• To prepare the anaesthetic; clove oil must be mixed with anhydrous ethyl alcohol 1: 9. It is best to prepare 50mL at a time so that a stock solution is always on hand. Keep the solution away from light and refrigerate.
• The final anaesthetic solution should contain between 40mg-60mg/L of clove oil.
• Thus, a 50mg/L solution would contain 0.05mL of clove oil, 0.45mL ethanol and 999.5mL artificial seawater.
• 2mL of the stock solution per 3.5L of artificial seawater works very well for anaesthetizing 2 hagfish (a concentration of 57mg/L). The anaesthetic should be added to moving sea water (i.e. on the stir plate). If the water is not moving the oil will simply form a thin layer on the surface.
• The hagfish should be immobilized within a few minutes (10-30)
• If more than two hagfish are being anaesthetized in a bucket the same concentration will work. However, the time for the anaesthetic to take hold is greatly increased. Thus, the dose can be increased by 0.5mL (stock solution) for 3-4 hagfish and 1mL for 5 hagfish. It is not recommended that more than 5 hagfish be anaesthetized in the same bucket. The concentration of clove oil is 85.7mg/L if 3mL of stock solution is added to 3.5L of seawater.
• The lethal limit for using clove oil as an anaesthetic is much greater than MS-222, but the recovery time is much longer as well. Thus, anaesthetizing hagfish in smaller numbers and lower doses results in a quick knock out time and much quicker recovery time, ultimately reducing the stress placed on the animal.

Note: The active ingredient of clove oil is eugenol. The addition of clove oil to ethanol extracts the eugenol and increases the effectiveness of clove oil as an anesthetic.

submitted by Tim Winegard June 2008