Validating a sexual development test using the 3-spined stickleback for addressing the 3Rs in fish toxicity testing

Project background

Endocrine disruptors (EDs) are substances, man-made and natural, able to impact on the endocrine system. This can result in adverse effects to the developmental, neurological, reproductive, and immune systems, as well as increasing the risk of carcinogenesis. These effects are not limited to humans and environmental endocrine disruption impacts on the sexual development and reproduction of birds, mammals and other wildlife, including fish. Fish, in particular, are sensitive to environmental EDs and this property means they are commonly used to assess substances for any suspected impacts on the endocrine system. The Fish Sexual Development Test (FSDT) exposes fish to potential EDs until the fish attain sexual maturity with species such as the zebrafish and the fathead minnow typically used in aquatic ecotoxicological studies.

Why we funded it

This Project Grant aims to reduce the numbers of fish required for toxicity testing by adopting the 3-spined stickleback as the preferred model species for the FSDT.   

FSDT guidelines provided by the OECD suggest a minimum of three different treatment levels, plus a control group, with four replicates for each treatment level. Each replicate requires at least 30 fish resulting in a total of 480 fish required per chemical tested. The use of the 3-spine stickleback is estimated to reduce fish requirements by up to 66% to between 160-240 fish depending on the number of replicates required for statistical power.

Research methods

Unlike the zebrafish and fathead minnow, the 3-spined stickleback possesses a genetic sex marker, which allows the genetic sex of the stickleback to be determined using a PCR assay. The sex ratio of fish in the FSDT is assumed to be 50:50 between males and females, with any deviation from this ratio attributed to the chemical tested. However, sex ratio in fish can be influenced by a number of external factors potentially resulting in a biased starting sex ratio (e.g. 30% male to 70% female). Without a genetic sex marker, large numbers of fish must be tested to confirm any biased sex ratios are due to the tested chemical, rather than as a result of random variation. A genetic sex marker allows both the genetic sex and phenotypic sex of each fish to be determined, and where a difference is found this can be unequivocally attributed to the substance being tested. This attribution also allows for a better definition as to whether the tested ED is an androgen or oestrogen. 

Prokkola JM et al. (2016). Microarray analysis of di-n-butyl phthalate and 17α ethinyl-oestradiol responses in three-spined stickleback testes reveals novel candidate genes for endocrine disruption. Ecotoxicol Environ Saf. 124:96-104. doi: 10.1016/j.ecoenv.2015.09.039

Sebire M et al. (2015). Prozac affects stickleback nest quality without altering androgen, spiggin or aggression levels during a 21-day breeding test. Aquat Toxicol. 168:78-89. doi: 10.1016/j.aquatox.2015.09.009

Aoki K et al. (2011). Evidence suggesting that di-n-butyl phthalate has antiandrogenic effects in fish. Environmental Toxicology and Chemistry 30(6):1338-45. doi: 10.1002/etc.502

Van Aggelen G et al. (2010). Integrating Omic Technologies into Aquatic Ecological Risk Assessment and Environmental Monitoring: Hurdles, Achievements and Future Outlook. Environmental Health Perspectives 118(1):1-5. doi: 10.1289/ehp.0900985

Katsiadaki I et al. (2010). Hepatic transcriptomic and metabolomic responses in the Stickleback (Gasterosteus aculeatus) exposed to ethinyl-estradiol. Aquatic Toxicology 97(3):174-187. doi: 10.1016/j.aquatox.2009.07.005

Jolly C et al. (2009). Detection of the anti-androgenic effect of endocrine disrupting environmental contaminants using in vivo and in vitro assays in the three-spined stickleback. Aquatic Toxicology 92:228-239. doi: 10.1016/j.aquatox.2009.02.006

Sebire M et al. (2009). Further refinement of the non-invasive procedure for measuring steroid production in the male three-spined stickleback, Gasterosteus aculeatusJournal of Fish Biology 75:2082-2094. doi: 10.1111/j.1095-8649.2009.02409.x

The data from this Project Grant have been published in the OECD validation report providing a case for the reduction of fish in the FSDT.



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Project grant



Principal investigator

Dr Ioanna Katsiadaki


Centre for Environment Fisheries and Aquaculture Science (CEFAS)

Grant reference number


Award date

Oct 2008 - Sep 2011

Grant amount