The role of water chemistry in zebrafish welfare and reproducibility of research studies

Lead Research Organisation: University of Exeter
Department Name: Biosciences


The present proposal will result in improvements relating to the Reduction and Refinement components of the 3Rs:


1) Zebrafish research facilities will need to produce fewer stock animals to fulfil experimental numbers as the animals produced will be higher quality when water chemistry is optimised.

2) Optimal water chemistry is also expected to improve the consistency of the quality of stock fish (i.e. less inter-individual variability). This will also reduce animal use overall because:

a) less replication within studies required to achieve the same statistical power in detecting treatment differences,


b) the reproducibility between studies and different facilities will be improved.

3) Optimising water quality will also extend the timeframe of peak embryo production by adults, which in turn will reduce the frequency of producing new broodstock lines. This window currently varies from 4 to 9 months, so consistently working at the upper end of this range could halve the number of new broodstock lines required.


This study will also minimise pain, suffering, distress or lasting harm associated with sub-optimal water quality. In particular elevated CO2 is known to impair brain function and the ability of fish to respond appropriately to sensory stimuli (olfactory, auditory and visual) that are part of their normal life and social interactions, and impairs learning and cognitive function, and can induce anxiety. The costs of coping with high CO2 and acid-base regulation also detract from energy spent on healthy growth, development, reproduction, immune function and disease resistance. The proposed study will provide evidence to optimise CO2 itself, as well as variables known to limit the ability of fish to achieve acid-base homeostasis associated everyday challenges of digestion/exercise/husbandry stresses and high CO2. The latter includes freshwater Na+ and Cl- levels which limit the rate of acid and base excretion by the gills, and calcium that directly regulates skin and gill permeability to all molecules. Water bicarbonate also determines the tolerance of fish to high CO2, and recovering their acid-base balance. The proposed experiments will therefore offer the opportunity to significantly reduce the detrimental impacts of water sub-optimal quality in zebrafish research facilities throughout the world.


Until the research is completed it is difficult to precisely quantify the anticipated reduction of animals used and suffering experienced. However, zebrafish are the number one model species used in fish studies globally, with more than 3,250 institutes in 100 countries estimated to conduct zebrafish research. Zebrafish represent the 2nd most used species of any vertebrate in animal research, with >5 million fish used per annum. Therefore, even if only 1 % of all the global zebrafish research facilities adopted the guidelines we aim to produce, then at least 50,000 fish per year would benefit from improved welfare (which should include physiological homeostasis, growth, behaviour and immune function). Even if this 1 % figure only applied to the UK, then >4,000 zebrafish would still benefit per year. In reality the 1 % figure is likely to be a very conservative estimate of improved welfare for zebrafish globally. This will be due to fewer fish being required per study due to better consistency of production (i.e. lower inter-individual variability), and due to improved reproducibility between studies and facilities. In turn, this will translate into more robust and reliable conclusions regarding the outcomes of research (e.g. efficacy of drug treatments etc.). Regarding the "Refinement" component, if high quality guidelines are produced and disseminated effectively there is potential to reduce animal suffering on a truly global scale and improve the cumulative lifetime experience of millions of animals per year.

Technical Summary

Water chemistry varies greatly across zebrafish culture facilities, with no guidelines or even basic advice available on many parameters important for healthy populations. This is very surprising as we have known for decades that the many aspects of freshwater chemistry have profound and varied influences on the physiology and health of fish. Hardness ions (especially calcium) control gill and skin permeability and epithelial integrity. Sodium and chloride are the major ions that freshwater fish must actively take up from the water for survival and are also vital counter-exchange ions required for regulating internal acid-base balance in response to the challenges of daily life (feeding, muscular activity, handling stress, high CO2 etc.). We are also ignorant concerning the health impacts of elevated CO2 on zebrafish. Climate change research has recently demonstrated a wide range of detrimental impacts of relatively small elevations in CO2 in aquatic animals. Many of these have clear welfare implications (e.g. reduced ability or even inability to respond to positive or negative odours, sounds, and visual stimuli, impaired learning, increased anxiety, and also reduced immune response and resistance to disease). At the same time recirculating aquarium systems (as used for zebrafish culture) have notoriously high CO2 levels, but this can be improved quite cheaply with effort and appropriate technical knowledge.

