Early Life Stages Of Zebrafish Research Articles

The Embryotoxicity of Some Phenol Derivatives on Zebrafish, Danio rerio

The existence of toxicants in ecosystems has been increased dramatically in recent years, especially in aquatic environments. Phenols and chlorinated phenol derivatives are toxic industrial compounds. Phenols and derivatives are known to be environmental contaminants. In the present study, 2,4-Dichlorophenol, 2-Chlorophenol and substituted phenol were tested for embryotoxicity and mortality in a four-day period using zebrafish, Danio rerio embryos. Tested phenol derivatives caused teratogenicity and embryo mortality in the embryos. The semi static 48-h LC50 (median lethal concentration) value for Substituted-phenol was 13.850 mg L-1; the corresponding values for 2-Chlorophenol and 2,4-Dichlorophenol were 8.378 mg L-1 and 6.558 mg L-1, respectively. The endpoints are incomplete eyes, head and tail, heart and chorda deformity, yolk sac edema, tail curvature, shrunken eyes, lordosis, delayed hatching, weak pigmentation, heart edema and non-pigmentation after exposure to the compounds. 2,4-Dichlorophenol was found to be more toxic than the others. This paper is the first to describe the relative toxicity of a suite of phenols in the early life stages of zebrafish.

Analysis of transcriptional response in zebrafish eleutheroembryos exposed to climbazole: Signaling pathways and potential biomarkers.

Climbazole is an antifungal active ingredient used in personal care products. After application this chemical reaches the aquatic environment and may pose a risk to fish. In the present study, we measured the transcriptional effects of essential genes related to a wide range of signaling pathways on zebrafish eleutheroembryos exposed to climbazole at environmentally relevant and predicted worst-case environmental concentrations, and explored the potential biomarkers via partial least squares discriminant analysis. Transcription analysis covering up to 73 genes revealed significant down-regulation of circadian rhythm- and steroidogenesis-related genes in zebrafish embryos and larvae after exposure to environmentally relevant concentrations of climbazole. This topical antifungal agent also modulated the transcripts of genes involved in inflammation, oxidative stress, oocyte maturation, and sexual differentiation at predicted worst-case environmental concentrations. In addition, mprα, igf3, nr1d1, nr1d2b, cyp19a1a, vtg1, il-1β, and il-8 were chosen as potential biomarkers in embryonic zebrafish following exposure to climbazole. These findings can help us understand the remarkable transcriptional response to climbazole in the early life stage of zebrafish. Future research should elucidate whether the transcriptional modulation translates into metabolic phenotypes associated with the corresponding signaling pathways. Environ Toxicol Chem 2019;38:794-805. © 2019 SETAC.

2,4-Dichlorophenoxyacetic acid herbicide effects on zebrafish larvae: development, neurotransmission and behavior as sensitive endpoints.

Assessment of pesticides toxicity using zebrafish early life stages is relevant for aquatic systems safety. This study aimed to evaluate the short-term effects of 2,4-dichlorophenoxyacetic acid (2,4-D) on zebrafish (Danio rerio) embryos from 3h post fertilization to 96hpf. A set of 2,4-D concentrations ranging from 0.32 to 80mg/L were tested and median lethal concentration (LC50) at 96-h was calculated as 2.86mg/L. A sub-teratogenic concentrations range from 0.02 to 0.8mg/L was then used to assess effects at ontogenic, biochemical, and behavioral levels. The main developmental defects were tail deformities and pericardial edema at concentrations equal or above 0.32mg/L. Cholinesterase activity (at 96hpf) and larvae swimming behavior (at 120hpf) were affected even at the lowest tested dose (0.02mg/L). The behavior analysis was a sensitive endpoint, with a decrease in the swimming distance of exposed larvae during light period. The effect of 2,4-D in ChE was translated by an inhibition of the enzyme activity in all treated groups. These findings demonstrate that 2,4-D can alter the cholinergic system by affecting ChE activity which may be involved in the locomotion reduction of exposed larvae and emphasize the potential of neurotransmission and behavioral endpoints as early warning signs of herbicides contamination in aquatic ecosystems.

