THE IMPACTS OF 2,4-D HERBICIDE DMA® 4 IVM ON REPRODUCTIVE HEALTH AND GENE EXPRESSION ALONG THE HYPOTHALAMIC-PITUITARY-GONAD-LIVER [HPGL] AXIS IN THE FATHEAD MINNOW (PIMEPHALES PROMELAS)

Abstract

Aquatic herbicides are commonly used to control a wide variety of non-native and nuisance plants. One common active ingredient used in commercial herbicide formulations globally is 2,4- dichlorophenoxyacetic acid (2,4-D). Due to the stability of 2,4-D in aquatic environments, many non-target aquatic species experience prolonged exposure throughout critical developmental life stages that could have adverse health consequences. To our knowledge, no studies have investigated cellular, biochemical, and transcriptional effects of 2,4-D exposure on fathead minnows (Pimephales promelas) throughout reproductive development (the juvenile life stage). Additionally, no mechanism of action has been described for 2,4-D as an endocrine disrupting chemical in fishes. By investigating changes in biochemistry, histology, and gene expression, we are can create a snapshot of the reproductive health of fish. Therefore, we conducted a series of experiments to expand upon previous studies by investigating the chronic effects of 2,4-D exposure on the developing reproductive system in juvenile fathead minnows. Herein, we observed significantly decreased plasma testosterone concentration in male fish exposed to environmentally relevant concentrations (0.50, 2.00, and 4.00 mg/L) of 2,4-D, but no histopathological impacts in the male gonads at the same concentrations. We observed a significant increase in ovarian severity grading and oocyte atresia in female fish exposed to 4.00 mg/L. Of differential expression analysis in fish exposed to 2.00 mg/L 2,4-D in DMA4, we identified significantly decreased expression of the steroid hormone receptors ESR1, ESR2b, and AR in male liver tissue while AR expression was significantly decreased in the female liver. ESR2a expression was significantly increased in male liver suggesting the presence of compensatory receptor expression. We observed significant increases in expression of male steroidogenic genes 3βHSD in the testis and 11βHSD2 in the liver of male fish while 17βHSD4

was significantly decreased in the female liver. Of genes regulating steroid metabolism, testis SULT1st2 and liver CBR1l were significantly increased in males while gonadal SULT2a1 was significantly decreased in both males and females. Altogether, these data suggest that 2,4-D could act as an endocrine disrupting chemical that alters expression of primary genes regulating hormone receptors, steroidogenesis, and steroid metabolism along the hypothalamic-pituitary- gonad-liver (HPGL) axis. The use of 2,4-D herbicides for weed control in aquatic ecosystems could present risks to the reproductive health of non-target aquatic species.