Early Life Stages Of Zebrafish Research Articles

Developmental toxicity, immunotoxicity and cardiotoxicity induced by methidathion in early life stages of zebrafish

Methidathion is a highly effective organophosphorus pesticide and is extensively utilized for the control of insects in agricultural production. However, there is little information on the adverse effects and underlying mechanisms of methidathion on aquatic organisms. In this work, embryonic zebrafish were exposed to methidathion at concentrations of 4, 10, and 25 mg/L for 96 h, and morphological changes and activities of antioxidant indicators alterations were detected. In addition, the locomotor behavioral abilities of zebrafish exposed to methidathion were also measured. To further explore the mechanism of the toxic effects of methidathion, gene expression levels associated with cardiac development, cell apoptosis, and the immune system were tested through qPCR assays. The findings revealed that methidathion exposure could induce a decrease in survival rate, hatchability, length of body, and increase in abnormality of zebrafish, as well as cardiac developmental toxicity. The LC50 value of methidathion in zebrafish embryos was determined to be about 30.72 mg/L at 96 hpf. Additionally, methidathion exposure triggered oxidative stress in zebrafish by increasing SOD activity, ROS, and MDA content. Acridine orange (AO) staining indicated that methidathion exposure led to apoptosis, which was mainly distributed in the pericardial region. Furthermore, significant impairments of locomotor activity in zebrafish larvae were induced by methidathion exposure. Lastly, the expression of pro-inflammatory factors including IFN-γ, IL-6, IL-8, CXCL-clc, TLR4, and MYD88 significantly up-regulated in exposed zebrafish. Taken together, the results in this work illustrated that methidathion caused developmental toxicity, cardiotoxicity, and immunotoxicity in embryogenetic zebrafish.

Developmental Toxicity and Cardiotoxicity Induced by PFOS and its Novel Alternative OBS in Early Life Stage of Zebrafish (Danio rerio)

Developmental Toxicity and Cardiotoxicity Induced by PFOS and its Novel Alternative OBS in Early Life Stage of Zebrafish (Danio rerio)

Comparison of toxicity of silver nanomaterials and silver nitrate on developing zebrafish embryos: Bioavailability, osmoregulatory and oxidative stress

The mechanisms of toxicity of engineered nanomaterials (ENMs) to the early life stages of freshwater fish, and the relative hazard compared to dissolved metals, is only partially understood. In the present study, zebrafish (Danio rerio) embryos were exposed to lethal concentrations of silver nitrate (AgNO3) or silver (Ag) ENMs (primary size 42.5 ± 10.2 nm). The 96 h-LC50 for AgNO3 was 32.8 ± 0.72 μg Ag L−1 (mean ± 95% CI) compared to 6.5 ± 0.4 mg L−1 of the whole material for Ag ENMs; with the ENMs being orders of magnitude less toxic than the metal salt. The EC50 for hatching success was 30.5 ± 1.4 μg Ag L−1 and 6.04 ± 0.4 mg L−1 for AgNO3 and Ag ENMs, respectively. Further sub-lethal exposures were performed with the estimated LC10 concentrations for both AgNO3 or Ag ENMs over 96 h, where about 3.7% of the total Ag as AgNO3 was internalised, as measured by Ag accumulation in the dechorionated embryos. However, for the ENM exposures, nearly all (99.8%) of the total Ag was associated with chorion; indicating the chorion as an effective barrier to protect the embryo in the short term. Calcium (Ca2+) and sodium (Na+) depletion was induced in embryos by both forms of Ag, but hyponatremia was more pronounced in the nano form. Total glutathione (tGSH) levels declined in embryos exposed to both Ag forms, but a superior depletion occurred with the nano form. Nevertheless, oxidative stress was mild as superoxide dismutase (SOD) activity stayed uniform and the sodium pump (Na+/K+-ATPase) activity had no appreciable inhibition compared to the control. In conclusion, AgNO3 was more toxic to the early life stage zebrafish than the Ag ENMs, still differences were found in the exposure and toxic mechanisms of both Ag forms.

Advances in PAH mixture toxicology enabled by zebrafish.