Exposure to dilute concentrations of bupropion affects zebrafish early life stages

Psychiatric pharmaceuticals are one of the most prescribed active substances globally. Bupropion (BPP) is an antidepressant that acts via inhibition of norepinephrine and dopamine reuptake. It has been found in various water matrices, and thus its effects on aquatic organisms must be studied. The present study aimed to evaluate the acute toxic effects of BPP on zebrafish (Danio rerio) early life stages. For developmental analysis, organisms were exposed for 168 h to concentrations ranging from 0 to 82000 μg/L. Two other experiments were performed by exposing embryos to a wide range of concentrations (from 0 to 50000 μg/L) in order to evaluate BPP effects on embryonic behavior, using the Zebrabox and testing at the biochemical level (acetylcholinesterase, glutathione-S-transferase, lactate dehydrogenase and catalase). Developmental analysis indicated that BPP had low acute toxicity with a calculated 168 h-LC50 of 50346 μg/L. Concentrations equal to or above 44800 μg/L elicited several effects such as hatching delay, edemas and tail deformities. However, concentrations from 7300 μg/L upwards elicited equilibrium alteration. Behavioral analysis showed that BPP affected zebrafish locomotor behavior by decreasing activity at 0.6 μg/L, increasing activity at 8.8 and 158 μg/L, and decreasing activity at 50000 μg/L. Biochemical analysis showed an increase of AChE activity at 158 and 2812 μg/L, an increase in GST at the highest concentrations, CAT alteration and increase of LDH at 0.6, 2812 and 50000 μg/L. We can conclude that BPP affects zebrafish early life stages at environmental concentrations.

Evaluating the physiological significance of hypoxic hyperventilation in larval zebrafish (Danio rerio).

In water-breathing fishes, the hypoxic ventilatory response (HVR) represents an increase in water flow over the gills during exposure to lowered ambient O2 levels. The HVR is a critical defense mechanism that serves to delay the negative consequences of hypoxia on aerobic respiration. However, the physiological significance of the HVR in larval fishes is unclear as they do not have a fully developed gill and rely primarily on cutaneous gas transfer. Using larval zebrafish (4, 7, 10 and 15 days post-fertilization; dpf), we examined HVR under three levels of hypoxia (25, 45 and 60 mmHg). The larvae exhibited widely different HVRs as a function of developmental age and level of the hypoxia. Yet, critical O2 tensions (Pcrit) remained constant (30-34 mmHg) over the same period of development. Micro-optrode O2 sensors were used to measure a significant decrease in buccal cavity water O2 tensions in 4 and 7 dpf larvae compared with the water they inspired, demonstrating significant extraction of O2 from the buccal cavity. To assess the physiological significance of the HVR, ventilatory water flow was prevented in larvae at 4 and 7 dpf by embedding their heads in agar. An increase in Pcrit was observed in larvae at 7 dpf but not 4 dpf, suggesting that buccal ventilation is important for O2 extraction by 7 dpf. Combined, these data indicate that branchial/buccal gas transfer plays a significant role in O2 uptake during hypoxia, and supports a physiological benefit of the HVR in early life stages of zebrafish.

Assessment of environmentally contaminated sediment using a contact assay with early life stage zebrafish (Danio rerio)

Lake Saint-Louis, a shallow fluvial lake near the western tip of the island of Montreal, QC, Canada is an important spawning ground for many species of fish. Sediments in certain areas of the lake are known to be contaminated with high levels of metals and legacy organic chemicals. To improve our understanding of risk to native fish populations, we conducted a study evaluating levels of sediment contamination and potential effects on early life stage fish. Concentrations of PAHs, PCBs, PCDDs and PCDFs were several orders of magnitude higher at two industrial sites (B1 and B2) than at a nearby reference site (IP). Concentrations of 32 metals and metalloids were at least 5-fold higher at B1 and B2 than at IP. Moreover, all available interim sediment quality guidelines (ISQGs) were exceeded at the two contaminated sites, while none were exceeded at the reference site. Biological effects were evaluated using a sediment contact assay. Zebrafish (Danio rerio) embryos were exposed to clean water (control), or to sediment from IP, B1, and B2 until 120 h post fertilization (hpf). Mortality was significantly elevated in fish exposed to the B1, but not the B2 sediment. The frequency of deformities increased with increasing contamination, but this trend was not statistically significant (p > 0.05). Genes that are implicated in the response to PAHs, PCBs, dioxins and furans (cyp1a, cyp1b1, ahr2) were significantly elevated in the 120 hpf larvae exposed to the B1 and B2 sediments. Global DNA methylation, and mRNA expression of genes related to oxidative stress (maft, cat, hmox1, sod2), embryonic development (bmp2b, baf60c), metal exposure (mt2), and DNA repair (gadd45b) were unaffected. Our results suggest that the Beauharnois sector of Lake Saint-Louis is poor quality spawning habitat due to high levels of contamination, and the potential for harmful effects on early life stage fish.

