Table 2.

Zebrafish exposure methods used to test toxicity of purified CYN. Embryos were exposed by either immersion into medium containing CYN, or by micro-injection directly into embryos, as described in Section 2. Different media were used, and media were supplemented with 2% DMSO to facilitate passage of CYN across membranes. Additionally, mechanical dechorionation was used to circumvent possible impermeability of the chorion. CYN was dissolved in water for immersion exposure, or Hank’s balanced salt solution +0.04% phenol red for microinjection, and these were used for appropriate vehicle controls. Observed toxicity, specifically identified by mortality or deformity of zebrafish embryos, is indicated for each exposure method, and further discussed in the text.

Exposure method	Medium used	2% DMSO?	Dechorionation?a	Concentration rangeb	Toxic?
Immersion	E3c	No	No	Up to 50 μg/mL	No
Immersion	ERSd	No	No	Up to 50 μg/mL	No
Immersion	Distilled water	No	No	Up to 50 μg/mL	No
Immersion	E3 medium	Yes	No	Up to 50 μg/mL	No
Immersion	Distilled water	Yes	No	Up to 50 μg/mL	No
Immersion	E3 medium	Yes	Yes	Up to 50 μg/mL	No
Immersion	Distilled water	Yes	Yes	Up to 50 μg/mL	No
Microinjectione	E3 medium	No	No	1.7–843 fmol/embryo (calculated dose injected)	Yesf

^aChorion removed mechanically with forceps.

^bFor negative controls, vehicle added to the medium, except for microinjection exposures for which vehicle injected identically to treated embryos.

^cBrand et al. (2002).

^d“Embryo rearing solution” described in Berry et al. (2007).

^eInjection into the yolk through the animal pole at 4–8 cell stage (see Section 2).

^fToxic results shown in Fig. 2.