Performance evaluation and methods comparison of transcriptomic-based novel approach methods (tNAMs) for the characterization of wastewater treatment effluent

ABSTRACT: The complexity of wastewater treatment effluents (WWTE) complicates efforts to fully characterize the risk they pose to aquatic ecosystems. Transcriptomic-based novel approach methods (tNAMs) may provide the necessary resolution, sensitivity, and breadth to provide a more comprehensive characterization of these mixtures; however, a rigorous evaluation of tNAM performance is lacking. To address this, the current study aimed to systematically evaluate the consistency of the gene expression responses over time and under varying experimental approaches. Two-day post hatch (dph) fathead minnow (FHM) were exposed to clean laboratory water (moderately hard reconstituted water; MHRW) or one of four grab samples taken at an upstream site or within a WWTE in the morning and afternoon for two successive days. Concurrently, 2-dph FHM were also deployed at each site. RNA-seq analysis was conducted on all exposed fish. Differential expression analysis was conducted on fish from exposures to individual grab samples as well as a count data pooled across grab samples, providing both a discrete and time integrated analysis. For all effluent exposed fish, differentially expressed genes (DEGs) were identified relative to the fish exposed to the MHRW control or to the upstream site. The size of the DEG sets was used as an indicator of WWTE potency and the degree of overlap among respective DEG sets as a metric of consistency. With either comparator (MHRW or upstream), clear time dependent differences were observed across individual grab samples from exposures to the effluent but not the upstream site, with morning samples having much larger DEG set sizes than afternoon. With the MHRW comparator, considerable DEG overlap was observed among grab samples, with near complete overlap in morning samples. However, DEG set sizes were greatly reduced with the upstream comparator, with effectively no DEGs identified in afternoon sampling events and morning samples having minimal overlap. Deployed fish and the pooled grab sample composite exposures had similar results when the upstream site served as the comparator. In contrast, DEG sets were highly consistent among composite approaches with the MHRW comparator. DEG sets overlapped significantly between the upstream and effluent sites when compared to the MHRW, suggesting some consistency in their composition. This was confirmed using non-targeted analysis (NTA) for the first morning sample and a targeted analytical method for all grab samples. Overall, results indicate that gene expression is highly consistent over time and across exposure regimens; however, this is only the case with the use of the MHRW control. Direct contrasts between fish exposed to the WWTE and the upstream site displayed minimal consistency, which do not appear to reflect effluent composition. These results suggest that gene expression, if paired with NTA data, may provide a powerful tool to evaluate the risk posed by WWTE and to link gene expression with specific chemicals.

ORGANISM(S): Pimephales promelas

PROVIDER: GSE300878 | GEO | 2026/08/01

REPOSITORIES: GEO