There are large differences in the approaches used to building new or refurbishing old zebrafish facilities, as well as in the resources available, husbandry experience of facility managers and the 'drivers' of research within each facility. This likely contributes to the lack of standardisation/guidelines for water chemistry in zebrafish culture. However, it is vital to determine ideal ranges of the major water parameters for zebrafish culture. This will directly aid in minimising mortalities, wastage, and improving production of higher quality embryos.

Planned Impact

The primary objective of this project is to provide evidence-based advice and guidelines on optimum water chemistry for culturing stock zebrafish and carrying out experimental research. By enhancing these environmental conditions we aim to improve the health and welfare of zebrafish in many ways including development, physiology, behaviour, immunity, and general growth and productivity of zebrafish stocks. By doing so this contribution to Refinement will directly improve the welfare of very large numbers of these fish (the second most used vertebrate in animal studies) throughout their life in research facilities. By improving the quality of zebrafish production our project should also contribute to Reduction, as fewer animals will need to be bred in each facility if the consistency of the offspring quality is enhanced (i.e. fewer fish will be wasted). Finally, our project also addresses the Reproducibility crisis in animal research. This is because variation in water chemistry (as clearly occurs both between and within facilities and between and within studies) is known to greatly alter the development, physiology, behaviour, growth and immune function of fish generally, and we anticipate that this will be true for zebrafish too. Thus, evidence-based guidelines on water chemistry have the potential to dramatically reduce this variability, improving reproducibility, and in doing so will further contribute to Reduction of the global numbers of zebrafish used in research (as confidence in results being reproducible recovers).

3Rs Benefits - Our project has the potential to impact welfare and 3Rs for millions of zebrafish per year. We conservatively estimated that even if only 1 % of all the >3250 zebrafish research facilities across 100 countries globally adopted the guidelines we aim to produce, then at least 50,000 fish per year used in research studies would benefit from improved welfare. Given the potential for very clear improvements in health and welfare of zebrafish (based on work published on other fish species), we are hopeful that this 1% figure is a major underestimate. However, the actual impact will depend on both the quality of our research outcomes and the clarity of their message. Importantly it will also hugely depend on dissemination of these guidelines and knowledge transfer to the global zebrafish research community, and the subsequent uptake of these recommendations by aquatic resource managers and researchers in their facilities. This will not be a straightforward task as researchers can be conservative creatures, with a desire to stick with previously published protocols for consistency, even if their animal care technicians are keen to try new ideas to promote animal welfare, and in this case the quality of zebrafish productivity at the facility level. So our intention is to primarily focus on the dissemination/knowledge transfer element within this Pathways to Impact.

Benefit implementation - International trust and buy-in regarding improvements in welfare and reproducibility will benefit greatly from parallel trials conducted by one Project Partner (Chris Lawrence) at the Boston Children's Hospital in the USA (at no cost to NC3Rs) - one of the largest zebrafish research programs in the world. This will provide a direct test of the improvements to Reproducibility that we suggest will be a key outcome of our research. In addition, we plan to promote dissemination and knowledge transfer throughout the global zebrafish research community by hosting a 2 day workshop at Exeter in Year 2. This will be organised and run by another Project Partner (Dr. Penny Hawkins - RSPCA) that specifically targets zebrafish facility managers and research leaders in the UK and internationally. We will also engage industries for the technical side of zebrafish culture facilities (e.g. Tecniplast our industrial Project Partner) to facilitate improvements in new build and retro-fitting existing zebrafish facilities.


10 25 50