Sequential assessment via daphnia and zebrafish for systematic toxicity screening of heterogeneous substances

Environment and organisms are persistently exposed by a mixture of various substances. However, the current evaluation method is mostly based on an individual substance's toxicity. A systematic toxicity evaluation of heterogeneous substances needs to be established. To demonstrate toxicity assessment of mixture, we chose a group of three typical ingredients in cosmetic sunscreen products that frequently enters ecosystems: benzophenone-3 (BP-3), ethylhexyl methoxycinnamate (EHMC), and titanium dioxide nanoparticle (TiO₂ NP). We first determined a range of nominal toxic concentration of each ingredient or substance using Daphnia magna, and then for the subsequent organismal level phenotypic assessment, chose the wild-type zebrafish embryos. Any phenotype change, such as body deformation, led to further examinations on the specific organs of transgenic zebrafish embryos. Based on the systematic toxicity assessments of the heterogeneous substances, we offer a sequential environmental toxicity assessment protocol that starts off by utilizing Daphnia magna to determine a nominal concentration range of each substance and finishes by utilizing the zebrafish embryos to detect defects on the embryos caused by the heterogeneous substances. The protocol showed additive toxic effects of the mixtures. We propose a sequential environmental toxicity assessment protocol for the systematic toxicity screening of heterogeneous substances from Daphnia magna to zebrafish embryo in-vivo models. - Highlights: • Contemporary ecological evaluation protocols are mostly based on an individual substance's toxicity. • This study proposes a systematic toxicity screening method for heterogeneous substances by demonstrating a proof-of-concept. • The systematic environmental toxicity assessment protocol used two in-vivo models, Daphnia magna and zebrafish embryo. • The combined toxicity effects of the mixtures were additive. - Toxicity of heterogeneous substances can be evaluated by the sequential assessment platform which starts from Daphnia to zebrafish embryos.