Toxicological Assessment and Physiological Responses to Dichloro‐Octylisothiazolinone and Octylisothiazolinone in Zebrafish Larvae

The widespread presence of isothiazolinones in aquatic environments has raised concerns regarding their potential effects on nontarget organisms. In this study, the toxicity of 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT) and 2-octyl-2H-isothiazol-3-one (OIT), tested individually and in combination, was investigated using the zebrafish (Danio rerio) fish embryo toxicity (FET) test coupled with targeted gene expression analysis. Acute toxicity was evaluated over 96 h after fertilization, and median lethal concentrations (LC₅₀) were determined. DCOIT exhibited higher intrinsic toxicity (LC₅₀ = 0.015 mg/L) compared to OIT (LC₅₀ = 0.14 mg/L), while the mixture showed increased toxicity (LC₅₀ = 0.006 mg/L). Sublethal developmental effects included pericardial and yolk sac edema following DCOIT exposure and depigmentation in larvae exposed to higher concentrations of OIT. To gain insight into potential molecular responses, the expression of selected genes related to inflammation, oxidative stress, and apoptosis was assessed by qRT-PCR at sublethal concentrations. Exposure to both single compounds and their mixture resulted in the modulation of pro-inflammatory markers (tnf-α and il-1β), antioxidant enzymes (sod-1 and cat), and apoptosis-related genes (casp3 and tp53), with generally stronger responses observed at higher concentrations and under mixture exposure. Overall, the results indicate that early life stages of zebrafish are sensitive to both DCOIT and OIT, and that combined exposure can enhance toxicity and molecular responses. Moreover, early developmental exposure to isothiazolinones is associated with the activation of inflammation-related molecular pathways, highlighting their potential risk for aquatic ecosystems.