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Fish in vivo screening methods to detect endocrine active substances, specifically interacting with the hypothalamic–pituitary–gonadal axis, have been developed by both the Organization for Economic Co-operation and Development (OECD) and United States Environmental Protection Agency (US-EPA). In application of these methods, i.e. regulatory testing, this paper provides a proposal on the setting of test concentrations using all available acute and chronic data and also discusses the importance of avoiding the confounding effects of systemic toxicity on endocrine endpoints. This guidance is aimed at reducing the number of false positives and subsequently the number of inappropriate definitive vertebrate studies potentially triggered by effects consequent to systemic, rather than endocrine, toxicity. At the same time it provides a pragmatic approach that maximizes the probability of detecting an effect, if it exists …