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A Framework for Ecotoxicity Testing in the 21 st Century: Ecotox21  
[PERSON](r), [PERSON] *, [PERSON], [PERSON], [PERSON] and [PERSON]  
State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing 100012, China; [EMAIL_ADDRESS] (Z.-g.Y. ); [EMAIL_ADDRESS] (F.G.); [EMAIL_ADDRESS] (J.-t.F. ); [EMAIL_ADDRESS] (S.-p.W. ); [EMAIL_ADDRESS] (L.-x.Y)  
* Correspondence: [EMAIL_ADDRESS]; Tel. : +86-10-84915174 check for updates  
Received: 24 December 2018; Accepted: 17 January 2019; Published: 28 January 2019  
To reduce the considerable investments of toxicity testing and protecting animal welfare, a new toxicity testing strategy based on response pathways of human cell lines has been proposed in the United States to evaluate the chemical exposure risks to human health. However, the in vitro high-throughput assays have not yet been fully applied in ecotoxicity testing. This paper proposes a framework for high-efficiency ecotoxicity testing strategies to evaluate the ecological risk of chemicals. It consists of pathway-based toxicity testing, embryo-based toxicity testing, and predictive toxicology and data extrapolation, etc., according to different situations. The results of ecotoxicity testing or data mining are analyzed together with physicochemical properties, environmental fate, and exposure data of chemicals to conduct a comprehensive risk assessment of chemicals. The framework provides valuable points to establish high-efficiency ecotoxicity testing strategies in the 21 st century. framework; ecotoxicity testing; high-throughput assay; ecological risk assessment  
Toxicity testing serves as an important approach to evaluate the health effects of chemicals. However, conventional animal-based tests are constrained by the considerable investments of animals, time, and capitals. Meanwhile, using in vivo animal tests to extrapolate human health predictions remains controversial due to a number of assumptions requiring scrutiny. As articulated in the 2007 National Research Council (NRC) report, a new toxicity testing strategy based on response pathways of human cell lines was proposed by the National Institute of Environmental Health Sciences (NIEHS) and the U.S. Environmental Protection Agency (US EPA) [1]. It envisioned a pathway-based foundation to evaluate risks of the chemical exposure. Scientists are attaching increasing importance to this approach [2]. To date, several new initiatives began to refine information from toxicity pathway tests for use in safety evaluations of substances [3]. High-throughput screening assays based on cellular response pathways are generally expected as the future of toxicity testing. As knowledge continues to evolve, all routine toxicity testing could finally be conducted in cells or cell lines. This approach arose in the area of toxicology over the last decade, but was mainly used to assess the health effects of toxicants. Chemical exposures not only pose threats to human well-being, but also damage the ecosystems with adverse effects on fish, birds, and other wild organisms. In this case, ecotoxicity testing is also at a pivotal juncture in identifying toxic substances and assessing risks of chemical exposures. However, the in vitro high-throughput assays have not yet been fully applied in the ecotoxicity testing. The ecotoxicity testing herein differs from the toxicity testing above, and it focuses upon effects of stressors on wild species rather than human well-being. Species vary from one another, and their sensitivities to environmental agents are significantly different. In high-throughput toxicity tests for human health assessments, cell lines of human origin are used to substitute laboratory animals. Similarly, cell lines of wild species are in demand for ecotoxicity tests. Nevertheless, cell lines of the most species have not been established, which results in the limitation of applying high-throughput screening assays.