Dr Ioanna Katsiadaki, from the Centre for Environment, Fisheries and Aquaculture Science, has used an NC3Rs project grant to validate a sexual development test using the stickleback. The method enables a 50% reduction in the number of fish used.
Principal Investigator: Ioanna Katsiadaki, Science Leader - Aquatic Animal Health
Organisation: Centre for Environment, Fisheries and Aquaculture Science (Cefas)
Award: £398,680, in 2008, over 36 months
Title: Validating a sexual development test using the 3-spined stickleback for addressing the 3Rs in fish toxicity testing
Read more about Dr Katsiadaki's research
Endocrine disrupting chemicals can affect sexual development
Endocrine disruptors are chemicals that when absorbed into the body either mimic or block hormones. Many chemicals, including pharmaceuticals, pesticides and plasticisers are endocrine disruptors. There is concern that exposure to endocrine disrupting chemicals (EDCs) in the environment affects the sexual development of humans and wildlife. EDCs are of major concern for fish, as the aquatic environment is an important sink for chemicals and sewage waste. Steroidal oestrogens such as ethinyl-oestradiol – the active ingredient of the human contraceptive pill – are implicated, albeit not exclusively, as causing the widely observed sexual disruption in wild fish.
Sexual development in fish is used as a screen for endocrine disruptors
Sexual development in fish is easily altered by endocrine disruptors and consequently they are used for screening chemicals for this purpose. Between 2007 and 2011, under the management of the Organisation for Economic Co-operation and Development (OECD), the Fish Sexual Development Test (FSDT) was assessed as a potential test guideline for detecting early life-stage effects and potential adverse consequences of putative EDCs on sexual development.
In the test, fish are exposed to the test chemical, from the fertilised egg stage until the completion of sexual differentiation. The fish are then euthanised. Two core endpoints are measured post-mortem as indicators of endocrine-associated developmental aberrations. This includes the concentration of vitellogenin (a precursor protein of egg yolk) and the proportions of each sex determined via histological examination of the gonads (phenotypic sex ratio). Whether the chemical causes full or partial sex reversal is determined by comparing the sex ratios in groups of fish exposed to the chemical with control groups.
Phenotypic sex ratios in fish can be highly variable
In the FSDT if the genetic (actual) sex of the fish is unknown it is assumed that the normal sex ratio of males to females is approximately 50:50, with any deviation being a result of the test chemical. However, sex ratio in fish is naturally highly flexible and subject to modification by external factors, including temperature. It is not unusual to have a sex ratio of 70:30 males to females or vice versa, which means that in the absence of information on the original sex ratio a large number of fish must be tested to provide conclusive information to attribute any change to chemical exposure rather than random variation.
The availability of a genetic marker of sex would allow fewer animals to be used by providing unequivocal information on the original sex ratio and therefore an improved basis to determine whether any change in ratio is a result of the test chemical. It would also have the advantage of allowing better definition of the mode of action of the chemical as an oestrogen or an androgen for example, since any differences between the genetic sex and the phenotypic (developed) sex can be attributed to chemical exposure.
Around 90% of fish do not have a known genetic marker for sexual determination. This includes the zebrafish – the original species of choice for validation of the FSDT. A sex determining region has been mapped to linkage group 19 in the three-spined stickleback and in 2007, Dr Ioanna Katsiadaki, Cefas, was awarded an NC3Rs grant to validate this species for the FSDT.
Validation of three-spined stickleback as a suitable test species
In order to establish that the three-spined stickleback could be used for the FSDT, Dr Katsiadaki, with collaborators from Brunel University and Leicester University, tested a series of endocrine-active reference chemicals including oestrogens, androgens and anti-androgens. The stickleback test showed high reproducibility between laboratories and comparable sensitivity in detecting compounds with endocrine activity to other fish species using the FSDT. The majority of chemicals tested in the three-spined stickleback caused gonadal intersex (that is the presence of both male and female gametes in a single gonad). This was not observed in the control fish increasing the overall reliability of the test compared to species where gonadal intersex is more commonly seen in controls.
International regulatory acceptance with reduced fish use
The FSDT was published as an official OECD test guideline (TG234) in 2011. The three-spined stickleback is one of the three recommended species, together with the zebrafish and Japanese medaka.
The study design for the OECD validation exercise tested five concentrations of each chemical, with four replicates of 40 fish for each concentration. Each study therefore used 800 fish. Based on experience of the test during validation, Dr Katsiadaki was able to provide a case to reduce the number of fish regardless of species by 25% to 600 per chemical and this is incorporated in the final test guideline. The use of the three-spined stickleback allows the number to be reduced by a further 25% per chemical.
The full impact of the FSDT will be realised with the REACH chemicals legislation which is driving the testing of chemicals for endocrine disruption. As of September 2013, 10,000 chemicals were registered with the European Chemicals Agency for the purposes of REACH. If only 0.5% of these are tested, the FSDT will avoid the use of around 10,000 fish (and 20,000 if the stickleback is used).
The results from this grant have been published in the OECD validation report for the FSDT.
This case study was published in a review of our research portfolio in November 2013.