A systematic scoping review of the neurological effects of COVID-19.

BACKGROUND: The global coronavirus 2019 (COVID-19) pandemic began in early 2020, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In mid-2020 the CIAO (Modelling the Pathogenesis of COVID-19 Using the Adverse Outcome Pathway Framework) project was established, bringing together over 75 interdisciplinary scientists worldwide to collaboratively investigate the underlying biological mechanisms of COVID-19 and consolidate the data using the Adverse Outcome Pathway (AOP) Framework. Neurological symptoms such as anosmia and encephalitis have been frequently reported to be associated with infection with SARS-CoV-2. OBJECTIVE: Within CIAO, a working group was formed to conduct a systematic scoping review of COVID-19 and its related neurological symptoms to determine which key events and modulating factors are most commonly reported and to identify knowledge gaps. DESIGN: LitCOVID was used to retrieve 86,075 papers of which 10,244 contained relevant keywords. After title and abstract screening, 2,328 remained and their full texts were reviewed based on predefined inclusion and exclusion criteria. 991 studies fulfilled the inclusion criteria and were retrieved to conduct knowledge synthesis. RESULTS: The majority of publications reported human observational studies. Early key events were less likely to be reported compared to middle and late key events/adverse outcomes. The majority of modulating factors described related to age or sex. Less recognised COVID-19 associated AO or neurological effects of COVID-19 were also identified including multiple sclerosis/demyelination, neurodegeneration/cognitive effects and peripheral neuronal effects. CONCLUSION: There were many methodological and reporting issues noted in the reviewed studies. In particular, publication abstracts would benefit from clearer reporting of the methods and endpoints used and the key findings, to ensure relevant papers are included when systematic reviews are conducted. The information extracted from the scoping review may be useful in understanding the mechanisms of neurological effects of COVID-19 and to further develop or support existing AOPs linking COVID-19 and its neurological key events and adverse outcomes. Further evaluation of the less recognised COVID-19 effects is needed.

Per- and Polyfluoroalkyl Substances: Impacts on Morphology, Behavior and Lipid Levels in Zebrafish Embryos.

The presence of per- and polyfluoroalkyl substances (PFASs) in aquatic environments is often persistent and widespread. Understanding the potential adverse effects from this group of chemicals on aquatic communities allows for better hazard characterization. This study examines impacts on zebrafish (Danio rerio) embryo physiology, behavior, and lipid levels from exposure to perfluorooctanoic acid (PFOA), perfluorohexane sulfonate (PFHxS), and heptadecafluorooctanesulfonic acid (PFOS). Embryos were exposed to lethal and sublethal levels of each chemical and monitored for alterations in physiological malformations, mortality, lipid levels, and behavior (only PFOA and PFHxS). The predicted 50% lethal concentrations for 120 hpf embryos were 528.6 ppm PFOA, 14.28 ppm PFHxS, and 2.14 ppm PFOS. Spine curvature and the inability of the 120 hpf embryos to maintain a dorsal-up orientation was significantly increased at 10.2 ppm PFHxS and 1.9 ppm PFOS exposure. All measured 120 hpf embryo behaviors were significantly altered starting at the lowest levels tested, 188 ppm PFOA and 6.4 ppm PFHxS. Lipid levels decreased at the highest PFAS levels tested (375 PFOA ppm, 14.4 PFHxS ppm, 2.42 ppm PFOS). In general, the PFAS chemicals, at the levels examined in this study, increased morphological deformities, embryo activity, and startle response time, as well as decreased lipid levels in 120 hpf zebrafish embryos.

Survival, Growth, and Reproduction Responses in a Three-Generation Exposure of the Zebrafish (Danio rerio) to Perfluorooctane Sulfonate.

