This study addresses a fundamental question in fish welfare: are the anaesthetics used for fish aversive? Despite years of routine general use of many agents, within both scientific research and aquaculture, there is a paucity of information regarding their tolerance and associated behavioural responses by fish. This study examined nine of the most commonly used fish anaesthetic agents, and performed preference tests using adult mixed sex zebrafish (Danio rerio), the most commonly held laboratory fish. Video tracking software quantified swimming behaviour related to aversion for each anaesthetic at 50% of its standard recommended dose compared with clean water in a flow-through chemotaxic choice chamber. Results suggest that several commonly used anaesthetics were aversive, including two of the most commonly recommended and used: MS222 (ethyl 3-aminobenzoate methanesulphate) and benzocaine. For ethical best practice, it is recommended that compounds that are aversive, even at low concentration, should no longer be used routinely for anaesthesia or indeed the first step of humane euthanasia of adult zebrafish. Two agents were found not to induce aversive behavioural responses: etomidate and 2,2,2 tribromoethanol. For the millions of adult zebrafish used in laboratories and breeding worldwide, etomidate appears best suited for future routine humane use.

A survey was conducted regarding zebrafish Danio rerio use for scientific research with a focus on: anaesthesia and euthanasia; housing and husbandry; breeding and production; refinement opportunities. A total of 98 survey responses were received from laboratories in 22 countries in Europe, North America, South America, Asia and Australia. There appears a clear and urgent need to identify the most humane methods of anaesthesia and euthanasia. Aversive responses to MS‐222 were widely observed raising concerns about the use of this anaesthetic for D. rerio. The use of anaesthesia in fin clipping for genetic identification is widely practised and there appears to be an opportunity to further develop less invasive methods and refine this process. Optimization (and potentially standardization) of feeding is an area for further investigation. Given that diet and body condition can have such profound effects on results of experiments, differences in practice could have significant scientific implications. Further research into transition between dark and light phases in the laboratory appears to represent an opportunity to establish best practice. Plants and gravel were not considered practical by many laboratories. The true value and benefits need to be established and communicated. Overproduction is a concern both from ethical and financial viewpoints. There is an opportunity to further reduce wastage of D. rerio. There are clear concerns and opportunities for the scientific community to work together to further improve the welfare of these important laboratory models.

There is a need to ensure that the care and welfare for fish maintained in the laboratory are to the highest standards. This extends to the use of anaesthetics for both scientific study, humane killing and euthanasia at end of life. An anaesthetic should not induce negative behaviours and fish should not seek to avoid the anaesthetic. Surprisingly little information is available to facilitate a humane choice of anaesthetic agent for fish despite over 100 years of use and the millions of fish currently held in thousands of laboratories worldwide. Using a chemotaxic choice chamber we found different species specific behavioural responses among four closely related fish species commonly held in the laboratory, exposed to three widely used anaesthetic agents. As previously found for zebrafish (Danio rerio), the use of MS-222 and benzocaine also appears to induce avoidance behaviours in medaka (Oryzias latipes); but etomidate could provide an alternative choice. Carp (Cyprinus carpio), although closely related to zebrafish showed avoidance behaviours to etomidate, but not benzocaine or MS-222; and rainbow trout (Oncorhynchus mykiss) showed no avoidance to the three agents tested. We were unable to ascertain avoidance responses in fathead minnows (Pimephales promelas) and suggest different test paradigms are required for that species.

Millions of laboratory animals are killed each year worldwide. There is an ethical, and in many countries also a legal, imperative to ensure those deaths cause minimal suffering. However, there is a lack of consensus regarding what methods of killing are humane for many species and stages of development. In 2013, an international group of researchers and stakeholders met at Newcastle University, United Kingdom to discuss the latest research and which methods could currently be considered most humane for the most commonly used laboratory species (mice, rats and zebrafish). They also discussed factors to consider when making decisions about appropriate techniques for particular species and projects, and priorities for further research. This report summarises the research findings and discussions, with recommendations to help inform good practice for humane killing.

An improved knowledge and understanding of the fundamental biological requirements is needed for many of the species of fish held in captivity and, without this knowledge it is difficult to determine the optimal conditions for laboratory culture. The aim of this paper is to review the key issues concerning environmental enrichment for laboratory-held fish species and identify where improvements are required. It provides background information on environmental enrichment, describes enrichment techniques currently used in aquatic ecotoxicology studies, identifies potential restrictions in their use and discusses why more detailed and species-specific guidance is needed.