S6.2.1 Noninhaled Agents

Immersion (1 step)—Intentional overdose via immersion in anesthetic solutions is a common method of euthanasia for fish.298,302–304 Some species exhibit aversive responses to particular anesthetic agents, while other species do not.305,306 Through preference and approach-avoidance testing, many anesthetic agents currently used for euthanasia have been identified as being aversive to varying degrees. Despite some evidence of distress and aversion, immersive anesthetics continue to be administered to fish because the benefits associated with their use outweigh any distress and aversion they may cause, similar to use of inhaled agents for air-breathing animals. Fish should be left in the anesthetic solution for a minimum of 30 minutes after cessation of opercular movement.251,298,302 A recent study307 demonstrated that use of buffered MS 222 in a 1-step immersion technique was inadequate for euthanasia of goldfish (Carassius auratus), a hypoxia-tolerant species. Results from this study suggest that a 2-step method may be required for euthanasia of hypoxia-tolerant species,: step 1, immersion to render the fish unconscious; step 2, a secondary adjunctive method to complete euthanasia (such as decapitation, pithing, or freezing). Options for immersion agents include the following:
(1) Benzocaine or benzocaine hydrochloride, buffered. Solutions for immersion should be prepared in concentrations ≥ 250 mg/L and should be buffered.304
(2) Carbon dioxide. Immersion in CO2-saturated water causes narcosis and loss of consciousness after several minutes.251,298 Some species may exhibit hyperactivity prior to loss of consciousness.302 Purity and concentration of CO2 are important for effectiveness. Only CO2 from a source that allows for careful regulation of concentration, such as from cylinders, is acceptable. Care must be taken when using CO2 to prevent exposure to personnel (ie, euthanasia must be conducted in
well-ventilated areas).
(3) Ethanol. Ethanol has been suggested as an acceptable alternative method for fish euthanasia.308 The depressive effects of ethanol on the CNS are well described,309 and exposure of zebrafish via
immersion has become a model for behavioral and molecular responses to alcohol, at concentrations from 10 to 30 mL of 95% ethanol/L.310–312 At this dose, alcohol induces anesthesia, and prolonged immersion produces death via respiratory depression causing anoxia. This is not equivalent to immersing fish directly into preservative concentrations of ethanol (70%), which is not acceptable as a euthanasia method.
(4) Eugenol, isoeugenol, and clove oil. Whenever possible, products with standardized, known concentrations of essential oils should be used so that accurate dosing is possible. Concentrations required for anesthesia will vary depending on species and other factors, but may be as low as 17 mg/L for some species. Greater concentrations (10 times the upper range for anesthesia) will be required for euthanasia.298,313–315 These oils are not very water soluble; injecting the solution through a syringe and fine-gauge needle under the water in the container used for euthanizing is helpful in ensuring dispersal in the water. Fish should be left in the anesthetic solution for a minimum of 10 minutes after cessation of opercular movement. These compounds are equivocal or known carcinogens according to the National
Toxicology Program.316 Some studies in rodents indicate this group of anesthetics may cause paralysis in addition to having anesthetic effects, and analgesic properties are unknown.70,317–319 The FDA strictly prohibits the use of clove oil and eugenol as anesthetics in fish having the potential to enter the food chain, except under Investigational New Animal Drug exemptions.320 Isoeugenol is a potential carcinogen316 so human safety in the application of that agent is of concern.
(5) Isoflurane, sevoflurane. These concentrated liquid anesthetics can be added to water, although they are generally not very water soluble.302 Injecting the solution through a syringe and fine gauge needle under the water in the container used for euthanizing is helpful in ensuring dispersal in the water. Doses of > 5 to 20 mL/L can be used (10 times the upper range for anesthesia). However, because both anesthetics are highly volatile, human safety is of concern and use in a well-ventilated area is imperative.
(6) Quinaldine sulfate. Solutions for immersion should be prepared in concentrations ≥ 100
mg/L.321 Quinaldine sulfate will acidify water; therefore, buffering is required to prevent distress from acute drop in pH.
