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Fish cell spheroids are promising 3D culture models for vertebrate replacement in ecotoxicology. However, new alternative ecotoxicological methods must be adapted for applications in industry and for regulatory purposes; such methods must be cost-effective, simple to manipulate and provide rapid results. Therefore, we compared the effectiveness of the traditional hanging drop (HD), orbital shaking (OS), and HD combined with OS (HD+OS) methods on the formation of zebrafish cell line spheroids (ZFL and ZEM2S). Time in HD (3-5 days) and different 96-well plates [flat-bottom or ultra-low attachment of round-bottom (ULA-plates)] in OS were evaluated. Easy handling, rapid spheroid formation, uniform-sized spheroids, and circularity were assessed to identify the best spheroid protocol. Traditional HD alone did not result in ZFL spheroid formation, whereas HD (5 days)+OS did. When using the OS, spheroids only formed on the ULA-plate. Both HD+OS and OS were reproducible in size (177.50 ± 2.81 µm and 225.62 ± 19.20 µm, respectively) and circularity (0.83 ± 0.02 and 0.80 ± 0.01, respectively) of ZFL spheroids. Nevertheless, HD+OS required a considerable time to completely form spheroids (10 days) and intensive handling, whereas the OS was fast (5 days of incubation) and simple. OS also yielded reproducible ZEM2S spheroids in 1 day (226.23 ± 0.57 µm diameter and 0.80 ± 0.01 circularity). In conclusion, OS in ULA-plate is an effective and simple spheroid protocol for high-throughput ecotoxicity testing. This study contributes to identify a fast, reproducible, and simple protocol of single piscine spheroid formation in 96-well plates and supports the application of fish 3D model in industry and academia., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

In this study, we analyzed samples of water from a river and a lake located near a hospital waste landfill with respect to physico-chemical parameters and conducted bioassays of ecotoxicity using Vibrio fischeri and Daphnia magna, which are species commonly used to evaluate the water toxicity. We also evaluated damage to the genetic material of fish (Astyanax sp. B) that were exposed (96 h) to water from these two sites that were located near the tank ditch, using the alkaline comet assay and the piscine micronucleus test. Parameters including aluminum, manganese, biochemical oxygen demand, sulfide, conductivity, phenol, total coliforms and Escherichia coli counts, were above acceptable levels that have been established in environmental legislation. However, the toxicity bioassays that we carried out in Vibrio fischeri and Daphnia magna and the piscine micronucleus test in fish showed no immediate risk due to acute effects. Based on the results of the comet assay, however, it was possible to detect damage to genetic material in fish that were acutely exposed in the laboratory to water samples from the river and lake that are located near the trench septic tank. Thus, our results suggest that tests beyond those usually employed to test water toxicity, such as the comet assay we used in the fish, are required to assess the toxicity of water with greater accuracy.