A prior multigenerational perfluorooctane sulfonic acid (PFOS) exposure investigation in zebrafish reported adverse effects at 0.734 µg/L, among the lowest aquatic effect levels for PFOS reported to date. The present three-generation PFOS exposure quantified survival, growth, reproduction, and vitellogenin (VTG; egg yolk protein) responses in zebrafish, incorporating experimental design and procedural improvements relative to the earlier study. Exposures targeting 0.1, 0.6, 3.2, 20, and 100 µg/L in parental (P) and first filial (F1) generations lasted for 180 days post fertilization (dpf) and the second filial generation (F2) through 16 dpf. Survival decreased significantly in P and F2 generation exposures, but not in F1, at the highest PFOS treatment (100 µg/L nominal, 94-205 µg/L, measured). Significant adverse effects on body weight and length were infrequent, of low magnitude, and occurred predominantly at the highest exposure treatment. Finally, PFOS had no significant effects on P or F1 egg production and survival or whole-body VTG levels in P or F1 male fish. Overall, the predominance and magnitude of adverse PFOS effects at <1 µg/L reported in prior research were largely nonrepeatable in the present study. In contrast, the present study indicated a threshold for ecologically relevant adverse effects in zebrafish at 117 µg/L (SE 8 µg/L, n = 10) for survival and 47 µg/L (SE 11 µg/L, n = 19) for all statistically significant negative effects observed. Environ Toxicol Chem 2024;43:115-131. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

The Adverse Outcome Pathway Framework Applied to Neurological Symptoms of COVID-19.

Several reports have shown that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has the potential to also be neurotropic. However, the mechanisms by which SARS-CoV-2 induces neurologic injury, including neurological and/or psychological symptoms, remain unclear. In this review, the available knowledge on the neurobiological mechanisms underlying COVID-19 was organized using the AOP framework. Four AOPs leading to neurological adverse outcomes (AO), anosmia, encephalitis, stroke, and seizure, were developed. Biological key events (KEs) identified to induce these AOs included binding to ACE2, blood-brain barrier (BBB) disruption, hypoxia, neuroinflammation, and oxidative stress. The modularity of AOPs allows the construction of AOP networks to visualize core pathways and recognize neuroinflammation and BBB disruption as shared mechanisms. Furthermore, the impact on the neurological AOPs of COVID-19 by modulating and multiscale factors such as age, psychological stress, nutrition, poverty, and food insecurity was discussed. Organizing the existing knowledge along an AOP framework can represent a valuable tool to understand disease mechanisms and identify data gaps and potentially contribute to treatment, and prevention. This AOP-aligned approach also facilitates synergy between experts from different backgrounds, while the fast-evolving and disruptive nature of COVID-19 emphasizes the need for interdisciplinarity and cross-community research.

Chronic aquatic toxicity of perfluorooctane sulfonic acid (PFOS) to Ceriodaphnia dubia, Chironomus dilutus, Danio rerio, and Hyalella azteca.

Perfluorooctane sulfonic acid (PFOS) is a ubiquitous and persistent contaminant in aquatic ecosystems. Chronic toxicity information for aquatic organisms is limited, therefore we conducted chronic PFOS toxicity tests for four model organisms commonly used for freshwater toxicology assays: Chironomus dilutus (midge), Ceriodaphnia dubia (water flea), Hyalella azteca (amphipod) and Danio rerio (zebrafish). The 16-day survival test with C. dilutus resulted in the lowest PFOS exposure concentrations to cause significant impacts, with reduced survival at 1 µg/L, a LC50 of 7.5 µg/L, and a growth EC10 of 1.5 µg/L. D. rerio was the next most sensitive species, with a 30-day LC50 of 490 µg/L and reduced growth at 260 µg/L. Effects for C. dubia and H. azteca occurred at concentrations a thousand-fold higher than for C. dilutus. H. azteca had a 42-day LC50 of 15 mg/L, an EC50 of 3.8 mg/L for reproduction (neonates per female) and an EC50 of 4.7 mg/L for growth. C. dubia was similarly tolerant of PFOS, with a 6-day LC50 of 20 mg/L for survival and an EC50 of 7 mg/L for reproduction (neonates per adult). H. azteca, C. dubia, and, to a lesser extent, D. rerio, appear tolerant of PFOS concentrations typically found in the environment. However, in agreement with previous studies, C. dilutus was particularly sensitive to PFOS exposure, with lethal and sublethal effects occurring at concentration levels present at highly contaminated sites.

Male fathead minnow transcriptomes and associated chemical analytes in the Milwaukee estuary system.