Tebuconazole reduces basal oxidative respiration and promotes anxiolytic responses and hypoactivity in early-staged zebrafish (Danio rerio)

Triazole fungicides are increasingly used in North America to combat mold and fungi, in order to protect vegetables, citrus, ornamental plants and field crops. To determine the biological impacts of tebuconazole in non-target aquatic organisms, early life stage zebrafish were exposed to 0.1–100 μM tebuconazole for 120 h (5 dpf). There was a significant increase in mortality over time and at 100 μM, only 50% of the animals survived 96 h compared to >95% for all other experimental groups. There was evidence for increased hatching time with 10 μM tebuconazole compared to the control group (~7 h longer at 50% total hatch) or a lack of hatch observed with 100 μM. Oxidative respiration and behavior were evaluated to assess whether the fungicide impaired energy-associated processes. Oxygen consumption rates in embryos (exposed from ~6 hpf) were determined with exposure to 2.5, 25, 50, 100 μM tebuconazole for 24 h using the XFe24 Extracellular Flux Analyzer. Embryos treated with 100 μM showed a ~60% reduction in basal respiration, indicating impaired oxygen consumption and/or changes in resource allocation (e.g. anti-oxidant production, metabolite synthesis). Environmentally-relevant concentrations of tebuconazole did not affect oxidative phosphorylation. As behavior is a sensitive endpoint for toxicity, we measured the dark photokinesis response and conducted a light-dark preference test in 6 dpf larvae following a sub-chronic exposure to 0.1, 1 and 10 μM tebuconazole beginning with 6 hpf embryos. It was observed in two independent experiments for dark photokinesis that 10 μM tebuconazole reduced total distance moved (i.e. hypoactivity) in the dark period by ~25–35%. In the light-dark preference test, there was an increase for mean time in dark zone (~100% increase in the average time/visits per second) and frequency in dark zone (increase of ~35% in average number of visits) with tebuconazole, suggestive of anxiolytic behavior at environmentally-relevant doses. This study demonstrates that exposure to tebuconazole can affect survival, hatch time, oxidative phosphorylation, and behavioral activity of early-staged zebrafish. While survival, hatch time, and mitochondrial bioenergetics were not different than control fish at environmentally-relevant levels of tebuconazole, behavioral responses were detected at concentrations reported in some aquatic environments.

The toxicity of 2,6-dichlorobenzoquinone on the early life stage of zebrafish: A survey on the endpoints at developmental toxicity, oxidative stress, genotoxicity and cytotoxicity

2,6-dichlorobenzoquinone (2,6-DCBQ), an emerging disinfection by-production, frequently occurs in reclaimed water and drinking water. However, limited information was available regarding its toxicity. To evaluate its impact, zebrafish at early life stage were exposed to 0, 10, 30, 60, 90, or 120 μg L−1 2,6-BDCQ for 72 h. Our results indicated that 2,6-BDCQ decreased zebrafish's survival rate to 65% and 44% at 90 and 120 μg L−1 treatments and increased its aberration rate to 11% and 26% at 90 μg L−1 and 120 μg L−1 treatments. Besides, 2,6-BDCQ had adverse effect on its oxidative stress (elevated superoxide dismutase activity), lipid peroxidation (increased malondialdehyde levels), DNA damage (increased 8-hydroxydeoxyguanosine contents) and apoptosis (increased caspase-3 activity). Although lower concentrations (≤60 μg L−1) of 2,6-BDCQ didn't exhibit significant effect on its survival development or lipid peroxidation of zebrafish, they induced obvious DNA damage and apoptosis occurrence. These results revealed 2,6-BDCQ caused genotoxicity and cytotoxicity to zebrafish. This study provides novel insight into 2,6-DCBQ-induced toxicity in zebrafish.

The non-steroidal anti-inflammatory drug diclofenac sodium induces abnormal embryogenesis and delayed lethal effects in early life stage zebrafish (Danio rerio).

