Is seven days enough? Comparing a 7-day exposure to the classical 21-day OECD TG 229 fish short-term reproduction assay in fathead minnow

Abstract The OECD (Organisation for Economic Co-operation and Development) test guidelines (TG) 229 - fish short-term reproduction assay (FSTRA) is one of the gold standard methods used to identify endocrine disrupting chemicals (EDCs). While informative, the FSTRA’s 5 to 6-week duration makes it difficult to use routinely. Prior studies have shown that EDCs’ impact on fecundity, vitellogenin (VTG) and steroid levels can be detected after less than one week of exposure suggesting the FSTRA could be shortened. This study compares both 7- and 21-day FSTRAs using fathead minnows (Pimephales promelas) for three known EDCs: 17α-ethinylestradiol (EE2; 40 ng/L), 17β-trenbolone (TRB; 50 µg/L), and propiconazole (PRP; 500 µg/L). All three compounds led to arrested fertility after 24 h of exposure, except for the 7-day EE2 treatment which still decreased reproduction. Moreover, independently of time of exposure, EE2 induced VTG production in males, and decreased estrogen levels in females and testosterone levels in males. In contrast, TRB induced VTG production in males, while the levels were not different from controls in females even though testosterone levels increased, and masculinization was observed. Finally, PRP led to a decrease in VTG levels which was only significant during the 21-day exposure, and surprisingly, no effect on steroid levels were observed despite its known effects on steroidogenesis. Apart from minor differences, both times of exposure led to similar outcomes supporting the shortening of the FSTRA to seven days. This proposed 7-day FSTRA could be used to screen EDCs in routine monitoring of environmental samples.