Polycyclic aromatic hydrocarbons (PAHs) are a class of organic compounds produced by a variety of petrogenic and pyrogenic sources. PAHs inherently occur in the environment in complex mixtures. The early life-stage zebrafish model is a valuable tool for high-throughput screening (HTS) for toxicity of complex chemical mixtures due to its rapid development, high fecundity, and superb sensitivity to chemical insult. Zebrafish are amenable to exposure to surrogate mixtures as well as extracts of environmental samples and effect-directed analysis. In addition to its utility to HTS, the zebrafish has proven an excellent model for assessing chemical modes of action and identifying molecular initiating and other key events in an Adverse Outcome Pathway framework. Traditional methods of assessing PAH mixture toxicity prioritize carcinogenic potential and lack consideration of non-carcinogenic modes of action, assuming a similar molecular initiating event for all PAHs. Recent work in zebrafish has made it clear that while PAHs belong to the same chemical class, their modes of action can be divergent. Future research should use zebrafish to better classify PAHs by their bioactivity and modes of action to better understand mixture hazards.

Pyriproxyfen influences growth as well as thyroid hormone–related and gh/igf-1 gene expression during the early life stage of zebrafish (Danio rerio)

Pyriproxyfen is an agricultural chemical pesticide that has been detected in the aquatic environment. This study aimed to clarify the effects of pyriproxyfen on the growth as well as thyroid hormone– and growth-related gene expression of zebrafish (Danio rerio) during its early life stage. Pyriproxyfen exhibited a lethal effect in a concentration-dependent manner: the lowest and no effect concentrations were 250.7 and 111.7 μg/L, respectively. These concentrations were considerably higher than the residual environmental concentrations, indicating the low risk of this pesticide when present at such concentrations. In the zebrafish group treated with 56.6 μg/L pyriproxyfen, thyroid hormone receptor β gene expression levels remained unchanged, whereas thyroid-stimulating hormone β subunit, iodtyronin deiodinase 2, and thyroid hormone receptor α gene expression levels significantly decreased compared with the control group expression levels. In zebrafish treated with 111.7 or 250.7 μg/L pyriproxyfen, iodtyronin deiodinase 1 gene expression levels significantly increased. These results indicate that pyriproxyfen disrupts thyroid hormone activity in zebrafish. Furthermore, pyriproxyfen exposure inhibited zebrafish growth; consequently, we examined the expression of growth hormone (gh) and insulin-like growth factor-I (igf-1), which are important for growth. Pyriproxyfen exposure suppressed gh expression; however, the igf-1 expression levels remained unchanged. Therefore, growth inhibition due to pyriproxyfen exposure was attributed to the suppression of gh expression.

The Embryotoxicity of Alpha-Pinene to the Early Life Stages of Zebrafish (Danio Rerio Hamilton, 1822)

The Alpha-pinene (AP) is produced by pine trees and other plants. The AP exhibits various biological activities such as the development of antimicrobial and antiviral agents, flavors, fragrances, and fungicidal agents. The toxicity data for AP are limited. This study aimed to determine the embryotoxic effects of AP in zebrafish embryos. 1.5 hpf zebrafish embryos were exposed to different concentrations of AP for 72 hours. The LC50 and EC50 values for AP were determined as 441.360 mg/L and 367.795 mg/L, respectively. The embryos were not much affected by AP until hatching. In addition, AP showed teratogenic effects at high doses (320 and 640 mg/L). Typical lesions were an absence of somite (≤48 hpf), lordosis, yolk sac deformity, tail abnormality, cardiac edema, and eye shrinkage (≤ 72 hpf). However, survival rates of zebrafish embryos in the 20, 40, and 80 mg/L AP groups were greater than 80 % during the exposure period. Very low teratogenicity for zebrafish embryos was observed in the 160 mg/L AP group. The results show virtually no embryotoxicity and teratogenicity at 20 and 40 mg/L AP concentrations. No delay in developmental was also observed. Therefore, AP can be considered a safe compound in the concentrations. Keywords: Alpha-pinene, Embryotoxicity, EC50, LC50, Zebrafish

Insights into the modes of action of tritium on the early-life stages of zebrafish, Danio rerio, using transcriptomic and proteomic analyses

In the environment, populations are exposed to different kinds of ionizing radiation. Little is known about their modes of action on non-human species, and whether or not they are similar for alpha, beta and gamma radiations, considered as the reference. In this context, tritium effects (beta emitter) under the form of tritiated water (HTO) were investigated in zebrafish, a common model in toxicology and ecotoxicology with a fully sequenced genome. Experiments were conducted on early life stages, considered to be highly sensitive to pollutants, by exposing eggs to 0.4 mGy/h of HTO until 10 days post fertilization. Tritium internalization was quantified, and effects were investigated using a combined approach of transcriptomic and proteomic analyses. Results highlighted similarities in the biological pathways affected by HTO by both techniques, such as defence response, muscle integrity and contraction, and potential visual alterations. These results correlated well with previous data obtained on earlier developmental stages (1 and 4 dpf). Interestingly, HTO effects were partly overlapping those obtained after gamma irradiation, underlying potential common modes of action. This study, therefore, brought a body of evidence on the effects of HTO observed at the molecular level on zebrafish larvae. Further studies could investigate if the effects persist in adult organisms.

