Effects of the biocide cybutryne (irgarol) on freshwater invertebrates

Cybutryne (irgarol) is a triazine biocide formerly used in antifouling paints worldwide and still detected in freshwater ecosystems in Europe, Asia, and North America despite its ban in antifouling paints in Europe in January 2017 and in the USA in April 2021. While primary producers are considered the most sensitive targets due to cybutryne's mode of action as a photosystem II inhibitor, data on freshwater invertebrates-particularly from standardized chronic tests-remain scarce. In this study, we investigated acute and chronic effects of cybutryne on three freshwater invertebrates representing major taxonomic phyla: the midge Chironomus riparius (Arthropoda), the blackworm Lumbriculus variegatus (Annelida), and the freshwater mudsnail Potamopyrgus antipodarum (Mollusca). Acute toxicity tests showed low sensitivity, with immobilization observed only in C. riparius larvae at mg/L concentrations (48 h 50% effect concentration (EC₅₀) = 2.96 mg/L), while no acute effects occurred in L. variegatus or P. antipodarum up to five mg/L. In chronic sediment-water tests, C. riparius exhibited no significant effects on survival, emergence, development time, sex ratio, or reproduction at concentrations up to five µg/L. In L. variegatus, reproduction was the most sensitive endpoint, with a 28-day 10% effect concentration (EC₁₀) of 0.268 µg/L (95% confidence interval: 0-462 µg/L) and a lowest observed effect concentration (LOEC) of 0.8 µg/L. In contrast, chronic reproduction tests with P. antipodarum revealed pronounced and consistent increases in embryo numbers at all tested cybutryne concentrations (0.05-5 µg/L), resulting in inverted U-shaped concentration-response relationships that precluded derivation of a no observed effect concentration (NOEC) or EC₁₀ values using conventional monotonic concentration-response models. Exploratory evaluation of quadratic hormesis-type models confirmed the non-monotonic inverted U-shaped concentration-response relationship. However, robust benchmark-concentration estimates could not be derived. The reproductive responses resembled those induced by estrogenic positive controls (17α-ethinylestradiol and bisphenol A) but the present results do not provide direct mechanistic evidence for endocrine disruption. Our results demonstrate that acute cybutryne exposure did not result in negative effects for the test organisms in the applied concentration range of up to five mg/L, except for C. riparius, while chronic reproductive endpoints-particularly in mollusks-appeared to be highly sensitive and suggest the possibility of endocrine-related or other non-specific reproductive modulation mechanisms that require further mechanistic investigation. These findings highlight the importance of including sensitive invertebrate reproduction tests in environmental risk assessment of antifouling biocides.