Influence of exposure scenario on the sensitivity to caffeine.

The chorion acts as a protective barrier, restricting some chemical absorption into the embryo and the surrounding fluids. In this sense, larvae may only have direct contact with some chemicals after dechorionation. This study aimed to evaluate the effects of caffeine (CAF) (0, 13, 20, 44, 67, and 100 mg.L(-1)) under different exposure scenarios (embryos with chorion or embryos/larvae already hatched) and rank the stage sensitivity. Thus, three scenarios were investigated: from 2 to 120 hours post fertilization (hpf) (5 days of exposure- 5dE), from 72 to 120 hpf (2dE), and from 96 to 120 hpf (1dE). Heart rate (48 hpf) and energy reserves (120 hpf) were measured in the 5dE scenario, and behavior and acetylcholinesterase (AChE) activity were evaluated at 120 hpf in all scenarios (5dE, 2dE, and 1dE). At 120 hpf, some of the fish was transferred to clean medium for a 10 days depuration period (10dPE). Behavior and AChE activity were assessed after this period. In the 5dE scenario, CAF increased heartbeat (13, 20, and 30 mg.L(-1)) and reduced carbohydrates (67, and 100 mg.L(-1)), while inhibiting AChE activity (100 mg.L(-1)) in the 5dE, 2dE, and 1dE scenarios. CAF reduced the total distance moved in the 5dE (67, and 100 mg.L(-1)), 2dE (20, 30, 44, 67, and 100 mg.L(-1)), and 1dE fish (67, and 100 mg.L(-1)) and increased erratic movements. Based on the lowest observed effect concentration (LOEC) for total distance moved (20 mg.L(-1)) and higher inhibition of AChE activity (100 mg.L(-1)) (65%), 2dE fish appear to be more sensitive to CAF. After 10dPE, a recovery in behavior was detected in all scenarios (5dE, 2dE, and 1dE). AChE activity remained inhibited in the 2dE scenario while increasing in the 1dE scenario. This study demonstrated that the presence of the chorion is an important factor for the analysis of CAF toxicity. After the loss of the chorion, organisms show greater sensitivity to CAF and can be used to evaluate the toxicity of various substances, including nanomaterials or chemicals with low capacity to cross the chorion. Therefore, the use of hatched embryos in toxicity tests is suggested, as they allow a shorter and less expensive exposure scenario that provides similar outcome as the conventional scenario.