Intercalibration Exercise Using a Stickleback Endocrine Disrupter Screening Assay

Abstract

The Organisation for Economic Cooperation and Development (OECD) is currently validating a short-term fish screening protocol for endocrine disrupters (estrogens, androgens, and their antagonists and aromatase inhibitors), using three core species: fathead minnow, Japanese medaka, and zebrafish. The main endpoints proposed for the first phase of validation of the screen are vitellogenin (VTG) concentration, gross morphology (secondary sexual characteristics and gonado-somatic index), and gonadal histopathology. A similar protocol is concurrently being developed in the United Kingdom using the three-spined stickleback, with identical endpoints to those for the core species and, in addition, a unique androgen-specific endpoint in the form of spiggin (glue protein) induction. To assess the suitability of this species for inclusion in the OECD protocol alongside the core species, an intercalibration was conducted using 17␤-estradiol (a natural estrogen) and trenbolone (a synthetic androgen), thus mimicking a previous intercalibration with the core species. All three participating laboratories detected statistically significant increases in VTG in males after 14 d exposure to nominal concentrations of 100 ng/L 17␤-estradiol and statistically significant increases in spiggin in females after 14 d exposure to nominal concentrations of 5,000 ng/L trenbolone. The stickleback screen is reliable, possessing both relevant and reproducible endpoints for the detection of potent estrogens and androgens. Further work is underway to assess the relevance and suitability of the screen for weakly acting estrogens, anti-androgens, and aromatase inhibitors.

Molecular biomarkers of endocrine disruption in small model fish

“…Please note different scale of the x-axis for medaka (C). Allen et al, 2008;Andersen et al, 2006;Ankley et al, 2001; Ankley et Ankley et al, 2005;Bogers et al, 2006;Filby et al, 2007;Hahlbeck et al, 2004a,b;Hornung et al, 2004;Jensen and Ankley, 2006;Jensen et al, 2004;Jolly, 2007;Katsiadaki et al, 2002a,b;Kinnberg et al, 2007;Kiparissis et al, 2003;Makynen et al, 2000;Martinovic et al, 2008;Miracle et al, 2006;OECD, 2006;Örn et al, 2003;Örn et al, 2006;Panter et al, 2004;Parrott and Wood, 2002;Pawlowski et al, 2004;Sanchez et al, 2008;Seki et al, 2006;Thorpe et al, 2007;Villeneuve et al, 2007;Wester et al, 2003;Wester et al, 2003;Zerulla et al, 2002 Tables and figures Table 1 The fathead minnow (Pimephales promelas) is a cyprinid fresh water fish with broad distribution across North America. It is used for a number of different regulatory tests for the risk assessment of chemicals.…”

“…Estrogenic and anti-estrogenic chemicals and their effect levels (lowest observed effect concentrations, LOEC) for vitellogenin/choriogenin expression, various molecular endpoints, secondary sex characters, histopathology and reproduction in males and or females of the small model species fathead minnow (A), zebrafish (B), medaka (C) and three-spined stickleback (D). The data were extracted fromAllen et al, 2008; Andersen et al, 2004; …”

Mammalian glucocorticoid metabolites act as androgenic endocrine disruptors in the medaka (Oryzias latipes)

“…Experimental design largely followed the OECD draft fish screening assay for endocrine‐active substances 36, which was adopted most recently 39. The fish treated in this study were sexually mature male and spawning female wild‐type Japanese medaka ( Oryzias latipes ; 8–10 months old, arithmetic mean ± standard deviation; range of variation in brackets: 35.7 ± 1.9 31–42 mm total length; 0.43 ± 0.09 [0.24–0.74] g mean wet body mass at the end of exposure), bred from a laboratory stock maintained at the above‐mentioned department. Two weeks before the start of exposure, the fish were transferred into the test aquaria so that each test aquarium contained four male and four female fish as indicated by distinct external sexual characteristics.…”

“…Therefore, during the past decade, much effort has been placed into reviewing, developing, and validating an array of screening assays within the Organization for Economic Co‐operation and Deveopment (OECD) Endocrine Disruptor Testing and Assessment (36; see http://www.oecd.org/document/62/0,2340,en_2649_34377_2348606_1_1_1_1,00.html) and the U.S. Environmental Protection Agency (U.S. EPA) Endocrine Disruptor Screening Program (37; see http://www.epa.gov/endo/pubs/assayvalidation/index.htm). For fish, two major lines of action have been pursued, the 21‐d fish endocrine screening assay with fathead minnow ( Pimephales promelas ), zebrafish ( Danio rerio ), and medaka ( Oryzias latipes ), followed by the androgenized female stickleback screening assay with stickleback ( Gasterosteus aculeatus ) 36–38. In this standardized endocrine disruptors testing with fish, methyltestosterone and trenbolone have been most frequently employed as exogenous androgenic model test compounds (14, 23, 36, 38–40; see also http://www.oecd.org/document/62/0,2340,en_2649_34377_2348606_1_1_1_1,00.html#Stickleback).…”

“…For fish, two major lines of action have been pursued, the 21‐d fish endocrine screening assay with fathead minnow ( Pimephales promelas ), zebrafish ( Danio rerio ), and medaka ( Oryzias latipes ), followed by the androgenized female stickleback screening assay with stickleback ( Gasterosteus aculeatus ) 36–38. In this standardized endocrine disruptors testing with fish, methyltestosterone and trenbolone have been most frequently employed as exogenous androgenic model test compounds (14, 23, 36, 38–40; see also http://www.oecd.org/document/62/0,2340,en_2649_34377_2348606_1_1_1_1,00.html#Stickleback).…”