Chad A Blanksma USEPA Great Lakes Toxicology and Ecology Division Histotechnologist

6201 Congdon Blvd. Duluth MN 55804

blanksma.chad@epa.gov 0009-0001-8811-8083 This image is from an adult female fathead minnow from the control treatment in a toxicological test. Approximately 1/2 of the ovary was collected and processed to a glass slide. The section in the image comes from the middle region of the ovary in the longitudinal axis. The image was created by combining several 50x images together using an image stitching technique with image editing software. Vitellogenic oocytes Whole ovary Fathead minnow Cortical alveolar oocytes Perinucleolar oocytes Oogenesis Vitellogenesis Adult This information is released under the Creative Commons license - Attribution - CC BY (https://creativecommons.org/licenses/by/4.0/). The consumer of these data ("Data User" herein) is required to cite it appropriately in any publication that results from its use. The Data User should realize that these data may be actively used by others for ongoing research and that coordination may be necessary to prevent duplicate publication. The Data User is urged to contact the authors of these data if any questions about methodology or results occur. Where appropriate, the Data User is encouraged to consider collaboration or co-authorship with the authors. The Data User should realize that misinterpretation of data may occur if used out of context of the original study. While substantial efforts are made to ensure the accuracy of data and associated documentation, complete accuracy of data sets cannot be guaranteed. All data are made available "as is." The Data User should be aware, however, that data are updated periodically and it is the responsibility of the Data User to check for new versions of the data. The data authors and the repository where these data were obtained shall not be liable for damages resulting from any use or misinterpretation of the data. Thank you. 2012 Kingdom Animalia animals 202423 Subkingdom Bilateria triploblasts 914154 Infrakingdom Deuterostomia 914156 Phylum Chordata chordates 158852 Subphylum Vertebrata vertebrates 331030 Infraphylum Gnathostomata 914179 Superclass Actinopterygii ray-finned fishes 161061 Class Teleostei 161105 Superorder Ostariophysi 162845 Order Cypriniformes minnows 162846 Superfamily Cyprinoidea 638796 Family Leuciscidae 1198935 Subfamily Pogonichthyinae 1198937 Genus Pimephales bluntnose minnows 163515 Species Pimephales promelas Fathead Minnow 163517 Chad A Blanksma US EPA Great Lakes Toxicology and Ecology Division Histotechnologist

6201 Congdon Blvd. Duluth MN 55804

blanksma.chad@epa.gov 0009-0001-8811-8083 Image collection device SPOT Insight 4 Mp CCD Scientific Color Digital Camera Image collection software SPOT Advanced Software Image analysis software - scale bar Image-Pro Premier Graphics Editing Software Corel Photo-Paint Carlie A LaLone US EPA Great Lakes Toxicology and Ecology Division lalone.carlie@epa.gov Corresponding Author Spironolactone is a pharmaceutical that in humans is used to treat conditions like hirsutism, various dermatologic afflictions, and female-pattern hair loss through antagonism of the androgen receptor. Although not routinely monitored in the environment, spironolactone has been detected downstream of a pharmaceutical manufacturer, indicating a potential for exposure of aquatic species. Furthermore, spironolactone has been reported to cause masculinization of female western mosquitofish, a response indicative of androgen receptor activation. Predictive methods to identify homologous proteins to the human and western mosquitofish androgen receptor suggest that vertebrates would be more susceptible to adverse effects mediated by chemicals like spironolactone that target the androgen receptor compared with invertebrate species that lack a relevant homolog. In addition, an adverse outcome pathway previously developed for activation of the androgen receptor suggests that androgen mimics can lead to reproductive toxicity in fish. To assess this, 21-d reproduction studies were conducted with 2 fish species, fathead minnow and Japanese medaka, and the invertebrate Daphnia magna. Spironolactone significantly reduced the fecundity of medaka and fathead minnows at 50 μg/L, whereas daphnia reproduction was not affected by concentrations as large as 500 μg/L. Phenotypic masculinization of females of both fish species was observed at 5 μg/L as evidenced by formation of tubercles in fathead minnows and papillary processes in Japanese medaka. Effects in fish occurred at concentrations below those reported in the environment. These results demonstrate how a priori knowledge of an adverse outcome pathway and the conservation of a key molecular target across vertebrates can be utilized to identify potential chemicals of concern in terms of monitoring and highlight potentially sensitive species and endpoints for testing. C5-B-2_5x_stitched-cropped-sharpened-contrast-background-scalebar C5-B-2_5x_stitched-cropped-sharpened-contrast-background-scalebar.tif tif LaLone CA, Villeneuve DL, Cavallin JE, Kahl MD, Durhan EJ, Makynen EA, Jensen KM, Stevens KE, Severson MN, Blanksma CA, Flynn KM, Hartig PC, Woodard JS, Berninger JP, Norberg-King TJ, Johnson RD, Ankley GT. Cross-species sensitivity to a novel androgen receptor agonist of potential environmental concern, spironolactone. Environ Toxicol Chem. 2013 Nov;32(11):2528-41. doi: 10.1002/etc.2330. Epub 2013 Sep 6. PMID: 23881739. histology USEPA_GLTED_PP_SPIRL21D_C5B2 female adult 1 ovary unspecified 1A 4 microns Zeiss Axiovert 35