Background: Chlorinated phosphate esters (CPEs) are widely used as additive flame retardants for low-density polyurethane foams and have frequently been detected at elevated concentrations within indoor environmental media.Objectives: To begin characterizing the potential toxicity of CPEs on early vertebrate development, we examined the developmental toxicity of four CPEs used in polyurethane foam: tris(1,3-dichloro-2-propyl) phosphate (TDCPP), tris(2-chloroethyl) phosphate (TCEP), tris(1-chloro-2-propyl) phosphate (TCPP), and 2,2-bis(chloromethyl)propane-1,3-diyl tetrakis(2-chlorethyl) bis(phosphate) (V6).Methods: Using zebrafish as a model for vertebrate embryogenesis, we first screened the potential teratogenic effects of TDCPP, TCEP, TCPP, and V6 using a developmental toxicity assay. Based on these results, we focused on identification of susceptible windows of developmental TDCPP exposure as well as evaluation of uptake and elimination of TDCPP and bis(1,3-dichloro-2-propyl)phosphate (BDCPP, the primary metabolite) within whole embryos. Finally, because TDCPP-specific genotoxicity assays have, for the most part, been negative in vivo and because zygotic genome remethylation is a key biological event during cleavage, we investigated whether TDCPP altered the status of zygotic genome methylation during early zebrafish embryogenesis.Results: Overall, our findings suggest that the cleavage period during zebrafish embryogenesis is susceptible to TDCPP-induced delays in remethylation of the zygotic genome, a mechanism that may be associated with enhanced developmental toxicity following initiation of TDCPP exposure at the start of cleavage.Conclusions: Our results suggest that further research is needed to better understand the effects of a widely used and detected CPE within susceptible windows of early vertebrate development.

Firemaster 550 (FM550) is an additive flame retardant formulation of brominated and aryl phosphate ester (APE) components introduced as a major replacement product for the commercial polybrominated diphenyl ether mixture (known as PentaBDE) used primarily in polyurethane foam. However, little is known about the potential effects of FM550-based ingredients during early vertebrate development. Therefore, we first screened the developmental toxicity of each FM550 component using zebrafish as an animal model. Based on these initial screening assays, we found that exposure to the brominated components as high as 10µM resulted in no significant effects on embryonic survival or development, whereas exposure to triphenyl phosphate (TPP) or mono-substituted isopropylated triaryl phosphate (mono-ITP)-two APEs comprising almost 50% of FM550-resulted in targeted effects on cardiac looping and function during embryogenesis. As these cardiac abnormalities resembled aryl hydrocarbon receptor (AHR) agonist-induced phenotypes, we then exposed developing embryos to TPP or mono-ITP in the presence or absence of an AHR antagonist (CH223191) or AHR2-specific morpholino. Based on these studies, we found that CH223191 blocked heart malformations following exposure to mono-ITP but not TPP, whereas AHR2 knockdown failed to block the cardiotoxic effects of both components. Finally, using a cell-based human AHR reporter assay, we found that mono-ITP (but not TPP) exposure resulted in a significant increase in human AHR-driven luciferase activity at similar nominal concentrations as a potent reference AHR agonist (β-naphthoflavone). Overall, our findings suggest that two major APE components of FM550 induce severe cardiac abnormalities during early vertebrate development.

BackgroundIn this commentary we present the findings from an international consortium on fish toxicogenomics sponsored by the U.K. Natural Environment Research Council (Fish Toxicogenomics—Moving into Regulation and Monitoring, held 21–23 April 2008 at the Pacific Environmental Science Centre, Vancouver, BC, Canada).ObjectivesThe consortium from government agencies, academia, and industry addressed three topics: progress in ecotoxicogenomics, regulatory perspectives on roadblocks for practical implementation of toxicogenomics into risk assessment, and dealing with variability in data sets.DiscussionParticipants noted that examples of successful application of omic technologies have been identified, but critical studies are needed to relate molecular changes to ecological adverse outcome. Participants made recommendations for the management of technical and biological variation. They also stressed the need for enhanced interdisciplinary training and communication as well as considerable investment into the generation and curation of appropriate reference omic data.ConclusionsThe participants concluded that, although there are hurdles to pass on the road to regulatory acceptance, omics technologies are already useful for elucidating modes of action of toxicants and can contribute to the risk assessment process as part of a weight-of-evidence approach.

The fish early life-stage (FELS) test (OECD Test Guideline 210) is the primary test used internationally to estimate chronic fish toxicity in support of ecological risk assessments and chemical management programs. As part of an on-going effort to develop efficient and cost-effective alternatives to the FELS test, there is a need to identify and describe potential adverse outcome pathways (AOPs) relevant to FELS toxicity. To support this endeavor, we outline and illustrate an overall strategy for discovery and annotation of FELS AOPs. Key events represented by major developmental landmarks were organized into a preliminary conceptual model of fish development. Using swimbladder inflation as an example, a weight-of-evidence-based approach was used to support linkage of key molecular initiating events to adverse phenotypic outcomes and reduced young-of-year survival. Based on an iterative approach, we explored the feasibility of using key events as the foundation for expanding a network of plausible linkages and AOP knowledge and, in the process, identify important knowledge gaps. Given the scope and scale of the task, prioritization of AOP development was recommended and key research objectives were defined relative to factors such as current animal use restrictions in the European Union and increased demands for fish toxicity data in chemical management programs globally. The example and strategy described are intended to guide collective efforts to define FELS-related AOPs and develop resource-efficient predictive assays that address the toxicological domain of the OECD 210 test.

Although cell-based assays exist, rapid and cost-efficient high-content screening (HCS) assays within intact organisms are needed to support prioritization for developmental neurotoxicity testing in rodents. During zebrafish embryogenesis, spontaneous tail contractions occur from late-segmentation (∼19 h postfertilization, hpf) through early pharyngula (∼29 hpf) and represent the first sign of locomotion. Using transgenic zebrafish (fli1:egfp) that stably express eGFP beginning at ∼14 hpf, we have developed and optimized a 384-well-based HCS assay that quantifies spontaneous activity within single zebrafish embryos after exposure to test chemicals in a concentration-response format. Following static exposure of one embryo per well from 5 to 25 hpf, automated image acquisition procedures and custom analysis protocols were used to quantify total body area and spontaneous activity in live embryos. Survival and imaging success rates across control plates ranged from 87.5 to 100% and 93.3-100%, respectively. Using our optimized procedures, we screened 16 chemicals within the US EPA's ToxCast Phase-I library, and found that exposure to abamectin and emamectin benzoate-both potent avermectins-abolished spontaneous activity in the absence of gross malformations. Overall, compared to existing locomotion-based zebrafish assays conducted later in development, this method provides a simpler discovery platform for identifying potential developmental neurotoxicants.