Clean isn't always safe: zebrafish (Danio rerio) and the toxic truth of treated wastewater

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Abstract
Wastewater discharges pose a significant risk to aquatic ecosystems due to the complex mixtures of contaminants they contain. Although the toxicity of individual contaminants is well documented, knowledge regarding the interactions among contaminants in wastewater mixtures remains limited. Zebrafish (Danio rerio) are a well-established biological model due to their genetic similarity to humans, transparent embryonic development, and sensitivity to pollutants, making them suitable for assessing wastewater toxicity. This systematic review followed PRISMA guidelines and analyzed studies published between 2015 and 2025 in Scopus and Scielo. A total of 615 articles were screened, and 52 met all inclusion criteria. Most studies focused on treated wastewater (n = 31), followed by raw wastewater (n = 17), and frequently evaluated the embryonic stage (n = 37). Industrial, domestic, and hospital wastewater were the most common sources. The predominant toxicological outcomes included acute toxicity and teratogenesis, although reproductive, genotoxic, oxidative, and behavioral effects were also reported. Pharmaceuticals and potentially toxic elements were the most frequently measured contaminants, whereas groups such as flame retardants, pesticides, and polycyclic aromatic hydrocarbons were rarely addressed. The reviewed studies indicate that exposure to some raw and treated wastewater samples can induce adverse effects on zebrafish development, particularly during early life stages. Major gaps include limited evaluation of larval and adult stages, underrepresentation of certain wastewater sources, and insufficient characterization of complex contaminant mixtures. Addressing these gaps is essential to strengthen environmental risk assessment and protect aquatic ecosystems.