Considerations in Aquatic Invertebrate Euthanasia
American Association of Zoo Veterinarians Conference 2002
Nikki Hackendahl1; Ted Y. Mashima2, DVM, DACZM
1Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, VA, USA; 2Center for Government and Corporate Veterinary Medicine, Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, VA, USA



According to the 2001 American Zoo and Aquarium Association directory, 80% of accredited zoological institutions display invertebrates. This number will almost certainly increase as zoos continue to develop education topics that focus on ecosystem health and habitat conservation. In addition, invertebrate exhibits are relatively inexpensive and require little maintenance, making them an appealing exhibit for zoological institutions to send an ecologic message.

Despite their extensive use in research, the concept of welfare and invertebrates is relatively foreign in the United States, as they are not covered under U.S. laboratory animal guidelines. Nevertheless, invertebrate welfare is becoming a more prevalent issue in other countries. As recently as last year, the New Zealand Marine Science Society produced its own guidelines regarding the ethical conduct of experiments that involve invertebrates, because invertebrates are not included in governmental legislation. Recent changes in animal cruelty legislation in Australia now require that the commercial killing of crabs be done humanely. This has prompted at least one research group to investigate different methods of humanely immobilizing and killing crabs.1

The 2000 Report of the American Veterinary Medical Association Panel on Euthanasia lists 12 different criteria to consider when choosing a method of euthanasia. Although the report does not specifically refer to invertebrates, it is intended to be a reference for any veterinarian who carries out or oversees euthanasia of animals.

The Question of Pain in Invertebrates

The potential infliction of pain, and therefore prevention of pain, should be a strong consideration when choosing a method of euthanasia for any animal. Determining a definition of pain can be controversial, and the concept of invertebrates and pain is even more controversial. For animals within lower taxonomic groups, the response to painful or noxious stimuli is often seen as a reflex and is usually distinguished from central recognition of and response to pain.2,3

In non-human species, researchers often rely on changes in behavior to assess distress and pain perception.4,5 When using changes in behavior to evaluate the presence of pain, it can be difficult to distinguish between a reflex response to a noxious stimulus versus recognition or perception of the stimulus resulting in a centralized, coordinated response.6 This distinction becomes even more difficult given the anatomy of the invertebrate nervous system. Rather than a single, higher brain center, the invertebrate nervous system is composed of multiple ganglia of nerve cells that are connected by a ventral nerve cord or neural nets.7-10

Nevertheless, there is behavioral evidence of pain perception in invertebrates. Octopi have been shown to learn discrimination behavior using electric shocks as negative reinforcement. The assumption here is that there is some pain associated with the electric shocks such that the octopus learns to avoid them. In order for the octopus to learn to avoid the stimulus, it is likely that some level of recognition of the electric shock takes place. Fruit flies have also been shown to be able to learn this avoidance behavior.6 However, many other insect behaviors indicate a lack of the perception of pain. Examples include an insect with an injured leg that will continue to walk with undiminished force on the injured leg, or a locust that will continue to eat, despite being eaten itself.5 These behaviors indicate that in some insects the stimulus, or injury, is not recognized as painful.2,5

Conclusions drawn from behavioral studies are often met with contention. However, results from experiments studying biochemical aspects of pain require less interpretation. Research has demonstrated that there is a similar mechanism to opioid analgesia in vertebrates and invertebrates. Experiments with slugs (Limax maximus) and mantis shrimp (Squilla mantis) have shown that when morphine products were administered, there was a latency period in the reaction time that it took the slugs and shrimp to react to noxious or painful stimuli: heated surfaces and electric shocks, respectively. This analgesic state was then reversed with the administration of naloxone.11-13 These studies suggest that vertebrates and invertebrates use similar receptor pathways and biochemical mechanisms to register and react to aversive stimuli, and possibly in the modulation of pain control.14

Regardless of our ability to assess pain perception in invertebrate animals, they should be given the benefit of the doubt.15 Therefore, it is the responsibility of the veterinarian to choose a method of euthanasia that is as quick, painless, and effective as possible.

