on C-start escape responses of larval zebrafish Danio rerio

Zebrafish Danio rerio embryos were exposed to 0, 25, 50 or 75 ppb Hg²⁺ from 0 to 24 h post‐fertilization (hpf) then placed into Hg²⁺‐free water. Inductively coupled plasma‐mass spectrophotometer analysis of whole embryo Hg²⁺ content at 24 hpf showed a positive correlation with exposure regime (Pearson's one‐tailed, r²= 0·698, P < 0·01); at 5 days post‐hatch (dph), whole larval Hg²⁺ content was not detectable. Hg²⁺‐induced behavioural deficits in larvae were, therefore, due to changes during embryogenesis and not to residual Hg²⁺ in the larvae. At 5 dph, larvae were tested for responses to different frequencies but equal intensities of vibrational stimuli generated by a remotely controlled plastic hammer. Data were recorded by high‐speed videography and computer‐analysed for latency of response (ms), amplitude of the response as measured by maximum initial velocity [normalized as body (standard) lengths s^?1; V_max] and duration of behaviour from initial head movement to cessation of caudal tail movement (ms). A single mechanical stimulus resulted in behavioural outcomes that were related to embryonic Hg²⁺ uptake. Response latency increased with exposure level and displayed an increase of ×1·5–2·5 over control values (ANOVA, P < 0·01). The V_max decreased with exposure level to a low of 71% of control at the highest Hg²⁺ concentration (ANOVA, P < 0·01). Duration of behaviour displayed a biphasic response pattern in which exposure to 0, 50 or 75 ppb Hg²⁺ did not result in a significantly different response yet exposure to 25 ppb Hg²⁺ caused a significantly longer time of active response (ANOVA, P < 0·01). Repeated stimulation (1, 2 or 4 hits s^?1) resulted in a concentration‐dependent increase in response failures. Regardless of stimulation frequency, larvae exposed to 0 or 25 ppb Hg²⁺ as embryos maintained higher V_max levels for longer intervals during the testing period than those exposed as embryos to either 50 or 75 ppb Hg²⁺.     

Valproate-induced teratogenesis in Japanese rice fish (Oryzias latipes) embryogenesis

Fertilized eggs of Japanese rice fish (medaka) at three developmental stages (Iwamatsu stages 4-30) were exposed to waterborne valproic acid (VPA) (0-80 mM) in hatching solution for 48 h. The amount of valproate to cause 50% mortality (IC(50)) is found to be developmental stage-specific. The embryos were more sensitive to valproate at early stages of development (Iwamatsu stages 4-10) than in the embryos in late stages (Iwamatsu stages 17-30). Valproate exposed embryos have microcephaly and disrupted cardiovasculature with delayed vessel circulation, thrombus formation, and slow heart rate. The hatching efficiency is also reduced by valproate exposure due to developmental delay. The mRNA analysis of nine genes belong to oxidative stress (catalase, gsr, gst), neurogenesis (iro3, wnt1, shh, otx2, nlgn3b) and cell cycle regulation (ccna2) have been done. It was observed that the genes belong to oxidative stress remained unaltered after valproate exposure. However, some of the genes belong to neurogenesis (wnt1,shh, otx2 and nlgn3b) and cell cycle (ccna2) showed developmental stage-specific alteration after valproate exposure. This study indicates that valproate is able to induce some of the phenotypic features which are analogous to human fetal valproate syndrome (FVS). Modulation of genes expressed in neural tissues indicates that this fish can be used to analyze the mechanisms of many neurobehavioral disorders like Autism spectrum disorder (ASD) in human.     

Nitrate (NO3) and nitrite (NO2) are endocrine disruptors to downregulate expression of tyrosine hydroxylase and motor behavior through conversion to nitric oxide in early development of zebrafish

