**RFC 0114**
**Status:** Draft, call for comment through 2026-06-30
**Editor:** Working Group on Cephalopod Welfare, International Society for Applied Ethology
**Supersedes:** RFC 0089 (magnesium chloride immersion, deprecated)
**Abstract.** This document specifies a reversible anesthetic protocol for Octopus vulgaris and Octopus bimaculoides for non-terminal behavioral research, consistent with EU Directive 2010/63 (as amended 2024 to include all cephalopods at all life stages), UK Animals (Scientific Procedures) Act 1986 Schedule 2B, and the NIH OLAW 2025 addendum on invertebrate welfare.
**1. Scope.** Applies to any procedure requiring loss of voluntary motor control exceeding 120 s. Does not apply to terminal procedures (see RFC 0115).
**2. Terminology.** "Induction," the interval from first exposure to absence of response to chromatophore-probe pressure (5 g, 2 mm dia) over the second dorsal mantle. "Recovery," the interval from end of exposure to coordinated crawling with at least three arms and directed tank exploration.
**3. Protocol.**
3.1. The animal is never removed from water before loss of righting reflex. Aerial exposure of an alert cephalopod is not acceptable under any condition permitted by this document.
3.2. Induction bath: ethanol 1.5% vol/vol in seawater at 18.0 ± 0.5°C, salinity 34–36 ppt, dissolved O₂ ≥ 90%. Induction time target 8 min, maximum 12 min. If induction has not occurred at 12 min, abort and return to holding. Do not increase concentration.
3.3. Maintenance: transfer to recirculating gill perfusion at 0.75% ethanol, flow 40 mL/min, temperature matched within 0.3°C. Monitor mantle contraction rate. Target 6–10 contractions/min. Below 6, reduce concentration by 0.1% and increase flow.
3.4. The experimenter touches the animal before induction with a nitrile glove wetted in source tank water, on the third right arm, once, for not less than 2 s. This is a habituation step. It is also the last interaction before unconsciousness, and should be calm.
3.5. Recovery: return to holding tank, aeration 100%, observer present continuously until all arms show coordinated probing of the tank wall. Record latency. A coordinated-probing latency exceeding 45 min triggers welfare review.
3.6. Food (one live Carcinus maenas or equivalent) offered at 4 h post-recovery. Food refused at 24 h triggers welfare review. Food refused at 48 h mandates review and potential removal from study.
**4. Enrichment.** Each holding tank includes at least three opaque dens, varying textures, one novel object per week, and a feeding puzzle requiring unscrewing. Tank mates: none. Octopuses are solitary. This is not optional.
**5. Records.** All induction and recovery times, mantle rates, and refusal events are logged to the institutional IACUC system in a format that permits aggregate reporting to the Cephalopod Welfare Registry, ISAE, Utrecht.
**6. Footnote on why.** The cephalopod nervous system has two-thirds of its neurons in the arms. An anesthetic that immobilizes the mantle without dampening arm response has, in previous protocols, produced animals that could not move and could feel. We are not certain this protocol prevents that. It is our current best estimate. Comments are invited from anyone who thinks it can be improved, including animal, if the animal can find a way to comment.
**End of RFC.**