Prediction of acute fish toxicity (AFT) and fish embryo toxicity (FET) tests by cytotoxicity assays using liver and embryo zebrafish cell lines (ZFL and ZEM2S).

Fish cell-based assays represent potential alternative methods to vertebrates' use in ecotoxicology. In this study, we evaluated the cytotoxicity of thirteen chemicals, chosen from OECD guidelines 236 and 249, in two zebrafish cell lines (ZEM2S and ZFL). We aimed to investigate whether the IC50 values obtained by viability assays (alamar blue, MTT, CFDA-AM, and neutral red) can predict the LC50 values of Acute Fish Toxicity (AFT) test and Fish Embryo Toxicity (FET) test. There was no significant difference between the values obtained by the different viability assays. ZFL strongly correlated with AFT and FET tests (R2AFT = 0.73-0.90; R2FET48h = 0.79-0.90; R2FET96h = 0.76-0.87), while ZEM2S correlated better with the FET test (48h) (R2 = 0.70-0.86) and weakly with AFT and FET tests (96h) (R2AFT = 0.68-0.74 and R2FET96h = 0.62-0.64). The predicted LC50 values allowed the correct categorization of the chemicals in 76.9% (AFT test) - 90.9% (FET test) using ZFL and in 30.7% (AFT test) - 63.6% (FET test) using ZEM2S considering the US EPA criterion for classifying acute aquatic toxicity. ZFL is a promising cell line to be used in alternative methods to adult fish and fish embryos in ecotoxicity assessments, and the method performed in 96-well plates is advantageous in promoting high-throughput cytotoxicity assessment.

The development and application of a novel hazard scoring tool for assessing impacts of cosmetic ingredients on aquatic ecosystems: A case study of rinse-off cosmetics.

The cosmetic industry has been committed to promoting less hazardous products to reduce the environmental impacts of cosmetic ingredients. This requires identifying safer cosmetic ingredients for developing cosmetic formulations that are less harmful to the environment. However, one of the challenges in developing eco-friendly cosmetics relies on integrating all environmental hazard (EH) information of cosmetic ingredients to select the most eco-friendly ones (i.e., ingredients least harmful to the aquatic environment). Thus, we developed a hazard scoring tool (IARA matrix), which integrates data on biodegradation, bioaccumulation, and acute aquatic toxicity, providing a hazard index to classify cosmetic ingredients (raw materials) into categories of EH (low, moderate, high, or very high). The classification of the IARA was based on parameters established by Cradle to Cradle (C2C), the US Environmental Protection Agency (USEPA), and European Regulation 1272/2008, considering the most conservative values of each source. The Leopold matrix was employed as a model for the tool, using a numerical scale from 0 to 6 (lowest to highest EH). According to the IARA, we have successfully demonstrated that ultraviolet (UV) filter ingredients have the highest EH out of 41 cosmetic ingredients commonly used for rinse-off products. In addition to UV filters, triclosan (bactericide) and dimethicone (emollient) presented the second-highest EH for aquatic ecosystems, and humectants presented the lowest hazard index. By applying the IARA in the case study of rinse-off products, we have estimated that the aquatic hazard of cosmetic products can be reduced 46% by identifying less hazardous ingredients and combining them into a cosmetic formulation. In summary, the IARA tool allows the estimation of the EH of cosmetic ingredients, provides safer products, and helps achieve sustainability for cosmetic products. Integr Environ Assess Manag 2023;19:1619-1635. © 2023 SETAC.

A 3D Culture Method of Spheroids of Embryonic and Liver Zebrafish Cell Lines.

Fish cell lines are promising in vitro models for ecotoxicity assessment; however, conventional monolayer culture systems (2D culture) have well-known limitations (e.g., culture longevity and maintenance of some in vivo cellular functions). Thus, 3D cultures, such as spheroids, have been proposed, since these models can reproduce tissue-like structures, better recapturing the in vivo conditions. This article describes an effective, easy, and fast 3D culture protocol for the formation of spheroids with two zebrafish (Danio rerio) cell lines: ZEM2S (embryo) and ZFL (normal hepatocyte). The protocol consists of plating the cells in a round-bottom, ultra-low attachment, 96-well plate. After 5 days under orbital shaking (70 rpm), a single spheroid per well is formed. The formed spheroids present stable size and shape, and this method avoids the formation of multiple spheroids in a well; thus, it is not necessary to handpick spheroids of similar sizes. The ease, speed, and reproducibility of this spheroid method make it useful for high-throughput in vitro tests.

Cytotoxicity Assays with Zebrafish Cell Lines.

Fish cell lines have become increasingly used in ecotoxicity studies, and cytotoxicity assays have been proposed as methods to predict fish acute toxicity. Thus, this protocol presents cytotoxicity assays modified to evaluate cell viability in zebrafish (Danio rerio) embryo (ZEM2S) and liver (ZFL) cell lines in 96-well plates. The cytotoxicity endpoints evaluated are mitochondrial integrity (Alamar Blue [AB] and MTT assays), membrane integrity via esterase activity (CFDA-AM assay), and lysosomal membrane integrity (Neutral Red [NR] assay). After the exposure of the test substances in a 96-well plate, the cytotoxicity assays are performed; here, AB and CFDA-AM are carried out simultaneously, followed by NR on the same plate, while the MTT assay is performed on a separate plate. The readouts for these assays are taken by fluorescence for AB and CFDA-AM, and absorbance for MTT and NR. The cytotoxicity assays performed with these fish cell lines can be used to study the acute toxicity of chemical substances on fish.