Although the effects of pharmaceuticals on aquatic organisms have been widely investigated during the last decades,
toxic effects, especially delayed toxicity, during the developmental stage at environmental relevant concentrations
were rarely known. In this study, a sensitive assay based on behavioral alterations was used for
studying the delayed toxicity during the developmental stage on zebrafish embryos. Eight pharmaceuticals
thatwere frequently detected with concentrations ranging fromng/l to μg/l were screened for this study. Behavioral
alterations of zebrafish at 118 hpf (hours post fertilization) after exposing to eight single pharmaceuticals
with concentrations in the ranges of environmental detected and their mixtures during embryonic development
(2–50 h post fertilization, hpf) were observed.Multiple endpoints, includingmortality, hatching rate, swimming
speed and angular velocity were evaluated. Results showed that behavioral profile alterations in zebrafish larvae
are promising for predicting delayed sublethal effects of chemicals. Delayed hatchwas observed at 72 hpf following
embryonic exposure to triclosan (1 μg/l) and carbamazepine (100 μg/l) up to 50 hpf. The zebrafish larval locomotor
behavior following embryonic exposure to 0.1 μg/l triclosan and 1 μg/l caffeine in the early stages of

development (2–50 hpf)was altered. Furthermore, the effects of the mixture of 8 pharmaceuticals eachwith the
highest environmental concentration on larval behavior were observed during embryonic development. Generally,
this study showed that the effects of pharmaceuticals singly or their mixtures in surface waters cannot be
ignored.