Zn-Al layered double hydroxides induce embryo malformations and impair locomotion behavior in Danio rerio

Layered double hydroxides (LDHs) are stimuli-responsive anionic nanoclays. The vast possibilities of using LDHs can lead to their existence in the ecosystem, raising a question of potential ecological concern. However, little is known about the effect of these nanomaterials on freshwater organisms. The present study aimed to assess the ecotoxicological effects of Zinc-Aluminium LDH-nitrate (ZnAl LDH-NO3) in zebrafish (Danio rerio) early life stages. The endpoints measured were mortality, malformations and hatching rate after exposure of D. rerio embryos and larvae to ZnAl LDH-NO3 following the OECD 236 guideline. The behavioral, biochemical (markers of oxidative stress and neurotoxicity), and molecular (at DNA level) alterations were also assessed using sub-lethal concentrations. No observable acute effects were detected up to 415.2 mg LDH/L while the 96 h-LC50 was estimated as 559.9 mg/L. Tested LDH caused malformations in D. rerio embryos, such as pericardial edema, incomplete yolk sac absorption and tail deformities (96 h-EC50 = 172.4 mg/L). During the dark periods, the locomotor behavior in zebrafish larvae was affected upon ZnAl LDH-NO3 exposure. However, no significant biochemical and molecular changes were recorded. The present findings suggest that ZnAl LDH-NO3 can be regarded as a non-toxic nanomaterial towards D. rerio (E/LC50 > > 100 mg/L) although impairment of the locomotion behavior on zebrafish embryos can be expected at concentrations below 100 mg/L.

Tritiated Thymidine Internalization in Zebrafish Early Life Stages: Joint Use of Experimental Procedures and Microdosimetry.

Tritium is found in the environment under three forms: free in the water, gaseous, and bound to organic matter. Once internalized in living organisms, it can be found in two forms: tissue free water tritium (TFWT) and organically bound tritium (OBT). This study aims to better understand OBT internalization in living organisms and to show the complementarity between experimental procedures and microdosimetry simulations that have often been used to obtain more information on imparted energy to cell nuclei. To do so, tritiated thymidine, an organic form of tritium, was chosen and zebrafish embryos [3.5 h post fertilization (hpf)] were exposed to a range of activity concentrations (2.21 × 103 to 5.95 × 105 Bq/mL). First, individual zebrafish embryos were sampled after different exposure times (1 to 96 h) to qualify the internalization kinetics. Then, the barrier role of the chorion was assessed after 2 days of exposure. Lastly, individual zebrafish embryos were sampled after 1 and 4 days of exposure to measure the internalization in the whole fish and its DNA, but also to highlight a possible link between the internal dose rate and the external activity concentration. Microdosimetry simulations were also made to quantify the imparted energy that could occur in the zebrafish cells after exposure to tritium. Results showed that when bound to thymidine, tritium rapidly incorporates in zebrafish early life stages, with the internalization being almost complete after 24 h. Results also showed that while the chorion acted as a barrier to prevent thymidine from entering the embryos, significant levels could still be measured in the whole organisms as well as in DNA. This study also highlighted that when the external activity concentration increased, the internal dose rate increased as well, following a sigmoidal trend. Microdosimetry simulations highlighted that the size and shape of the cell matters, and that the smallest cells seem to be at the greater risk, with only low-energy electrons inducing energy depositions. A linear fit was also found between the mean energy deposited and the logarithm of the radius of the cell, thus showing that the quantity of deposited energy is proportional to the radius of the cell. While this study highlighted important internalization pattern, it will also be used as the starting point of a study focusing on the toxic effects of tritiated thymidine on zebrafish in its early life stages.