Invertebrate animals have nervous systems and respond to noxious stimuli, and therefore must also be treated humanely (AALAS 1987).

Practical Considerations and Recommendations

Verification of death of invertebrate animals can be quite challenging. For certain larger species, ultrasound evaluation of cardiac function can be attempted.8 In cephalopods, paling of the skin may be a useful indicator.16 However, in some animals it may be necessary to verify sedation by checking for a response to stimulus, and then when anesthetized, destroying the individual to a point with which life is incompatible.

There are both physical and chemical methods for invertebrate euthanasia, which frequently need to be combined. Often a method may only be considered humane if the animal is anesthetized first. The goal of this section of the paper is to review and suggest methods of invertebrate euthanasia that are accessible to a zoological veterinarian.

Several physical methods are available, which require little equipment or expertise. However, with most of these methods, prior sedation or anesthesia is ideal. Some of the problems associated with physical methods of euthanasia are that they may not be very esthetic, there is a decreased return on the postmortem exam, and there is potential for dissemination of infectious agents.

The easiest method is the classic method of invertebrate euthanasia: whole body crushing. This can be done without anesthesia on small animals that can be destroyed effectively and quickly in a single instance. For larger animals, anesthesia may be required first, which can be followed up by some method of destruction.

Decapitation alone would not be a suitable method of euthanasia for most invertebrates, because the nervous system of invertebrates is so diffuse and there is a higher degree of somatic responses that come from local nervous tissue rather than centralized nervous tissue.7,8 Techniques to kill certain crabs have been described as ticking specific ganglia. However, the technique requires training and is specific to certain species of crab, making it difficult to apply to other species.8,9

When conducted under specific recommendations, boiling is a method that may be considered humane in crustaceans without prior sedation.9 Gradual heating of an animal has been shown to cause great distress and is considered inhumane.1

Cooling and freezing of animals is controversial. It is unclear as to whether analgesic effects occur with hypothermia. Consequently, the freezing process may be painful as ice crystals form.17 Freezing of an animal diminishes the ability to perform an adequate postmortem workup, especially histology and electron microscopy. Many cold-water species become sedate only after extended periods of time at temperatures close to freezing, and will often recover rapidly after being removed.1 Cooling may be a useful technique to sedate an animal prior to a rapid killing procedure. Conversely, an animal may be anesthetized first and then placed into a deep freeze.18

Freshwater baths have been the standard killing method for many crustacean industries; however, several studies involving crustaceans have shown adverse reactions to freshwater baths, including increased activity, autotomy (dropping limbs), and tearing at their abdomens and walking legs.1,19 This is not recommended as a humane method of euthanasia.

Carbon dioxide has been utilized in the past by pumping gas directly into a chamber, putting dry ice in a container for animals that can be out of the water or adding Alka-Seltzer tablets in the water for species that must be left in water.20 Although carbon dioxide was previously considered humane, one study revealed that hypercapnic seawater resulted in high levels of distress in crabs.1

Chemical methods provide an alternative to the physical methods that result in large degrees of tissue destruction. Many of the chemical methods of euthanasia are terminal extensions of anesthetic protocols that have an increase in concentration or time of exposure.20 There are several sources regarding anesthesia of aquatic invertebrates that can be referred to as well.1,7,10,15,18,21-25

Tricaine methane sulfonate, or MS-222, in solution is a standard anesthetic and euthanasia agent for fish and some amphibians.15,25 However, its effectiveness with aquatic invertebrates tends to be unreliable and has a high degree of variability between different species, whether exposure is through immersion or intracoelomic injection.1,10,21,22 The mode of action of MS-222 in vertebrates is the disruption of the sodium permeability within the nerve membrane, thereby preventing impulse conduction.1,25 It has been speculated that the unpredictable effects may be related to the absence of acetylcholine at some of the nerve terminals in invertebrates.1 MS-222 is not generally recommended for euthanasia of aquatic invertebrates.