Diclofenac sodium, a non-steroidal anti-inflammatory drug widely used in both human and veterinary medicine, has been detected in aquatic environments; therefore, its ecotoxicological effects on aquatic organisms need to be clarified. Recently, toxicity testing using zebrafish (Danio rerio) embryos has been recommended from the point of view of animal welfare; therefore, we investigated the suitability of using sub-lethal endpoints observed during embryogenesis for predicting lethal effects in early life stage zebrafish exposed to diclofenac sodium. After exposure to diclofenac sodium (0.4-7.0mg/L) from 2hours post-fertilization to 30days post-hatching, abnormal embryogenesis, characterized by the presence of edema and body curvature, was observed in the 7.0mg/L exposure group but not in any other groups including controls. The body curvature was found to be the result of abnormal development of the spine. All abnormal embryos hatched without delay, but died within 1week after hatching, suggesting that the combination of the sub-lethal endpoints of edema and abnormal development of the spine during embryogenesis may predict lethal effects in early life stage zebrafish exposed to diclofenac sodium. Further investigations to verify these findings are needed. The value of the no observed effect concentrations for the embryogenesis, survival and growth endpoints were 3.5, 1.8 and >3.5mg/L, respectively.

Down-Regulation of hspb9 and hspb11 Contributes to Wavy Notochord in Zebrafish Embryos Following Exposure to Polychlorinated Diphenylsulfides.

It is hypothesized that key genes, other than ahr2, are present and associated with the development of a unique type of notochord malformation known as wavy notochord in early life stages of zebrafish following exposure to polychlorinated diphenylsulfides (PCDPSs). To investigate the potential mechanism(s), time-dependent developmental morphologies of zebrafish embryos following exposure to 2500 nM 2,4,4',5-tetra-CDPS, 2,2',4-tri-CDPS or 4,4'-di-CDPS were observed to determine the developmental time point when notochord twists began to occur (i.e., 21 h-postfertilization (hpf)). Simultaneously, morphometric measurements suggested that PCDPS exposure did not affect notochord growth at 21 or 120 hpf; however, elongation of the body axis was significantly inhibited at 120 hpf. Transcriptome analysis revealed that the retardation of body growth was potentially related with dysregulation of transcripts predominantly associated with the insulin-associated Irs-Akt-FoxO cascade. Moreover, knockdown and gain-of-function experiments in vivo on codifferentially expressed genes demonstrated that reduced expression of hspb9 and hspb11 contributed to the occurrence of wavy notochord. The results of this study strongly support the hypothesis that the notochord kinks and twists are triggered by the down-regulation of hspb9 and hspb11, and intensified by body growth retardation along with normal notochord length in PCDPS-exposed zebrafish embryos.

DNA methylation and gene expression alterations in zebrafish early-life stages exposed to the antibacterial agent triclosan

There is increasing evidence that toxicant exposure can alter DNA methylation profile, one of the main epigenetic mechanisms, particularly during embryogenesis when DNA methylation patterns are being established. In order to investigate the effects of the antibacterial agent Triclosan on DNA methylation and its correlation with gene expression, zebrafish embryos were exposed during 7 days post-fertilization (starting at maximum 8-cells stage) to 50 and 100 μg/l, two conditions for which increased sensitivity and acclimation have been respectively reported. Although global DNA methylation was not significantly affected, a total of 171 differentially methylated fragments were identified by Reduced Representation Bisulfite Sequencing. The majority of these fragments were found between the two exposed groups, reflecting dose-dependant specific responses. Gene ontology analysis revealed that pathways involved in TGF-β signaling were enriched in larvae exposed to 50 μg/l, while de novo pyrimidine biosynthesis functions were overrepresented in fish exposed to 100 μg/l. In addition, gene expression analysis revealed a positive correlation between mRNA levels and DNA methylation patterns in introns, together with significant alterations of the transcription of genes involved in nervous system development, transcriptional factors and histone methyltransferases. Overall this work provides evidence that Triclosan alters DNA methylation in zebrafish exposed during embryogenesis as well as related genes expression and proposes concentration specific modes of action. Further studies will investigate the possible long-term consequences of these alterations, i.e. latent defects associated with developmental exposure and transgenerational effects, and the possible implications in terms of fitness and adaptation to environmental pollutants.