Lead acetate induces cartilage defects and bone loss in zebrafish embryos by disrupting the GH/IGF-1 axis

Skeletal system toxicity due to lead exposure has attracted extensive attention in recent years, but few studies focus on the skeletal toxicity of lead in the early life stages of zebrafish. The endocrine system, especially the GH/IGF-1 axis, plays an important role in bone development and bone health of zebrafish in the early life. In the present study, we investigated whether lead acetate (PbAc) affected the GH/IGF-1 axis, thereby causing skeletal toxicity in zebrafish embryos. Zebrafish embryos were exposed to lead PbAc between 2 and 120 h post fertilization (hpf). At 120 hpf, we measured developmental indices, such as survival, deformity, heart rate, and body length, and assessed skeletal development by Alcian Blue and Alizarin Red staining and the expression levels of bone-related genes. The levels of GH and IGF-1 and the expression levels of GH/IGF-1 axis-related genes were also detected. Our data showed that the LC50 of PbAc for 120 h was 41 mg/L. Compared with the control group (0 mg/L PbAc), after PbAc exposure, the deformity rate increased, the heart rate decreased, and the body length was shortened at various time periods, in the 20-mg/L group at 120 hpf, the deformity rate increased by 50 fold, the heart rate decreased by 34%, and the body length shortened by 17%. PbAc altered cartilage structures and exacerbated bone loss in zebrafish embryos; in addition, PbAc exposure down-regulated the expression of chondrocyte (sox9a, sox9b), osteoblast (bmp2, runx2) and bone mineralization-related genes (sparc, bglap), and up-regulated the expression of osteoclast marker genes (rankl, mcsf). The GH level increased and the IGF-1 level declined significantly. The GH/IGF-1 axis related genes (ghra, ghrb, igf1ra, igf1rb, igf2r, igfbp2a, igfbp3, igfbp5b) were all decreased. These results suggested that PbAc inhibited the differentiation and maturation of osteoblasts and cartilage matrix, promoted the formation of osteoclasts, and ultimately induced cartilage defects and bone loss by disrupting the GH/IGF-1 axis.

Sodium valproate exposure influences the expression of pparg in the zebrafish model.

Valproic acid (VPA) is an anti-epileptic drug used alone or in combination with other medications to treat seizures, mania, and bipolar disorder. VPA recognized as a teratogenic chemical can cause severe birth defects mainly affecting the brain and spinal cord when administered during pregnancy. However, the potential mechanisms of developmental toxicity are still less studied, and in the present study, the influence of VPA exposure was evaluated on zebrafish early-life stages. Zebrafish were exposed to two sublethal concentrations of sodium valproate (SV) (0.06 mM and 0.15 mM) from 24 hours post-fertilization (hpf) to 96 hpf and the SV teratogenic potential was investigated through morphometric analysis of zebrafish larvae combined with the evaluation of cartilage profile. Moreover, the effect of SV on the transcription level of pparg was also performed. The results of the study showed the teratogenic potential of SV, which disrupts the morphometric signature of the head and body. The marked distortion of cartilage structures was paralleled to a malformation of telencephalon and optic tectum in both concentrations suggesting a high teratogen effect of SV on the brain. These data were further confirmed by the increased expression of pparg in the zebrafish head. Overall, the present study confirms the teratogenic activity of SV in the zebrafish model and, for the first time, points out the potential protective role of pparg in the SV dose-dependent toxicity.

Differences in toxicity and accumulation of metal from copper oxide nanomaterials compared to copper sulphate in zebrafish embryos: Delayed hatching, the chorion barrier and physiological effects