An ethanol solution has been recommended for the anesthesia of aquatic invertebrates, although it has been shown to cause stress in cephalopods.21,23 Again, the concentration, or time spent in the solution, could be increased to result in euthanasia.

Magnesium chloride (MgCl2) and potassium chloride (KCl) both block nerve transmission by interfering with cellular depolarization.7,8,10 Magnesium salts in solution are a common anesthetic for aquatic invertebrates.21,23,24 However, it is suspected that immersion in MgCl2 solution merely acts as a paralytic and not an anesthetic, making it an inhumane method of euthanasia.7,10 Intracoelomic administration of MgCl2 may provide sedation or anesthesia along with paralysis if the solution comes into contact with the ganglia.10 Similarly, KCl is not considered a humane method of euthanasia in vertebrates when used alone, because it affects cardiac function without crossing the blood-brain barrier. However, injections of KCl (1 g/kg) directly into the hemolymph sinus of lobsters has been shown to inhibit the nervous system prior to having an effect on cardiac function, allowing KCl to be considered a humane method of euthanasia.8

Immersion in clove oil solution (≥0.125 ml/L) has been shown to be a good choice for euthanasia of crustaceans. It worked at reasonable doses, had a quick onset time (as short as 16 minutes), is inexpensive, and is likely to work on a variety of species.1

Chemical solution baths can be utilized for most species of aquatic invertebrates, but there are several injectable protocols that are likely to work with species that have an open system with a large hemocoel, such as crustaceans and some molluscs. Researchers have noted that injectable dosages for invertebrates are often higher than for vertebrates. Several hypotheses have been suggested as to why this may be.8,13

A few injectable drugs have been tested for anesthesia or euthanasia of crustaceans. For example, pentobarbital and xylazine have been administered by direct injection into the hemocoel.21 Additionally, injections of both xylazine and ketamine into the hemolymph sinus, as well as ketamine administered intramuscularly, have proven to work well for crustaceans.1,22

In summary, invertebrate welfare is an emerging issue. Given this issue and the increased presence of invertebrates in zoological settings, invertebrates are beginning to receive increasing amounts of veterinary attention. Even though there is quite a bit of controversy as to whether invertebrates are capable of perceiving pain, the ethics of euthanasia apply to invertebrate species just as they do to vertebrate species. Therefore, it is the veterinarian obligation to make the euthanasia as quick and as painless as possible. Several methods are discussed, although many have been tested only on a preliminary basis. A combination of more than one method may result in an improved protocol, analogous to the concept of neuroleptic anesthesia. With the increase in prevalence of invertebrates within veterinary medicine, it is likely that standard protocols will evolve.

While pain and suffering in another being is, strictly speaking, unknowable, yet for practical purposes there are a number of lines of evidence of their presence and intensity which deserve attention (Paton, 1984).2

Literature Cited

1.  Gardner, C. 1997. Options for humanely immobilizing and killing crabs. J. Shellfish Res. 16 (1): 219–224.

2.  National Research Council. 1992. Recognition of Pain and Distress in Laboratory Animals. National Academy Press: Washington, D.C.

3.  Kavaliers, M. 1988. Evolutionary and comparative aspects of nociception. Brain Res. Bull. 21: 923–931.

4.  Chapman, C.R., K.L. Casey, R. Dubner, K.M. Foley, R.H. Gracely, and A.E. Reading. 1985. Pain measurement: an overview. Pain. 22: 1–31.

5.  Eisemann, C.H., W.K. Jorgensen, D.J. Merritt, M.J. Rice, B.W. Cribb, P.D. Webb, and M.P. Zalucki. 1984. Do insects feel pain? A biological view. Experientia. 40: 164–167.