Exposure to low concentration of fluoxetine affects development, behaviour and acetylcholinesterase activity of zebrafish embryos

Fluoxetine (FLX) is a selective serotonin reuptake inhibitor (SSRI) antidepressant widely used in clinics and very often found in environmental samples of urban aquatic ecosystems in concentrations ranging from ng/L to μg/L. Fish populations might be especially susceptible to FLX due to the presence of conserved cellular receptors of serotonin. Neurotoxic effects on fish biota of polluted water bodies may be expected, but there are no sufficient studies in the current literature to elucidate this hypothesis. Batteries of embryo larval assays with zebrafish were performed to evaluate the potential effects of FLX exposure, including environmentally relevant concentrations. Evaluated parameters included survival, development, behaviour and neuronal biochemical markers. Regarding acute toxicity, a 168 h-LC50 value of 1.18 mg/L was obtained. Moreover, hatching delay and loss of equilibrium were observed, but at a concentration level much higher than FLX measured environmental concentrations (>100 μg/L). On the other hand, effects on locomotor and acetylcholinesterase activity (≥0.88 and 6 μg/L, respectively) were found at levels close to the maximum reported FLX concentration in surface waters. Altogether, these results suggest that FLX is neurotoxic to early life stages of zebrafish, in a short period of time causing changes in important ecological attributes which can probably be linked from molecular to population level.

Effects of silver nanoparticles in early life-stage zebrafish are associated with particle dissolution and the toxicity of soluble silver

The aim of the present study was to investigate and compare the acute and sub-lethal toxicity of silver nanoparticles (Ag NPs) with soluble Ag (as AgNO3) in early life-stage zebrafish using an experimental design suited to separating the nanoscale specific effects of NPs from the toxicity of released metal ions. Early life-stage zebrafish were exposed to AgNO3 and Ag NPs in four separate acute toxicity exposures, each of 24 h duration, in increments from 24 to 120 hours post fertilisation (hpf). The data showed that the sensitivity of zebrafish to both materials increased from 24 to 120 hpf. Overall, the Ag NPs were much less toxic than AgNO3 but the relative magnitudes of change in median lethal concentrations (LC50) across each increment of development were similar. The physiological targets of both materials were therefore likely to be the same and a radiotracer (22Na+) flux technique confirmed the main acute toxic mechanism of both materials to be inhibition of Na+ uptake. Sub-lethal exposures at one quarter of the LC50 (i.e. the same level of effect) showed that concentrations of total Ag in zebrafish were greater from Ag NP exposure than AgNO3. But, the NPs were unlikely to have been accumulated intracellularly or have contributed to the toxicity observed; expression of the metal ion chaperone metallothionein (mt2) and genes involved in oxidative stress pathways were the same at equal relative concentrations of Ag NPs and AgNO3. In conclusion, these data indicated that toxicity of Ag NPs in early life-stage zebrafish was caused by NP dissolution and the release of soluble Ag.

Exposure to ayahuasca induces developmental and behavioral alterations on early life stages of zebrafish

Ayahuasca is a psychoactive concoction prepared from the plants Banisteriopsis caapi and Psychotria viridis which are used ancestrally by Amazonian Indian populations and more recently, by Christian religious groups in Brazil and other countries. The aims of the present study were to identify the effects of ayahuasca on zebrafish embryo development and neurobehavior. Toxicity and developmental endpoints for zebrafish embryos were assessed from 0 to 1000 mg/L over 96 h of exposure. The effects on locomotor activity of zebrafish larvae were assessed using a video tracking system (ZebraBox) from 0 to 20 mg/L and after 120 and 144 h of exposure. The LC50 of ayahuasca in zebrafish was determined as 236.3 mg/L. Ayahuasca exposure caused significant developmental anomalies in zebrafish embryos, mainly at the highest concentration tested, including hatching delay, loss of equilibrium, edema and the accumulation of red blood cells. Embryo behavior was also significantly affected, with decreased locomotor activity at the highest concentration tested. These results are in accordance with data obtained in mammal studies highlighting the possible risks of uncontrolled use of ayahuasca. Further research employing more specific behavior analysis could provide additional data on both therapeutic benefits and possible toxicological risk of ayahuasca.