The mechanisms of toxicity of engineered nanomaterials (ENMs) to the early life stages of freshwater fish, and the relative hazard compared to dissolved metals, is only partially understood. In the present study, zebrafish embryos were exposed to lethal concentrations of copper sulphate (CuSO4) or copper oxide (CuO) ENMs (primary size ∼15 nm), and then the sub-lethal effects investigated at the LC10 concentrations over 96 h. The 96 h-LC50 (mean ± 95% CI) for CuSO4 was 303 ± 14 µg Cu L−1 compared to 53 ± 9.9 mg L−1 of the whole material for CuO ENMs; with the ENMs being orders of magnitude less toxic than the metal salt. The EC50 for hatching success was 76 ± 11 µg Cu L−1 and 0.34 ± 0.78 mg L−1 for CuSO4 and CuO ENMs respectively. Failure to hatch was associated with bubbles and foam-looking perivitelline fluid (CuSO4), or particulate material smothering the chorion (CuO ENMs). In the sub-lethal exposures, about 42% of the total Cu as CuSO4 was internalised, as measured by Cu accumulation in the de-chorionated embryos, but for the ENMs exposures, nearly all (94%) of the total Cu was associated with chorion; indicating the chorion as an effective barrier to protect the embryo from the ENMs in the short term. Both forms of Cu exposure caused sodium (Na+) and calcium (Ca2+), but not magnesium (Mg2+), depletion from the embryos; and CuSO4 caused some inhibition of the sodium pump (Na+/K+-ATPase) activity. Both forms of Cu exposure caused some loss of total glutathione (tGSH) in the embryos, but without induction of superoxide dismutase (SOD) activity. In conclusion, CuSO4 was much more toxic than CuO ENMs to early life stage zebrafish, but there are subtle differences in the exposure and toxic mechanisms for each substance.

Neurodevelopmental Toxicity of Emamectin Benzoate to the Early Life Stage of Zebrafish Larvae (Danio rerio).

Emamectin benzoate (EMB) is a widely used pesticide and feed additive in agriculture and aquaculture. It easily enters the aquatic environment through various pathways, thus causing adverse effects on aquatic organisms. However, there are no systematic studies regarding the effects of EMB on the developmental neurotoxicity of aquatic organisms. Therefore, the aim of this study was to evaluate the neurotoxic effects and mechanisms of EMB at different concentrations (0.1, 0.25, 0.5, 1, 2, 4 and 8 μg/mL) using zebrafish as a model. The results showed that EMB significantly inhibited the hatching rate, spontaneous movement, body length, and swim bladder development of zebrafish embryos, as well as significantly increased the malformation rate of zebrafish larvae. In addition, EMB adversely affected the axon length of motor neurons in Tg (hb9: eGFP) zebrafish and central nervous system (CNS) neurons in Tg (HuC: eGFP) zebrafish and significantly inhibited the locomotor behavior of zebrafish larvae. Meanwhile, EMB induced oxidative damage and was accompanied by increasing reactive oxygen species in the brains of zebrafish larvae. In addition, gene expression involvement in oxidative stress-related (cat, sod and Cu/Zn-sod), GABA neural pathway-related (gat1, gabra1, gad1b, abat and glsa), neurodevelopmental-related (syn2a, gfap, elavl3, shha, gap43 and Nrd) and swim bladder development-related (foxa3, pbxla, mnx1, has2 and elovlla) genes was significantly affected by EMB exposure. In conclusion, our study shows that exposure to EMB during the early life stages of zebrafish significantly increases oxidative damage and inhibits early central neuronal development, motor neuron axon growth and swim bladder development, ultimately leading to neurobehavioral changes in juvenile zebrafish.

Combined toxic effects of T-2 toxin and propiconazole on the early life stages of zebrafish (Danio rerio)

BackgroundAgricultural products are frequently co-contaminated by mycotoxins and pesticides. Most studies have only focused on the single toxicities of these chemicals, while their combined effects are neglected. Therefore, we investigated the combined toxic effects of T-2 toxin (T-2) and propiconazole (PRO) on zebrafish (Danio rerio).ResultsOur data exhibited that T-2 had higher toxicity to embryonic fish (96-h LC50 value of 0.39 mg a.i. L−1) than PRO (96-h LC50 value of 17.16 mg a.i. L−1). The mixture of T-2 and PRO showed an acute synergistic effect on zebrafish. Meanwhile, indicators associated with oxidative stress (SOD, Mn-sod, and cat) displayed significant variations in most exposures to T-2 and PRO mixtures (MTP) compared with the single exposures. The expressions of apoptosis-related genes cas3 and cas9 were also substantially elevated in the high-dose MTP exposure compared with the corresponding T-2 exposure. Besides, the expressions of endocrine system-related genes (TRβ, tsh, crh, cyp19a, and vtg1) were markedly varied in most MTP exposures compared with the corresponding single exposures. Our present results suggested that the mixture of T-2 and PRO could cause enormous effects on oxidative stress, cellular apoptosis, and the hypothalamic–pituitary–thyroid/hypothalamic–pituitary–gonadal (HPG/HPT) axis of zebrafish.ConclusionsOur results provided new insights into the development of combined pollution standards for agricultural products. Taken together, the impact of the combined effects could be considered and regulated as priorities.