Contaminants of Emerging Concern (CECs) can be measured in waters across the United States, including the tributaries of the Great Lakes. The extent to which these contaminants affect gene expression in aquatic wildlife is unclear. This dataset presents the full hepatic transcriptomes of laboratory-reared fathead minnows (Pimephales promelas) caged at multiple sites within the Milwaukee Estuary Area of Concern and control sites. Following 4 days of in situ exposure, liver tissue was removed from males at each site for RNA extraction and sequencing, yielding a total of 116 samples from which libraries were prepared, pooled, and sequenced. For each exposure site, 179 chemical analytes were also assessed. These data were created with the intention of inviting research on possible transcriptomic changes observed in aquatic species exposed to CECs. Access to both full sequencing reads of animal samples as well as water contaminant data across multiple Great Lakes sites will allow others to explore the health of these ecosystems in support of the aims of the Great Lakes Restoration Initiative.

Reactive Oxygen Species in the Adverse Outcome Pathway Framework: Toward Creation of Harmonized Consensus Key Events.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are formed as a result of natural cellular processes, intracellular signaling, or as adverse responses associated with diseases or exposure to oxidizing chemical and non-chemical stressors. The action of ROS and RNS, collectively referred to as reactive oxygen and nitrogen species (RONS), has recently become highly relevant in a number of adverse outcome pathways (AOPs) that capture, organize, evaluate and portray causal relationships pertinent to adversity or disease progression. RONS can potentially act as a key event (KE) in the cascade of responses leading to an adverse outcome (AO) within such AOPs, but are also known to modulate responses of events along the AOP continuum without being an AOP event itself. A substantial discussion has therefore been undertaken in a series of workshops named "Mystery or ROS" to elucidate the role of RONS in disease and adverse effects associated with exposure to stressors such as nanoparticles, chemical, and ionizing and non-ionizing radiation. This review introduces the background for RONS production, reflects on the direct and indirect effects of RONS, addresses the diversity of terminology used in different fields of research, and provides guidance for developing a harmonized approach for defining a common event terminology within the AOP developer community.

Developmental, Behavioral and Transcriptomic Changes in Zebrafish Embryos after Smoke Dye Exposure.

(1) Background: Disperse Blue 14, Disperse Red 9, Solvent Red 169 and Solvent Yellow 33 have been used to color smoke; however, they have not been comprehensively assessed for their potential health hazards. (2) Methods: To assess the effects of these dyes, zebrafish embryos were exposed from 6 to 120 h post fertilization (hpf) to 10-55 µM Disperse Red 9, 1-50 µM Solvent Red 169, 7.5-13.5 µM Solvent Yellow 33 or 133-314 µM Disperse Blue 14. Embryos were monitored for adverse effects on gene expression at 48 hpf as well as for mortality, development and behavior at 120 hpf. The dyes were examined for their potential to cross the blood-brain barrier. (3) Results: Solvent Yellow 33 and Disperse Blue 14 impaired development and behavior at all concentrations. Disperse Red 9 impaired behavior at all concentrations and development at all concentrations except for 10 µM. Solvent Red 169 caused no effects. Mortality was only seen in Disperse Blue 14 at 261.5 and 314 µM. Gene expression indicated impacts on neurodevelopment and folate and retinol metabolism as potential mechanisms of toxicity. (4) Conclusions: Smoke dyes have a high potential for causing developmental changes and neurotoxicity and should be examined more closely using comprehensive approaches as used here.

From Qualitative to Quantitative AOP: A Case Study of Neurodegeneration.

Adverse outcome pathways (AOPs) include a sequence of events that connect a molecular-level initiating event with an adverse outcome at the cellular level for human health endpoints, or at the population level for ecological endpoints. When there is enough quantitative understanding of the relationships between key events in an AOP, a mathematical model may be developed to connect key events in a quantitative AOP (qAOP). Ideally, a qAOP will reduce the time and resources spent for chemical toxicity testing and risk assessment and enable the extrapolation of data collected at the molecular-level by in vitro assays, for example, to predict whether an adverse outcome may occur. Here, we review AOPs in the AOPWiki, an AOP repository, to determine best practices that would facilitate conversion from AOP to qAOP. Then, focusing on a particular case study, acetylcholinesterase inhibition leading to neurodegeneration, we describe specific methods and challenges. Examples of challenges include the availability and collection of quantitative data amenable to model development, the lack of studies that measure multiple key events, and model accessibility or transferability across platforms. We conclude with recommendations for improving key event and key event relationship descriptions in the AOPWiki that facilitate the transition of qualitative AOPs to qAOPs.

Integration of Adverse Outcome Pathways, Causal Networks and 'Omics to Support Chemical Hazard Assessment.

