Assessing the impact of wastewater treatment plant effluent on downstream drinking water-source quality using a zebrafish (Danio Rerio) liver cell-based metabolomics approach

Cell-based metabolomics was used in a proof-of-concept fashion to investigate the biological effects of contaminants as they traveled from a wastewater treatment plant (WWTP) discharge to a drinking water treatment plant (DWTP) intake in a surface-water usage cycle. Zebrafish liver (ZFL) cells were exposed to water samples collected along a surface-water flowpath, where a WWTP was located ∼14.5 km upstream of a DWTP. The sampling sites included: 1) upstream of the WWTP, 2) the WWTP effluent discharging point, 3) a proximal location downstream of the WWTP outfall, 4) a distal location downstream of the WWTP outfall, 5) the drinking water intake, and 6) the treated drinking water collected prior to discharge to the distribution system. After a 48-h laboratory exposure, the hydrophilic and lipophilic metabolites in ZFL cell extracts were analyzed by proton nuclear magnetic resonance (1H NMR) spectroscopy and gas chromatography-mass spectrometry (GC-MS), respectively. Multivariate statistical analysis revealed distinct changes in metabolite profiles in response to WWTP effluent exposure. These effects on the hydrophilic metabolome gradually diminished downstream of the WWTP, becoming non-significant at the drinking water intake (comparable to upstream of the WWTP, p = 0.98). However, effects on the lipophilic metabolome increased significantly as the river flowed from the distal location downstream of the WWTP to the drinking water intake (p < 0.001), suggesting a source of bioactive compounds in this watershed other than the WWTP. ZFL cells exposed to treated drinking water did not exhibit significant changes in either the hydrophilic (p = 0.15) or lipophilic metabolome (p = 0.83) compared to the upstream site, suggesting that constituents in the WWTP effluent were efficiently removed by the drinking water treatment process. Impacts on ZFL cells from the WWTP effluent included disrupted energy metabolism, a global decrease in amino acids, and altered lipid metabolism pathways. Overall, this study demonstrated the utility of cell-based metabolomics as an effective tool for assessing the biological effects of complex pollutant mixtures, particularly when used as a complement to conventional chemical monitoring.