Different developmental insecticide exposure windows trigger distinct locomotor phenotypes in the early life stages of zebrafish

Due to their extensive use and high biological activity, insecticides largely contribute to loss of biodiversity and environmental pollution. The regulation of insecticides by authorities is mainly focused on lethal concentrations. However, sub-lethal effects such as alterations in behavior and neurodevelopment can significantly affect the fitness of individual fish and their population dynamics and therefore deserve consideration. Moreover, it is important to understand the impact of exposure timing during development, about which there is currently a lack of relevant knowledge. Here, we investigated whether there are periods during neurodevelopment of fish, which are particularly vulnerable to insecticide exposure. Therefore, we exposed zebrafish embryos to six different insecticides with cholinergic mode of action for 24 h during different periods of neurodevelopment and measured locomotor output using an age-matched behavior assay. We used the organophosphates diazinon and dimethoate, the carbamates pirimicarb and methomyl as well as the neonicotinoids thiacloprid and imidacloprid because they are abundant in the environment and cholinergic signaling plays a major role during key processes of neurodevelopment. We found that early embryonic motor behaviors, as measured by spontaneous tail coiling, increased upon exposure to most insecticides, while later movements, measured through touch-evoked response and a light-dark transition assay, rather decreased for the same insecticides and exposure duration. Moreover, the observed effects were more pronounced when exposure windows were temporally closer to the performing of the respective behavioral assay. However, the measured behavioral effects recovered after a short period, indicating that none of the exposure windows chosen here are particularly critical, but rather that insecticides acutely interfere with neuronal function at all stages as long as they are present. Overall, our results contribute to a better understanding of risks posed by cholinergic insecticides to fish and provide an important basis for the development of safe regulations to improve environmental health.

Early Exposure to Environmental Pollutants: Imidacloprid Potentiates Cadmium Toxicity on Zebrafish Retinal Cells Death.

In the present study, we analyzed the combination of non-toxic concentrations per se, of Cd and a pesticide the imidacloprid (IMI) (10 and 50 μM for Cd and 195 μM for IMI), to highlight early developmental toxicity and possible damage to retinal cells. Co-exposure to Cd and IMI showed a toxic effect in zebrafish larval development, with lowered degrees of survival and hatching, and in some cases the induction of structural alterations and edema. In addition, co-exposure to 50 and 195 μM, respectively, for Cd and IMI, also showed increased apoptosis in eye cells, accompanied by up regulation of genes associated with antioxidant markers (cat, sod1, nrf2 and ho-1). Thus, the present study aims to highlight how the presence of multiple contaminants, even at low concentrations, can be a risk factor in a model of zebrafish (Danio rerio). The presence of other contaminants, such as IMI, can cause an enhancement of the toxic action of Cd on morphological changes in the early life stage of zebrafish, but more importantly disrupt the normal development of the retina, eventually triggering apoptosis.

Developmental toxicity of chlorpyrifos-methyl and its primary metabolite, 3,5,6-trichloro-2-pyridinol to early life stages of zebrafish (Danio rerio)

Chlorpyrifos-methyl (CPM) is one of the thiophosphate insecticides, and it is mainly metabolized to 3,5,6-trichloro-2-pyridinol (TCP) in the environment. As CPM is a strongly toxic and TCP is persistent in the environment, CPM and TCP need to be evaluate their toxicities using animal model organisms. With this regard, CPM and TCP were treated on zebrafish (Danio rerio) embryos and LC50 values were determined as over 2000 μg/L and 612.5 μg/L, respectively. For the hatchability, CPM did not exhibit any interference, while TCP showed weak inhibition. In the CPM-treated embryos, pericardial edema and bleeding were observed at 48 hpf, but recovered afterwards. The pericardial edema and yolk sac edema were observed in TCP-treated zebrafish embryos at the concentration of 500 μg/L after 72 hpf. TCP induced abnormal heart development and the heartbeat was dramatically decreased in Tg(cmlc2:EGFP) embryos at the level of 500 μg/L. The expression level of heart development-related genes such as gata, myl7, and cacna1c was significantly decreased in the TCP 500 μg/L-treated embryos at the 96 hpf. Taken together, TCP appears to be more toxic than the parent compound towards the zebrafish embryos. It is highly requested that TCP needs to be monitored with a strong public concern because it affects presumably heart development in early-stage aquatic vertebrates.

