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Effects of the fungicide imazalil on the fathead minnow (Pimephales promelas) reproductive axis a case study in 21st century toxicity testing
Citation:
Randolph, E., G. Ankley, B. Blackwell, J. Cavallin, W. Cheng, D. Feifarek, K. Jensen, M. Kahl, R. Milsk, S. Poole, T. Saari, AND Dan Villeneuve. Effects of the fungicide imazalil on the fathead minnow (Pimephales promelas) reproductive axis a case study in 21st century toxicity testing. SETAC Midwest Chapter, Minneapolis, MN, March 20 - 22, 2017.
Impact/Purpose:
The current research demonstrates that high throughput screening data along with adverse outcome pathways can be effectively applied to predict an apical hazard associated with a previously untested chemical. The pesticide imazalil was identified via the ToxCast data set as a chemical able to impair steroid biosynthesis, an activity of concern for the EDSP program. Based on an established adverse outcome pathway (AOP), it was predicted that imazalil would act as a reproductive toxicant in fish. That activity was confirmed and further supporting evidence for the AOP was established, further strengthening its predictive utility. Additionally, quantitative predictions of effect concentrations generated using a novel quantitative AOP construct, were tested in order to identify further model refinements that could improve predictive accuracy. Presentation of this case study will to highlight the utility of the AOP framework for 21st century chemical safety assessment to an audience of state and regional risk assessors and environmental managers, aiding adoption of tools and approaches under development in the CSS research program.
Description:
Since its introduction in 1983, imazalil has been used primarily as a fungicide on crops post-harvest. Its effectiveness lies in the ability to inhibit the fungal cytochrome P450 (cyp), lanosterol 14 á-demethylase. However, like other azole fungicides, imazalil can inhibit a range of cyp enzymes, including one or more involved in steroid biosynthesis in vertebrates. Previous in vitro assays showed that imazalil can cause aromatase inhibition and reduce 17â-estradiol (E2) production by H295R cells. In the present study, we evaluated the effects of imazalil in a 21 d Fish Short-Term Reproduction Assay (FSTRA) with fathead minnows (Pimephales promelas) to evaluate whether the chemical would elicit effects consistent with an adverse outcome pathway (AOP) linking inhibition of aromatase to reduced fecundity. Fish were sampled at two time points (10, 21 d) and several endpoints (including key events [KE]s) associated with the AOP were measured. Fecundity also was determined daily. These data, combined with results of two previous exposures of 24 h and 60 h, produce a full time-course of effects. Ex vivo ovarian E2 production in females exposed to imazalil was significantly lower (p<0.05) than controls after 24 h at concentrations of 100, 500, and 1580 µg/L and remained significantly lower at 60 h, 10 d, and 21 d in fish exposed to 200 µg/L. Plasma E2 concentrations from females exposed for 24 h were significantly lower at imazalil concentrations of 80 and 250 µg/L and remained significantly lower at 60 h when exposed to 200 µg/L. Real-time quantitative PCR analyses measuring relative abundance of hepatic vtg mRNA transcripts were significantly lower after 60 h in individuals exposed to 200 µg imazalil/L. Spawning within the FSTRA ceased in the high concentration (200 µg/L) within 2 days after exposure to imazalil began. A decrease in egg production was also initially observed at 63.2 µg/L, but the fish appeared to recover. All KE endpoints measured displayed temporal concordance with the proposed AOP, supporting the causal relationships between those events. Overall, data collected to date support the hypothesis that imazalil causes endocrine disruption in exposed female fathead minnows in a manner consistent with the established AOP and supports the broader utility of that AOP, in hazard assessment.