Several approaches have been used in an attempt to simplify and codify the events that lead to adverse effects of chemicals including systems biology, 'omics, in vitro assays and frameworks such as the Adverse Outcome Pathway (AOP). However, these approaches are generally not integrated despite their complementary nature. Here we propose to integrate toxicogenomics data, systems biology information and AOPs using causal biological networks to define Key Events in AOPs. We demonstrate this by developing a causal subnetwork of 28 nodes that represents the Key Event of regenerative proliferation - a critical event in AOPs for liver cancer. We then assessed the effects of three chemicals known to cause liver injury and cell proliferation (carbon tetrachloride, aflatoxin B1, thioacetamide) and two with no known cell proliferation effects (diazepam, simvastatin) on the subnetwork using rat liver gene expression data from the toxicogenomic database Open TG-GATEs. Cyclin D1 (Ccnd1), a gene both causally linked to and sufficient to infer regenerative proliferation activity, was overexpressed after exposures to carbon tetrachloride, aflatoxin B1 and thioacetamide, but not in exposures to diazepam and simvastatin. These results were consistent with known effects on rat livers and liver pathology of exposed rats. Using these approaches, we demonstrate that transcriptomics, AOPs and systems biology can be applied to examine the presence and progression of AOPs in order to better understand the hazards of chemical exposure.

Environmental levels of carbaryl impair zebrafish larvae behaviour: The potential role of ADRA2B and HTR2B.

The insecticide carbaryl is commonly found in indirectly exposed freshwater ecosystems at low concentrations considered safe for fish communities. In this study, we showed that after only 24 h of exposure to environmental concentrations of carbaryl (0.066-660 ng/L), zebrafish larvae exhibit impairments in essential behaviours. Interestingly, the observed behavioural effects induced by carbaryl were acetylcholinesterase-independent. To elucidate the molecular initiating event that resulted in the observed behavioural effects, in silico predictions were followed by in vitro validation. We identified two target proteins that potentially interacted with carbaryl, the α2B adrenoceptor (ADRA2B) and the serotonin 2B receptor (HTR2B). Using a pharmacological approach, we then tested the hypothesis that carbaryl had antagonistic interactions with both receptors. Similar to yohimbine and SB204741, which are prototypic antagonists of ADRA2B and HTR2B, respectively, carbaryl increased the heart rate of zebrafish larvae. When we compared the behavioural effects of a 24-h exposure to these pharmacological antagonists with those of carbaryl, a high degree of similarity was found. These results strongly suggest that antagonism of both ADRA2B and HTR2B is the molecular initiating event that leads to adverse outcomes in zebrafish larvae that have undergone 24 h of exposure to environmentally relevant levels of carbaryl.

Altered Larval Yellow Perch Swimming Behavior Due to Methylmercury and PCB126 Detected Using Hidden Markov Chain Models.

Fish swimming behavior is a commonly measured response in aquatic ecotoxicology because behavior is considered a whole organism-level effect that integrates many sensory systems. Recent advancements in animal behavior models, such as hidden Markov chain models (HMM), suggest an improved analytical approach for toxicology. Using both new and traditional approaches, we examined the sublethal effects of PCB126 and methylmercury on yellow perch (YP) larvae (Perca flavescens) using three doses. Both approaches indicate larvae increase activity after exposure to either chemical. The middle methylmercury-dosed larvae showed multiple altered behavior patterns. First, larvae had a general increase in activity, typically performing more behavior states, more time swimming, and more swimming bouts per second. Second, when larvae were in a slow or medium swimming state, these larvae tended to switch between these states more often. Third, larvae swam slower during the swimming bouts. The upper PCB126-dosed larvae exhibited a higher proportion and a fast swimming state, but the total time spent swimming fast decreased. The middle PCB126-dosed larvae transitioned from fast to slow swimming states less often than the control larvae. These results indicate that developmental exposure to very low doses of these neurotoxicants alters YP larvae overall swimming behaviors, suggesting neurodevelopment alteration.

COVID-19 through Adverse Outcome Pathways: Building networks to better understand the disease - 3rd CIAO AOP Design Workshop.