Lethal and sublethal toxicity of abamectin and difenoconazole (individually and in mixture) to early life stages of zebrafish

In recent years, the need for the development of alternative test methods for the conventional acute fish toxicity test (AFT) with adult fish has often been discussed. In addition, concerns have been raised on the potential risks related with environmentally realistic pesticide mixtures since risk evaluations have traditionally been based on individual pesticides. The insecticide/acaricide abamectin and the fungicide difenoconazole are the main pesticides that are intensively used in Brazilian strawberry crop and are hence likely to occur simultaneously in edge-of-field waterbodies. The aim of the present study was therefore to evaluate the lethal and sublethal toxicity of single and mixture exposures of these pesticides to zebrafish early life stages (embryos and juveniles). By comparing the derived toxicity data of the individual compounds with that previously determined for zebrafish adults, the order of life stage sensitivity was juvenile > adult > embryo. The pesticide mixture revealed a dose-level dependent deviation of the independent action model, with antagonism at low dose levels and synergism at high dose levels. Sublethal parameters (especially those related with locomotion) were considerably more sensitive than lethality. Subsequently, the inclusion of sublethal parameters may greatly improve the sensitivity of FET tests and hence its suitability as a substitution of adult fish testing in risk assessment evaluations.

Red disperse dyes (DR 60, DR 73 and DR 78) at environmentally realistic concentrations impact biochemical profile of early life stages of zebrafish (Danio rerio)

Dyes have been used for more than twenty thousand years in textile, pharmaceutical, food, cosmetic, and photographic industries, among others. Despite their importance in these applications, dyes can be toxic and resistant to many degradation processes used in wastewater treatment plants. Although a large number of dyes have been released in the environments in high amounts, studies into the environmental toxicity of these substances are still scarce. The aim of this study was to evaluate the potential toxic effects of textile dyes Disperse Red 60, Disperse Red 73 and Disperse Red 78 in zebrafish early life stages. To this end, biochemical biomarkers were selected to evaluate non-enzimatic antioxidant (Total Glutathione), antioxidant enzymes (Glutathione S-transferase and Catalase), oxidative stress (lipid peroxidation), neurotransmission (acetylcholinesterase) and energetic metabolism parameters (energy available and energy consumed) after 96 h exposure to these dyes. Our results demonstrated that these disperse dyes induce biochemical alterations in zebrafish embryos at environmental realistic concentrations and that the discharge of these disperse dyes into water bodies should be carefully evaluated. The selected biomarkers were sensitive as early-warning endpoints of disperse dyes toxicity on zebrafish embryos. Implications for risk assessment and indications for future research are discussed.

Toxicity of Ochratoxin to Early Life Stages of Zebrafish (Danio rerio).

Ochratoxin A (OTA) is a known contaminant in fish feed but its effect on fish health remains rather unknown. A study was conducted to investigate the effects of different concentrations of ochratoxin on early life stages of zebrafish (Danio rerio). The tests with ochratoxin A showed a correlation between the exposure to mycotoxin and the amount of damage. The mortality rate and the incidents of embryonal damage was increased by increasing ochratoxin concentrations. The calculations resulted in a lethal concentration for 50% of the embryos (LC50) of 0.29 mg/L and a concentration at which 50% of the animals showed impairment (EC50) of 0.36 mg/L after 96 h of exposure. During the test, reduced heart rates were also observed revealing a clear dose-response relationship. The EC50 determination for this endpoint was 1.26 mg/L after 72 h of exposure. The measurement of oxidative stress was proven to be the most sensitive system to indicate OTA effects on the zebrafish embryos with an EC50 value of 0.067 mg/L after 72 h of exposure. The test validity was given because the control test with 3,4-Dichloroaniline (3,4-D) showed a LC50 value of 2.88 mg after 96 h of exposure which is comparable to the available reference values. According to the current knowledge, these experimental doses did not exceed the environmental concentrations of this ochratoxin A. However, this study raises concerns about the effects of ochratoxin on fish.

Developmental toxicity of synthetic phenolic antioxidants to the early life stage of zebrafish