(7) Tricaine methanesulfonate, buffered (MS 222, TMS). An aversive response to MS 222 has been
demonstrated for zebrafish and medaka, while carp, fathead minnow, and rainbow trout showed
no aversion.305,306 Despite evidence of distress and aversion, immersive anesthetics continue to be administered to fish because the benefits associated with their use outweigh any distress and aversion they may cause. Solutions must be buffered, and concentrations required for euthanasia may vary depending upon the species, life stage, and water chemistry parameters. A concentration of 250 to 500 mg/L, or 5 to 10 times the anesthetic dosage, is effective for most species.298,304 MS 222 at a dose of 400 mg/L has been shown to be ineffective for a few species (eg, Gulf of Mexico sturgeon).298 A recent study307 demonstrated that use of buffered MS 222 in a 1-step immersion technique was inadequate for euthanasia of goldfish (C auratus), a hypoxia-tolerant species. Results from this study support the recommendation for use of a 2-step method for euthanasia of goldfish and some other hypoxia-tolerant species, including cichlids, with the first step of involving immersion to render the fish unconscious and the second involving application of an adjunctive method (such as decapitation, pithing, or freezing) to complete euthanasia. Fish that are too large for practical or cost-effective immersion in lethal doses of buffered MS 222 can be euthanized by applying the concentrated, buffered solution directly to the gills.298,302
(8) 2-phenoxyethanol. Solutions for immersion should be prepared in concentrations ≥ 0.5 to 0.6
mL/L or 0.3 to 0.4 mg/L.321
(9) Lidocaine. A buffered solution at 400 mg/L is effective for euthanasia of adult zebrafish,109 but response to lidocaine by immersion varies considerably across species. Injection—Injectable agents have been administered for euthanasia via IV, intracoelomic, IM, and intracardiac routes.298,308
(1) Pentobarbital (1 step). Sodium pentobarbital (60 to 100 mg/kg [27.3 to 45.5 mg/lb]) can be administered by IV, intracardiac, or intracoelomic routes for euthanasia.251 Pentobarbital may also be administered via intracardiac injection for anesthetized animals as the second step of a 2-step euthanasia procedure. Death usually occurs within 30 minutes.
(2) Ketamine (2 step). Ketamine may be administered at dosages from 66 to 88 mg/kg315 (30 to 40 mg/lb) via an IM injection followed by a lethal dose of pentobarbital. Observers should be advised about the possibility of ketamine-induced muscle spasms during induction.298
(3) Ketamine-medetomidine (2 step). A combination of ketamine, at dosages of 1 to 2 mg/kg, with
medetomidine, at dosages of 0.05 to 0.1 mg/kg (0.02 to 0.05 mg/lb), may be administered via IM injection followed by a lethal dose of pentobarbital.315(4) Propofol (2 step). A dose of 1.5 to 2.5 mg/kg (0.7 to 1.1 mg/lb) can be administered IV followed by an injection of a lethal dose of pentobarbital.315

S6.2.2 Physical Methods
The following methods can be applied for euthanasia, providing they are performed with the proper equipment by trained personnel who are regularly monitored for proficiency.
(1) Decapitation followed by pithing (2 step). Rapid severance of the head and brain from the spinal cord, followed by pithing of the brain, will cause rapid death and unconsciousness. Decapitation alone is not considered a humane approach to euthanasia, especially for species that may be particularly tolerant of low O2 concentrations. Pithing helps ensure rapid loss of brain function and death for those species.322
(2) Cervical transection using a knife or other sharp instrument inserted caudal to the skull to sever
the spinal cord and cervical vertebrae, followed by pithing (2 step). The rationale for this approach is similar to that for decapitation (destruction of connections between brain and spinal cord) and pithing (destruction of brain tissue), except that the head is still physically attached by musculature to the body.
(3) Manually applied blunt force trauma (cranial concussion; Figure 24) followed by pithing or exsanguination (2 step). Manually applied blunt force trauma (a rapid, accurately placed blow of sufficient energy to the cranium with an appropriate-sized club) can cause immediate unconsciousness and potentially death, but should be followed by pithing or exsanguination to ensure death. The fish’s size, species, and anatomy and characteristics of the blow (including its accuracy, speed, and club mass) will determine the efficacy of manually applied blunt force trauma. This procedure requires training and monitoring for proficiency. Anatomic features, such as the location of the eyes, can help serve as a guide to
the location of the brain.322,323
(4) Penetrating captive bolt or NPCB (Figure 25). These methods are usually applied to large fish
species.322
(5) Maceration (1 step). When applied correctly, using a well-maintained macerator specifically designed for the size of fish being euthanized, death is nearly instantaneous.324 The process is aesthetically unpleasant for some operators and observers.