Toxic Effects and Mechanism of Pefloxacin on the Early Life Stage of Zebrafish (Danio rerio)

Pefloxacin (PFLX) is a type of fluoroquinolone (FQ) that has been widely used in aquaculture and animal husbandry. It is also a new kind of pseudo-persistent pollutant, which has some toxic effects. In this study, zebrafish (Danio rerio) embryos were used as exposure models to analyze the toxicity of PFLX. The results showed that PFLX was absorbed on the outer membrane surface at the beginning, and then a few PFLX slowly entered the cytoplasm. The adsorption complied with the Langmuir isotherm adsorption model. The surface morphology of the embryonic membrane was changed. PFLX exposure had no effect on fetal autonomous movement, but it significantly inhibited the embryo hatching rate. After PFLX exposure, the embryo and larval survival rates showed a clear dose–effect relationship. Though PFLX exposure did not cause teratogenic toxicity, it still affected cardiovascular function. The potential toxicity mechanism and developmental toxicity of PFLX to zebrafish were revealed. These results could be important for studies concerning FQ toxicology.

Adapting classic paradigms to analyze alterations of shoal-wide behavior in early-life stages of zebrafish (Danio rerio) – A case study with fluoxetine

Given the strong increase in prescription of neuroactive pharmaceuticals, neurotoxicity has received growing concern in science and the public. Regulatory requirements stimulated the development of new methods to evaluate the risk of neurotoxic substances for humans and the environment, and, with respect to potential damage to aquatic ecosystems, a variety of behavior-based assays have been proposed for neurotoxicity testing, most of which, however, are restricted to changes in the behavior of individual fish. Since many fish species form shoals under natural conditions, this may cause important aspects of behavior to be overlooked and there is a need for behavior assays integrating individual behavior with behavior of the entire swarm. In order to combine more environmentally realistic sub-chronic exposure scenarios with undistorted social behavior and animal welfare considerations, two behavioral assays are proposed that might be integrated into early-life stage toxicity studies according to OECD TG 210, which are commonly run for a multitude of regulations: To this end, protocols for a novel tank test and a predator response assay were adapted to also record the behavior of free-swimming zebrafish (Danio rerio) juveniles within shoals. Comparisons of the diving response (novel tank) or the shoal's coherence and position relative to the stimulus (predator) with control groups allow conclusions about the anxiety state of the fish, which might well have an impact on survival chances in the wild. As a model substance, the antidepressant fluoxetine ((RS)-N-Methyl-3-phenyl-3-(4-trifluoromethylphenoxy)propylamine) produced adverse effects down to concentrations three orders of magnitude below the EC10 from acute fish embryo toxicity tests according to OECD TG 236. With the integration of such behavior tests into OECD TG 210, important population-relevant information on potential neurotoxicity can be collected without increasing the number of experimental animals.

Enantioselective toxic effects of the novel chiral antifungal agrochemical penthiopyrad in the early life stage of zebrafish (Danio rerio)

Penthiopyrad was extensively applied in agricultural production, however, the toxicities information of the penthiopyrad enantiomers on early life stages of aquatic organism were limited. This study investigated the enantioselective toxicity of penthiopyrad on the early life stage of zebrafish by acute toxicity, sublethal toxic effects and the mRNA relative expression levels of genes related to succinate dehydrogenase, cardiac development, and lipid metabolism. The results showed that the 96-h-LC50 of penthiopyrad racemate and enantiomers to zebrafish embryos were Rac-: 2.784 mg/L; R-(−)-: 3.528 mg/L; S-(+)-: 1.882 mg/L. Penthiopyrad exposure induced autonomous movement abnormalities, slowed heart rate and delayed hatching in zebrafish embryos, and caused developmental toxic effects such as pericardial edema and yolk sac edema. The mRNA relative expression levels results showed that penthiopyrad exposure induced significant enantioselectivity effect for the expression of the Sdha, Pr1 and Nkx2.5 with a 1.94–4.98-fold difference between different enantiomers, and significantly affected succinate dehydrogenase (energy metabolism), lipid metabolism and cardiac development-related genes expression. In general, S-(+)-penthiopyrad induced higher toxic effects in zebrafish embryos, and mitochondrial dysfunction may be an important cause of abnormal development. This study contributed to improve the comprehensive risk assessment and enantiomeric research system of penthiopyrad to early life stage of zebrafish.