Synthetic phenolic antioxidants (SPAs) have gained high concerns due to their extensive usages and unintended environmental release via various routes. Their contamination in water system could pose potential threat to aquatic organisms, therefore, the studies on the aquatic toxicology of this kind of chemicals are of high importance. In this research, the developmental toxicities of four commonly used SPAs, including butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tert-butyl hydroquinone (TBHQ), and 2,2′-methylenebis (6-tert-butyl-4-methylphenol) (AO2246) were investigated using the zebrafish embryo toxicity test (ZFET). The results showed that these four SPAs exerted different acute toxicities to zebrafish, and the toxic order, based on their 96 h LC50 values, was AO2246 > TBHQ > BHA > BHT, and decreased hatching rates were induced for the embryos in BHA, TBHQ and AO2246 exposure groups. Non-lethal exposures of BHA (≤20 μM), TBHQ (≤20 μM), BHT (≤200 μM) and AO2246 (≤2 μM) decreased the heart rates and body lengths of zebrafish in exposure concentration-dependent manners. Diverse morphological deformities, including uninflated swim bladder, pericardial edema, spinal curvature, severe yolk deformation, or abnormal pigmentation, were induced in zebrafish larvae upon SPA treatments. The transcriptional levels of the related genes, examined by quantitative PCR, indicated that the interferences of SPAs with hypothalamic-pituitary-thyroid axis (HPT axis), GH/PRL synthesis and Hedgehog (hh) pathway contributed to their developmental toxicities in zebrafish. The up-regulation of pluripotency biomarker, Oct4, caused the developmental retardation during the early stages of zebrafish embryos in BHA and TBHQ exposure groups. The results obtained herein provided important information on the developmental toxicity of SPAs, which could be very helpful in guiding the risk assessment on their aquatic toxicology.

Electrochemical remediation of amoxicillin: detoxification and reduction of antimicrobial activity

Amoxicillin (AMX) is one of the most commonly prescribed antibiotics around the world to treat and prevent several diseases in both human and veterinary medicine. Incomplete removal of AMX during wastewater treatment contributes to its presence in water bodies and drinking water. AMX is an emerging contaminant since its impact on the environment and human health remains uncertain. This contribution was aimed to evaluate the electrochemical oxidation (EO) of AMX using different anodes in tap water, NaCl or Na2SO4 solutions and to evaluate the potential toxicity of remaining AMX and its by-products on zebrafish early-life stages. Chemical intermediates generated after EO were determined by mass spectrometry and their resulting antimicrobial activity was evaluated. AMX did not induce significant mortality in zebrafish during extended exposure but affected zebrafish development (increased body length) from 6.25 mg/L to 25 mg/L and inhibited enzymatic biomarkers. Carbon modified with titanium oxide (TiO2@C) anode achieved complete AMX removal in just a few minutes and efficiency of the supported electrolytes occurred in the following order: 0.1 M NaCl > 0.1 M Na2SO4 > 0.01 M NaCl > tap water. The order of potential toxicity to zebrafish early life-stages related to lethal and sublethal effects was as follows: 0.1 M Na2SO4 > 0.1 M NaCl >0.01 M NaCl = tap water. Additionally, the EO of AMX using TiO2@C electrode with 0.01 M NaCl was able to inhibit the antimicrobial activity of AMX, reducing the possibility of developing bacterial resistance.

Effects of exposure to BPF on development and sexual differentiation during early life stages of zebrafish (Danio rerio)

Bisphenol F (BPF) has become a predominant bisphenol contaminant in recent years. It has significant estrogenic properties in both in vivo and in vitro studies. We have previously studied the disrupting mechanisms of BPF on the hypothalamic–pituitary–gonadal axis of adult zebrafish. However, the effects of BPF exposure on development and sexual differentiation of zebrafish embryos/larvae remain unclear. To determine the effects of BPF on the critical stage of sex differentiation in zebrafish, zebrafish embryos/larvae were exposed to 1, 10, 100, and 1000 μg/L BPF from fertilization to 60 days post-fertilization (dpf). Developmental malformations were induced by exposure to BPF from 2 h post-fertilization (hpf), with a LC50 of 10,030 μg/L at 96 hpf and 9391 μg/L at 120 hpf. Long-term exposure during sex differentiation tended to result in a female sex ratio bias. Histological analyses at 60 dpf indicated that the development of ovo-testes and immature ovaries was induced by 100 and 1000 μg/L BPF. Homogenate testosterone levels decreased and 17β-estradiol levels increased in zebrafish in a concentration-dependent manner. BPF exposure suppressed gene expression of double sex, Mab3-related transcription factor 1(dmrt1), fushi tarazu factor 1d (ff1d), sry-box containing gene 9a (sox9a) and anti-Mullerian hormone (amh); induced expression of the forkhead box L2 transcription factor (foxl2), leading to increased expression of aromatase (cyp19a1a), which promoted production of estrogens, and further caused phenotypic feminization of zebrafish. These results suggest that developmental exposure to BPF has adverse effects on sexual differentiation, and the results were useful for a BPF risk assessment.