Table 1. Assessment of the protocols carried out for pharmacological treatments.
| Drugs | Embryo stages | Duration of drug application | Final concentrations | Frequency of baths changes | Survival rate | Statistical analysis | ||
|---|---|---|---|---|---|---|---|---|
| p-value (Gauss) | p-value (Poisson) | p-value (Q-Poisson) | ||||||
| DEAB | 28 | 1 week | 10−4M/0.1% DMSO | Every 48 h | 100% (n = 4) | 0.799 | ||
| DEAB | 28 | 1 week | 5 × 10−5M/0.05% DMSO | Every 48 h | 100% (n = 12) | 0.631 | ||
| DEAB | 28 | 1 week | 10−5M/0.05% DMSO | Every 48 h | 92% (n = 12) | 0.953 | ||
| DEAB | 28 | 1 week | 10−3M/1% DMSO | Daily | 0% (n = 6) | ND | ||
| DEAB | 28 | 1 week | 10 −4 M/0.1% DMSO | Daily | 68% (n = 41) | 0.269 | ||
| DEAB | 31–32 | 3 weeks | 10−4M/0.1% DMSO | Daily | 0% (n = 9) | ND | ||
| DEAB | 31–32 | 3 weeks | 10−5M/0.01% DMSO | Daily | 10% (n = 29) | ND | ||
| DEAB | 31–32 | 3 weeks | 10−4M/0.1% DMSO | Every 48 h | 0% (n = 8) | ND | ||
| DEAB | 31–32 | 3 weeks | 5 × 10−5M/0.05% DMSO | Every 48 h | 12% (n = 8) | ND | ||
| DEAB | 31–32 | 3 weeks | 10−5M/0.01% DMSO | Every 48 h | 37% (n = 8) | ND | ||
| DEAB | 31–32 | 1 week | 5 × 10−5M/0.05% DMSO | Daily | 56% (n = 34) | 2.826 e−10 | ||
| DEAB | 31–32 | 1 week | 10 −5 M/0.01% DMSO | Daily | 70% (n = 30) | 0.22 | ||
| RA | 28 | 48 h | 10 −6 M/0.01% DMSO | Daily | 94% (n = 31) | 0.0015 | ||
| RA | 28 | 48 h | 10−7M/0.01% DMSO | Daily | 100% (n = 8) | 0.015 | ||
| RA | 28 | 48 h | 10−8M/0.01% DMSO | Daily | 100% (n = 8) | 2.232 e−5 | ||
| RA | 28 | 72 h | 10 −6 M/0.01% DMSO | Daily | 100% (n = 14) | 0.105 | ||
| RA | 31–32 | 1 week | 10 −6 M/0.01% DMSO | Daily | 58% (n = 33) | 1.664 e −9 | ||
Note:
Small-spotted catshark embryos were treated at stage 28 for scale development and stage late 31/early 32 for tooth development. Different treatment times, drug concentrations and bath change frequencies were tested. The p-values and the regression model used to evaluate the pharmacological effect on scale or tooth number are indicated. Significant p-values are indicated with an underline. We selected protocols that induced a morphological effect with the lower mortality effect (indicated in bold). An exposure to DEAB 10−4M was selected for stage 28 embryos even though no significative effect could be observed, a higher dose (10−3) being lethal. For stage 31/32 embryos, continuous exposure to DEAB 10−5or 5 × 10−5M for a week was selected, other protocols providing low survival rates. The dose of 10−6M and a short exposure to RA were selected for both 28 and 31/32 stages with acceptable survival rates and with a significant effect. Q-Poisson, Quasi-Poisson; ND, not determined; n, number of treated embryos.