With a view to consider the increasing concern over nitrogen pollution in the aquatic environment, we investigated effects of nitrate (NO₃⁻) and nitrite (NO₂⁻) on the activity of dopaminergic neuron in zebrafish embryos and larvae. Both nitrate and nitrite exposure decreased the expression of tyrosine hydroxylase (TH) in dopaminergic neurons at 48 hpf. Only nitrite decreased the response to tactile stimulation at 72 hpf, whereas both nitrate and nitrite decreased the swimming activity at 6 dpf. When the embryos were exposed to nitrate or nitrite together with an estrogen receptor blocker (ICI 182,780), the decreases in TH expression and motor behavior caused by nitrate or nitrite alone were reversed suggesting the effects of nitrate and nitrite were mediated through estrogen receptor (ER). The result of co-incubation with an oxidoreductase inhibitor, diphenyleneiodonium, indicated the conversion to nitric oxide (NO) is likely to be responsible for the effects of nitrate and nitrite, which was further supported by the increased staining for NO after exposure. The present study demonstrates that nitrate and nitrite are neurotoxicants acting as an endocrine disruptor possibly through conversion to NO to downregulate the activity of dopaminergic neuron in early development of zebrafish.     

The role of the visceral yolk sac in hyperglycemia-induced embryopathies in mouse embryos in vitro.

The adverse developmental effects of hyperglycemia to rodent embryos have been shown using whole embryo culture. Although, a mechanism by which hyperglycemia-induced effects occur is unknown, recent work has focused on the visceral yolk sac as a potential target tissue. Therefore, we have evaluated the developmental effects of hyperglycemia in early head fold stage mouse embryos in vitro and assessed the histiotrophic function of the visceral yolk sac. As has been previously shown in rodents, hyperglycemia produced neural tube closure defects in a concentration dependent manner at 33, 50, and 67 mM glucose using a 44 h exposure period. However, exposure times between 6 and 12 h were sufficient to alter embryonic development when the glucose concentration was 50 or 67 mM. In contrast, early somite stage embryos (4-6 somite stage) appear to be less sensitive to dysmorphogenesis and a 48 h exposure to 67 mM glucose but not 33 or 50 mM also produced neural tube defects. Hyperglycemia (67 mM) did not alter the uptake of 35S-methionine and 35S-cysteine-labeled hemoglobin (35S-Hb) in the visceral yolk sac (VYS) in early headfold staged embryos. However, the accumulation of 35S in the embryo was reduced by 16-18% at glucose concentrations of 50 or 67 mM during the last 12 h of a 44 h exposure period. No effect on VYS uptake or embryonic accumulation of 35S-labeled products was observed at shorter exposure periods (12-24 and 24-36 h).(ABSTRACT TRUNCATED AT 250 WORDS)     

Timing and duration of developmental nicotine exposure contribute to attenuation of the tadpole hypercapnic neuroventilatory response

The ability for air‐breathing vertebrates to adjust ventilation in response to increased CO₂ (hypercapnia) is fundamental to maintaining pH homeostasis. Developmental nicotine exposure has been shown to impair tadpole neuroventilatory responses to hypercapnia following 8–12 weeks of exposure. It is not clear, however, to what extent the timing of exposure during development and/or the duration over which the exposure takes place contribute to this impairment. Here, tadpoles were exposed to 30 μg/L of nicotine for 3‐ or 10‐week durations, either early or late in tadpole development. Correlates of tadpole lung neuroventilation were monitored during normocapnic (1.5% CO₂) and hypercapnic (5% CO₂) conditions of isolated brainstems. Preparations derived from early metamorphic tadpoles failed to increase lung neuroventilation in response to hypercapnia whether they had been exposed to nicotine for 3 or 10 weeks. Preparations derived from late metamorphic tadpoles failed to respond to hypercapnia after being exposed to nicotine for 10 weeks. These results suggest that both the developmental timing and duration of exposure are important when considering nicotine's effect on the hypercapnic neuroventilatory response. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

Development of chick embryos in 1 Hz to 100 kHz magnetic fields

Summary Chick embryos were exposed during their 48 first hours of development to sinusoidally oscillating magnetic fields. The frequencies 1 Hz, 10 Hz, 16.7 Hz, 30 Hz, 50 Hz, 1 kHz, 10 kHz and 100 kHz, and the field strengths 0.1, 1, 10 and 100 A/m were used. Each exposure group consisted of 20 eggs. After the exposure, the embryos were examined for abnormalities and classified by the developmental stage. The percentage of abnormal embryos (%AE) was significantly increased at frequencies from 16.7 Hz to 100 kHz. Above a threshold field strength of about 0.1 to 1 A/m, %AE was rather independent of the field strength, varying from 16% to 56% in different exposure groups. 13% of the sham-exposed control embryos (n = 150) were abnormal. Only the 0.1 A/m exposure group differed significantly from the controls at 1 Hz, and no significant effect was found at 10 Hz. The developmental stage was in general not affected by the magnetic fields, but some abnormal embryos showed retarded development.     