On April 28-29, 2021, 50 scientists from different fields of expertise met for the 3rd online CIAO workshop. The CIAO project "Modelling the Pathogenesis of COVID-19 using the Adverse Outcome Pathway (AOP) framework" aims at building a holistic assembly of the available scientific knowledge on COVID-19 using the AOP framework. An individual AOP depicts the disease progression from the initial contact with the SARS-CoV-2 virus through biological key events (KE) toward an adverse outcome such as respiratory distress, anosmia or multiorgan failure. Assembling the individual AOPs into a network highlights shared KEs as central biological nodes involved in multiple outcomes observed in COVID-19 patients. During the workshop, the KEs and AOPs established so far by the CIAO members were presented and posi­tioned on a timeline of the disease course. Modulating factors influencing the progression and severity of the disease were also addressed as well as factors beyond purely biological phenomena. CIAO relies on an interdisciplinary crowd­sourcing effort, therefore, approaches to expand the CIAO network by widening the crowd and reaching stakeholders were also discussed. To conclude the workshop, it was decided that the AOPs/KEs will be further consolidated, inte­grating virus variants and long COVID when relevant, while an outreach campaign will be launched to broaden the CIAO scientific crowd.

U.S. Federal Agency interests and key considerations for new approach methodologies for nanomaterials.

Engineered nanomaterials (ENMs) come in a wide array of shapes, sizes, surface coatings, and compositions, and often possess novel or enhanced properties compared to larger sized particles of the same elemental composition. To ensure the safe commercialization of products containing ENMs, it is important to thoroughly understand their potential risks. Given that ENMs can be created in an almost infinite number of variations, it is not feasible to conduct in vivo testing on each type of ENM. Instead, new approach methodologies (NAMs) such as in vitro or in chemico test methods may be needed, given their capacity for higher throughput testing, lower cost, and ability to provide information on toxicological mechanisms. However, the different behaviors of ENMs compared to dissolved chemicals may challenge safety testing of ENMs using NAMs. In this study, member agencies within the Interagency Coordinating Committee on the Validation of Alternative Methods were queried about what types of ENMs are of agency interest and whether there is agency-specific guidance for ENM toxicity testing. To support the ability of NAMs to provide robust results in ENM testing, two key issues in the usage of NAMs, namely dosimetry and interference/bias controls, are thoroughly discussed.

Translatable pathways classification (TransPath-C) for inferring processes germane to human biology from animal studies data: example application in neurobiology.

How to translate insights gained from studies in one organismal species for what is most likely to be germane in another species, such as from mice to humans, is a ubiquitous challenge in basic biology as well as biomedicine. This is an especially difficult problem when there are few molecular features that are obviously important in both species for a given phenotype of interest. Neuropathologies are a prominent realm of this complication. Schizophrenia is complex psychiatric disorder that affects 1% of the population. Many genetic factors have been proposed to drive the development of schizophrenia, and the 22q11 microdeletion (MD) syndrome has been shown to dramatically increase this risk. Due to heterogeneity of presentation of symptoms, diagnosis and formulation of treatment options for patients can often be delayed, and there is an urgent need for novel therapeutics directed toward the treatment of schizophrenia. Here, we present a novel computational approach, Translational Pathways Classification (TransPath-C), that can be used to identify shared pathway dysregulation between mouse models and human schizophrenia cohorts. This method uses variation of pathway activation in the mouse model to predict both mouse and human disease phenotype. Analysis of shared dysregulated pathways called out by both the mouse and human classifiers of TransPath-C can identify pathways that can be targeted in both preclinical and human cohorts of schizophrenia. In application to the 22q11 MD mouse model, our findings suggest that PAR1 pathway activation found upregulated in this mouse phenotype is germane for the corresponding human schizophrenia cohort such that inhibition of PAR1 may offer a novel therapeutic target.

Transcriptomic response patterns of hornyhead turbot (Pleuronichthys verticalis) dosed with polychlorinated biphenyls and polybrominated diphenyl ethers.

To evaluate the impact of environmental contaminants on aquatic health, extensive surveys of fish populations have been conducted using bioaccumulation as an indicator of impairment. While these studies have reported mixtures of chemicals in fish tissues, the relationship between specific contaminants and observed adverse impacts remains poorly understood. The present study aimed to characterize the toxicological responses induced by persistent organic pollutants in wild-caught hornyhead turbot (P. verticalis). To do so, hornyhead turbot were interperitoneally injected with a single dose of PCB or PBDE congeners prepared using environmentally realistic mixture proportions. After 96-hour exposure, the livers were excised and analyzed using transcriptomic approaches and analytical chemistry. Concentrations of PCBs and PBDEs measured in the livers indicated clear differences across treatments, and congener profiles closely mirrored our expectations. Distinct gene profiles were characterized for PCB and PBDE exposed fish, with significant differences observed in the expression of genes associated with immune responses, endocrine-related functions, and lipid metabolism. Our findings highlight the key role that transcriptomics can play in monitoring programs to assess chemical-induced toxicity in heterogeneous group of fish (mixed gender and life stage) as is typically found during field surveys. Altogether, the present study provides further evidence of the potential of transcriptomic tools to improve aquatic health assessment and identify causative agents.

