Using Zebrafish to Screen Developmental Toxicity of Per- and Polyfluoroalkyl Substances (PFAS).

Per- and polyfluoroalkyl substances (PFAS) are found in many consumer and industrial products. While some PFAS, notably perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS), are developmentally toxic in mammals, the vast majority of PFAS have not been evaluated for developmental toxicity potential. A concentration-response study of 182 unique PFAS chemicals using the zebrafish medium-throughput, developmental vertebrate toxicity assay was conducted to investigate chemical structural identifiers for toxicity. Embryos were exposed to each PFAS compound (≤100 µM) beginning on the day of fertilization. At 6 days post-fertilization (dpf), two independent observers graded developmental landmarks for each larva (e.g., mortality, hatching, swim bladder inflation, edema, abnormal spine/tail, or craniofacial structure). Thirty percent of the PFAS were developmentally toxic, but there was no enrichment of any OECD structural category. PFOS was developmentally toxic (benchmark concentration [BMC] = 7.48 µM); however, other chemicals were more potent: perfluorooctanesulfonamide (PFOSA), N-methylperfluorooctane sulfonamide (N-MeFOSA), ((perfluorooctyl)ethyl)phosphonic acid, perfluoro-3,6,9-trioxatridecanoic acid, and perfluorohexane sulfonamide. The developmental toxicity profile for these more potent PFAS is largely unexplored in mammals and other species. Based on these zebrafish developmental toxicity results, additional screening may be warranted to understand the toxicity profile of these chemicals in other species.

Burn pit-related smoke causes developmental and behavioral toxicity in zebrafish: Influence of material type and emissions chemistry.

Combustion of mixed materials during open air burning of refuse or structural fires in the wildland urban interface produces emissions that worsen air quality, contaminate rivers and streams, and cause poor health outcomes including developmental effects. The zebrafish, a freshwater fish, is a useful model for quickly screening the toxicological and developmental effects of agents in such species and elicits biological responses that are often analogous and predictive of responses in mammals. The purpose of this study was to compare the developmental toxicity of smoke derived from the burning of 5 different burn pit-related material types (plywood, cardboard, plastic, a mixture of the three, and the mixture plus diesel fuel as an accelerant) in zebrafish larvae. Larvae were exposed to organic extracts of increasing concentrations of each smoke 6-to-8-hr post fertilization and assessed for morphological and behavioral toxicity at 5 days post fertilization. To examine chemical and biological determinants of toxicity, responses were related to emissions concentrations of polycyclic hydrocarbons (PAH). Emissions from plastic and the mixture containing plastic caused the most pronounced developmental effects, including mortality, impaired swim bladder inflation, pericardial edema, spinal curvature, tail kinks, and/or craniofacial deformities, although all extracts caused concentration-dependent effects. Plywood, by contrast, altered locomotor responsiveness to light changes to the greatest extent. Some morphological and behavioral responses correlated strongly with smoke extract levels of PAHs including 9-fluorenone. Overall, the findings suggest that material type and emissions chemistry impact the severity of zebrafish developmental toxicity responses to burn pit-related smoke.

Influence of Methylene Blue or Dimethyl Sulfoxide on Larval Zebrafish Development and Behavior.

The use of larval zebrafish developmental testing and assessment, specifically larval zebrafish locomotor activity, has been recognized as a higher throughput testing strategy to identify developmentally toxic and neurotoxic chemicals. There are, however, no standardized protocols for this type of assay, which could result in confounding variables being overlooked. Two chemicals commonly employed during early-life stage zebrafish assays, methylene blue (antifungal agent) and dimethyl sulfoxide (DMSO, a commonly used vehicle) have been reported to affect the morphology and behavior of freshwater fish. In this study, we conducted developmental toxicity (morphology) and neurotoxicity (behavior) assessments of commonly employed concentrations for both chemicals (0.6-10.0 µM methylene blue; 0.3%-1.0% v/v DMSO). A light-dark transition behavioral testing paradigm was applied to morphologically normal, 6 days postfertilization (dpf) zebrafish larvae kept at 26°C. Additionally, an acute DMSO challenge was administered based on early-life stage zebrafish assays typically used in this research area. Results from developmental toxicity screens were similar between both chemicals with no morphological abnormalities detected at any of the concentrations tested. However, neurodevelopmental results were mixed between the two chemicals of interest. Methylene blue resulted in no behavioral changes up to the highest concentration tested, 10.0 µM. By contrast, DMSO altered larval behavior following developmental exposure at concentrations as low as 0.5% (v/v) and exhibited differential concentration-response patterns in the light and dark photoperiods. These results indicate that developmental DMSO exposure can affect larval zebrafish locomotor activity at routinely used concentrations in developmental neurotoxicity assessments, whereas methylene blue does not appear to be developmentally or neurodevelopmentally toxic to larval zebrafish at routinely used concentrations. These results also highlight the importance of understanding the influence of experimental conditions on larval zebrafish locomotor activity that may ultimately confound the interpretation of results.

Inconsistencies in variable reporting and methods in larval zebrafish behavioral assays.

New approaches in developmental neurotoxicity (DNT) screening are needed due to the tens of thousands of chemicals requiring hazard assessments. Zebrafish (Danio rerio) are an alternative vertebrate model for DNT testing, but without a standardized protocol for larval behavioral assays, comparison of results among laboratories is challenging. To evaluate the congruence of protocols across laboratories, we conducted a literature review of DNT studies focusing on larval zebrafish behavior assays and cataloged experimental design consistencies. Our review focused on 51 unique method variables in publications where chemical exposure occurred in early development and subsequent larval locomotor evaluation focused on assays that included a light/dark photoperiod transition. We initially identified 94 publications, but only 31 exclusively met our inclusion criteria which focused on parameters that are important to an assay employed by our laboratory. No publication reported 100% of the targeted variables; only 51 to 86% of those variables were reported in the reviewed publications, with some aspects of the experimental design consistent among laboratories. However, no protocol was exactly the same for any two publications. Many of these variables had more than one parameter/design reported, highlighting the inconsistencies among methods. Overall, there is not only a strong need for the development of a standardized testing protocol for larval zebrafish locomotor assays, but there is also a need for a standardized protocol for reporting experimental variables in the literature. Here we include an extensive guideline checklist for conducting larval zebrafish developmental behavior assays.