Effects of Age and Exposure Duration on the Sensitivity of early life stage Pimephales promelas (Fathead Minnow) to Waterborne Propranolol Exposure

Propranolol is a heavily prescribed, non-specific beta-adrenoceptor (bAR) antagonist frequently found in wastewater effluents, prompting concern over its potential to adversely affect exposed organisms. Herein, the transcriptional responses of 4, 5, and 6 dpf +/- 1h fathead minnow (FHM), exposed for 6, 24, or 48hr to 0.66 or 3.3mg/L (nominal) propranolol were characterized using RNA-seq. The number of differentially expressed genes (DEGs) was used as an estimate of sensitivity. A trend toward increased sensitivity with age was observed; fish >7dpf at the end of exposure were particularly sensitive to propranolol. DEGs largely overlapped among treatment groups, suggesting a highly consistent response that was independent of age. Cluster analysis was performed using normalized count data for unexposed and propranolol exposed fish. Control fish clustered tightly by age, with fish >= 7dpf clustering away from younger fish, reflecting developmental differences. When clustering was conducted using exposed fish, in cases where propranolol induced a minimal or no transcriptional response, results mirrored those of the control fish and did not appreciably cluster by treatment. In treatment groups that displayed a more robust transcriptional response, the effects of propranolol were evident, however, fish < 7dpf clustered away from older fish, despite having similar numbers of DEGs. Increased sensitivity at 7dpf coincided with developmental milestones with the potential to alter propranolol pharmacokinetics or pharmacodynamics, such as the onset of exogenous feeding and gill functionality, as well as increased systemic expression of bAR. These results may have broader implications, as toxicity testing often utilizes fish < 4dpf, prior to the onset of these potentially important developmental milestones, which may result in an underestimation of risk for some chemicals.