Understanding COVID-19 through adverse outcome pathways - 2nd CIAO AOP Design Workshop.

The CIAO project (Modelling the Pathogenesis of COVID-19 using the Adverse Outcome Pathway framework) aims at a holistic assembly of knowledge to deliver a truly transdisciplinary description of the entire COVID-19 physiopathology starting with the initial contact with the SARS-CoV-2 virus and ending with one or several adverse outcomes, e.g., respiratory failure. On 27-28 January 2021, a group of 50+ scientists from numerous organizations around the world met in the 2nd CIAO AOP Design Workshop to discuss the depiction of the COVID-19 disease process as a series of key events (KEs) in a network of AOPs. During the workshop, 74 such KEs forming 13 AOPs were identified, covering COVID-19 manifestations that affect the respiratory, neurological, liver, cardiovascular, kidney and gastrointestinal systems. Modulating factors influencing the course and severity of the disease were also addressed, as was a possible extension of the investigations beyond purely biological phenomena. The workshop ended with the creation of seven working groups, which will further elaborate on the AOPs to be presented and discussed in the 3rd CIAO workshop on 28-29 April 2021.

Androgenic activation, impairment of the monoaminergic system and altered behavior in zebrafish larvae exposed to environmental concentrations of fenitrothion.

Fenitrothion is an organophosphorus insecticide usually found in aquatic ecosystems at concentrations in the range of low ng/L. In this manuscript we show that 24 h exposure to environmental concentrations of fenitrothion, from ng/L to low µg/L, altered basal locomotor activity, visual-motor response and acoustic/vibrational escape response of zebrafish larvae. Furthermore, fenitrothion and expression of gap43a, gfap, atp2b1a, and mbp exhibited a significant non-monotonic concentration-response relationship. Once determined that environmental concentrations of fenitrothion were neurotoxic for zebrafish larvae, a computational analysis identified potential protein targets of this compound. Some of the predictions, including interactions with acetylcholinesterase, monoamine-oxidases and androgen receptor (AR), were experimentally validated. Binding to AR was the most suitable candidate for molecular initiating event, as indicated by both the up-regulation of cyp19a1b and sult2st3 and the non-monotonic relationship found between fenitrothion and the observed responses. Finally, when the integrity of the monoaminergic system was evaluated, altered levels of L-DOPA, DOPAC, HVA and 5-HIAA were found, as well as a significant up-regulation of slc18a2 expression at the lowest concentrations of fenitrothion. These data strongly suggest that concentrations of fenitrothion commonly found in aquatic ecosystems present a significant environmental risk for fish communities.

Morphological and Behavioral Effects in Zebrafish Embryos after Exposure to Smoke Dyes.

Solvent Violet 47 (SV47) and Disperse Blue 14 (DB14) are two anthraquinone dyes that were previously used in different formulations for the production of violet-colored smoke. Both dyes have shown potential for toxicity; however, there is no comprehensive understanding of their effects. Zebrafish embryos were exposed to SV47 or DB14 from 6 to 120 h post fertilization (hpf) to assess the dyes' potential adverse effects on developing embryos. The potential ability of both dyes to cross the blood-brain barrier was also assessed. At concentrations between 0.55 and 5.23 mg/L, SV47 showed a dose-dependent increase in mortality, jaw malformation, axis curvature, and edemas. At concentrations between 0.15 and 7.54 mg/L, DB14 did not have this same dose-dependence but had similar morphological outcomes at the highest doses. Nevertheless, while SV47 showed significant mortality from 4.20 mg/L, there was no significant mortality on embryos exposed to DB14. Regardless, decreased locomotor movement was observed at all concentrations of DB14, suggesting an adverse neurodevelopmental effect. Overall, our results showed that at similar concentrations, SV47 and DB14 caused different types of phenotypic effects in zebrafish embryos.