Effects of Low‐Dose Embryonic Thyroid Disruption and Rearing Temperature on the Development of the Eye and Retina in Zebrafish

Thyroid hormones are required for vertebrate development, and disruption of the thyroid system in developing embryos can result in a large range of morphologic and physiologic changes, including in the eye and retina. In this study, our anatomic analyses following low‐dose, chronic thyroid inhibition reveal that both methimazole (MMI) exposure and rearing temperature affect eye development in a time‐ and temperature‐dependent fashion. Maximal sensitivity to MMI for external eye development occurred at 65 hr postfertilization (hpf) for zebrafish reared at 28°C, and at 69 hpf for those reared at 31°C. Changes in eye diameter corresponded to changes in thickness of two inner retinal layers: the ganglion cell layer and the inner plexiform layer, with irreversible MMI‐induced decreases in layer thickness observed in larvae treated with MMI until 66 hpf at 28°C. We infer that maximal sensitivity to MMI between 65 and 66 hpf at 28°C indicates a critical period of thyroid‐dependent eye and retinal development. Furthermore, our results support previous work that shows spontaneous escape from MMI‐induced effects potentially due to embryonic compensatory actions, as our data show that embryos treated beyond the critical period generally resemble controls     

硫代硫酸钠干扰斑马鱼胚胎发育并致畸

硫的衍生物潜在的威胁着胚胎的发育过程.斑马鱼被用于研究不同浓度（1×10-6～1 mol/L）的硫代硫酸钠（sodium thiosulfate, STS）对胚胎发育的影响,在解剖显微镜下实时观察斑马鱼胚胎发育的全过程.采用Western 印迹法检测乙酰化的微管蛋白——α-微管蛋白（acetylated tubulin, α-tubulin）和神经元增殖细胞核抗原（proliferating cell nuclear antigen,PCNA）的表达,分别检测STS暴露后胚胎的运动神经元功能,神经元的增殖状态.发育中的斑马鱼胚胎暴露于0.1～1 mol/L STS,呈现出严重的发育迟缓,并且伴随多脏器畸形;暴露于10 μmol/L～10 mmol/L STS,胚胎呈现循环系统,神经系统以及颌面部畸形.胚胎在48 hpf (hours post fertilization)时,对STS的暴露敏感高于24 hpf和96 hpf.STS可能干扰细胞的增殖及运动神经元的正常分化.STS可能干扰正常的细胞骨架结构,并在胚胎发育晚期影响细胞增殖,对胚胎神经系统、循环系统及颌面部有致畸作用.     

Lack of chick embryotoxicity after 20 kHz, 1.1 mT magnetic field exposure

This investigation was undertaken because biological studies to evaluate the effects of intermediate frequency magnetic fields are insufficient. White Leghorn fertile eggs (60/group) were either exposed to a 20 kHz, 1.1 mT(rms) sinusoidal magnetic field or sham‐exposed during the first 2, 7, or 11 days of embryogenesis. Lower dose exposures at 0.011 and 0.11 mT(rms) for 2 days were also conducted to elucidate possible dose–response relationships. Additional eggs given all‐trans‐retinoic acid, a teratogen, were exposed to the 1.1 mT(rms) magnetic field for the same periods to investigate the modification of embryotoxicity. After exposure, embryos were examined for mortality and developmental abnormalities. Developmental stage, number of somite pairs, and other developmental endpoints were also evaluated. Experiments were triplicated and conducted in a blind fashion. No exposure‐related changes were found in any of the endpoints in intact embryos exposed to1.1 mT(rms) or to the lower doses of 0.11 and 0.011 mT(rms) magnetic fields. Retinoic acid administration produced embryotoxic responses, which were embryonic death and developmental abnormalities, in 40–60% of embryos in the sham‐exposed groups. The magnitude of these responses was not changed significantly by the magnetic field exposures. Under the present experimental conditions, exposure to 20 kHz magnetic field up to 1.1 mT(rms) was not embryotoxic in the chick and did not potentiate the embryotoxic action of retinoic acid. Bioelectromagnetics 30:573–582, 2009. © 2009 Wiley‐Liss, Inc.