(6) Rapid chilling (hypothermic shock; 1 step or 2 step). It is acceptable for zebrafish (D rerio) to be euthanized by rapid chilling (2° to 4°C) until loss of orientation and operculum movements108,110,111 and subsequent holding times in ice-chilled water, specific to fish size and age. Zebrafish adults (approx 3.8 cm long) can be rapidly killed (10 to 20 seconds) by immersion in 2° to 4°C (36° to 39°F) water. Adult zebrafish should be exposed for a minimum of 10 minutes and fry 4 to 7 dpf for at least 20 minutes following loss of operculum movement to ensure death. Use of rapid chilling and use of buffered MS 222 alone have been shown to be unreliable euthanasia methods for zebrafish embryos < 3 dpf. To ensure embryonic lethality these methods should be followed with an adjunctive method such as use of dilute sodium or calcium hypochlorite solution at 500 mg/L.111,115 If necessary to ensure death of other life stages, rapid chilling may be followed by either an approved adjunctive euthanasia method or a humane killing method. Until further research is conducted, rapid chilling is acceptable with conditions for other small-bodied, similarly
sized tropical and subtropical stenothermic species. Species-specific thermal tolerance and body size will determine the appropriateness and effectiveness of rapid chilling for euthanasia of fish. Fish size is important because the rate of heat loss via thermal conduction from a body is proportional to its surface area. Based on these 2 factors, it has been suggested that rapid chilling in water associated with an ice slurry is a suitable killing method for small tropical and subtropical fish species 3.8 cm in length (tip of the snout to the posterior end of the last vertebra) or smaller, having lower lethal temperatures above 4°C.
To ensure optimal hypothermal shock (ie, rapid killing), transfer of fish into ice water must be completed as quickly as possible. This means rapid transitions from acclimatization temperature to 2° to 4°C must be achieved. This can be accomplished by using minimal water volume to transfer fish (ie, using a net to place fish in chilled water). In addition, fish should not be in direct contact with the ice in the water; rather a depression should be formed in the ice slurry to expose the entire surface of the fish to the chilled water. Full contact with cold water ensures optimal exposure and rapid chilling of the fish. Water temperature must not exceed 2° to 4°C. Well-insulated containers, such as coolers, will assist in maintaining the ice slurry and a probe thermometer can be used to confirm water temperature. This method of euthanasia is not appropriate for temperate, cool, or cold-water–tolerant fish, such as carp, koi, goldfish, or other species that can survive at 4°C and below. It is appropriate for zebrafish and other small-bodied (3.8-cm-long or smaller) tropical and subtropical stenothermic fish, for which the lower lethal temperature range is above 4°C.108,110,111 This method can also be acceptable for small to medium-sized (2.8- to 13.5-cm-long) Australian river gizzard shad, as long as secondary euthanasia methods are applied after fish are rendered nonresponsive.110 However, because of surface-to-volume considerations, use of this method is not appropriate in other medium- to large-bodied fish until data regarding its applicability to euthanasia for those species become available.

S6.2.3 Adjunctive Methods
Decapitation, pithing, exsanguination, freezing, and other physical or chemical methods for destroying brain function may be used as the second step of a 2-step procedure when fish have been rendered unconscious prior to their application by an acceptable or acceptable-with-conditions, first-step method. If necessary to ensure death, rapid chilling for specified groups may be followed by an approved adjunctive euthanasia method. Use of a dilute sodium hypochlorite or calcium hypochlorite solution may be an adjunctive method for early life stages of fish, including embryos and larvae.108,115

S6.2.4 Unacceptable Methods
The following are unacceptable methods of euthanasia in any situation. Flushing of fish into sewer, septic, or other types of outflow systems is unacceptable for many reasons. Water chemistry and quality may delay time to death and result in exposure to noxious compounds. For systems in close proximity
to and/or connected to natural waterways, pathogen release or transmission may occur from diseased or
carrier animals. Slow chilling or freezing of unanesthetized animals, including placing fish into a freezer without prior anesthesia, is also an unacceptable method. Similarly death by anoxia and desiccation
after removal from the water or by anoxia in water; any death due to exposure to caustic chemicals; and
death including prolonged traumatic injury prior to unconsciousness are unacceptable. While metomidate has been used for euthanasia of some finfish species, its listing in the Index of Legally Marketed Unapproved New Animal Drugs for Minor Species by the FDA (with a specified use for sedation and anesthesia) means that its extralabel use for euthanasia is currently illegal.