6.  Smith, J.A. A question of pain in invertebrates. 1991. ILAR News 33(1–2): 25–31.

7.  Ross, L.G., and B. Ross. 1999. Anaesthesia of aquatic invertebrates. In: Anaesthetic and Sedative Techniques for Aquatic Animals, 2nd ed. Blackwell Science: London. Pp 46–57.

8.  Battison, A., R. MacMillan, A. MacKenzie, P. Rose, R. Cawthorn, and B. Horney. 2000. Use of injectable potassium chloride for euthanasia of American lobsters (Homarus americanus). Comp. Med. 50(5): 545–550.

9.  Universities Federation for Animal Welfare. Humane Killing of Animals. UFAW. 1978.

10.  Clark, T.R., P.C. Nossov, J.P. Apland and M.G. Filbert. 1996. Anesthetic agents for use in the invertebrate sea snail, Aplysia californica. Contemp Top Lab Anim Sci. 35(5): 75–79.

11.  Kavaliers, M. and M. Hirst. 1986. Naloxone-reversible stress-induced feeding and analgesia in the slug Limax maximus. Life Sci. 38: 203–209.

12.  Kavaliers, M., M. Hirst, and G. Campbell Teskey. 1983. A functional role for an opiate system in snail thermal behavior. Science. 220: 99–101.

13.  Maldonado, H. and A. Miralto. 1982. Effect of morphine and naloxone on a defensive response of the mantis shrimp (Squilla mantis). J. Comp. Physiol. 147: 455–459.

14.  Fiorito, G. 1986. Is there pain in invertebrates? Behav. Processes. 12: 383–388.

15.  Cooper, J.E., R. Ewbank, C. Platt, and C. Warwick. 1989. Euthanasia of Amphibians and Reptiles. Universities Federation for Animal Welfare and World Society for the Protection of Animals. London.

16.  Universities Federation for Animal Welfare. The UFAW Handbook on the Care and Management of Laboratory Animals, 7th ed. Volume 2. Blackwell Science: London. 1999.

17.  AVMA Panel on Euthanasia. 2000 report of the AVMA panel on euthanasia. J. Am. Vet. Med. Assoc. 218(5): 669–696.

18.  Ingle, R.W. The UFAW Handbook on the Care and Management of Decapod Crustaceans in Captivity. UFAW, England. 1995.

19.  Speare, D.J., R.J. Cawthorn, B.S. Horney, R. MacMillan, and A.L. MacKenzie. Effects of formalin, chloramine-T, and low salinity dip on the behavior and hemolymph biochemistry of the American lobster. Can. Vet. J. 37: 729–734.

20.  Kesel, M.L. and T.Y. Mashima. 1995. Invertebrates. In: Rollin, B.E. and M.L. Kesel. The Experimental Animal in Biomedical Research, Vol II. CRC Press: Boca Raton, FL.

21.  National Resource Council, Institute of Laboratory Animal Resources. 1981. Laboratory Animal Management of Marine Invertebrates. National Academy Press: Washington, D.C.

22.  Brown, P.B., M.R. White, J. Chaille, M. Russell, and C. Oseto. 1996. Evaluation of three anesthetic agents for crayfish (Orconectes virilis). J. Shellfish Res. 15(2): 433–435.

23.  Gilbert, D.L., W.J. Adelman, Jr., and J.M. Arnold. 1990. Squid as Experimental Animals. Plenum Press: New York, London.

24.  Messenger, J.B., M. Nixon, and K.P. Ryan. 1985. Magnesium chloride as an anaesthetic for cephalopods. Comp. Biochem. Physiol. 82C: 203–205.

25.  Letcher, J. 1992. Intracelomic use of tricaine methanesulfonate for anesthesia of bullfrogs (Rana catesbeiana) and leopard frogs (Rana pipiens). Zoo Biol. 11: 243–251.


Speaker Information
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Nikki Hackendahl
Virginia-Maryland Regional College of Veterinary Medicine
Blacksburg, VA, USA

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