Detection of viral, bacterial, and protozoan pathogens and microbial source tracking markers in paired large- and small-volume water samples

Authors: Heffron, Joseph; STOKDYK, JOEL - Us Geological Survey (USGS); FIRNSTAHL, AARON - Us Geological Survey (USGS); Cook, Rachel; HRUBY, CLAIRE - Drake University; BORCHARDT, MARK - US Department Of Agriculture (USDA)

Submitted to: ES&T Water
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/11/2025
Publication Date: 11/25/2025
Citation: Heffron, J.A., Stokdyk, J.P., Firnstahl, A.D., Cook, R.M., Hruby, C., Borchardt, M.A. 2025. Detection of viral, bacterial, and protozoan pathogens and microbial source tracking markers in paired large- and small-volume water samples. ES&T Water. https://doi.org/10.1021/acsestwater.5c00639.
DOI: https://doi.org/10.1021/acsestwater.5c00639

Interpretive Summary: When testing water for biosecurity or to protect public health, researchers and water managers face the choice of collecting small volume (~1 L) grab samples or passing hundreds of liters of water through an ultrafilter. Standard methods recommend large volume ultrafilter sampling for detecting pathogens in groundwater and small volume grab samples for fecal indicator microbes, particularly in surface water. However, not all applications fit these prescriptions. Given that large volume sampling is costly and time-intensive, and requires trained personnel, stakeholders need to understand the trade-offs between different sampling methods based on their application and microbial target. We show that the main benefit of large-volume sampling is in the detection of low-concentration microbial targets. However, large volume sampling becomes less advantageous as the concentration of the microbe in the environment and the complexity of the water matrix increase. Researchers and facility managers can use our conclusions to tailor sampling to their purpose, rather than base their selection on best guesses and logistical constraints.

Technical Abstract: When sampling for waterborne microbes, researchers may diverge from recommended sample volumes due to logistical constraints, novel targets, or challenging matrices, with little guidance about the potential impact on results. In field studies, we measured bacteria, viruses, and protozoa (15 qPCR assays) in paired large and small volume samples to evaluate when and why a method’s performance was superior. Concordance between methods was generally low. Large volume ultrafiltration yielded more detections than small volume sampling, especially for pathogens in groundwater. Greater microbial concentrations, as observed for commensal microbes and surface water, were associated with more frequent detections in small volume samples and greater concordance between paired samples. Compared to grab sampling, time-integrated small volume sampling controlled for temporal heterogeneity of microbes but did not fully account for differences in method performance. In laboratory studies, recovery of microbes was poorer for large volume methods than small volume methods, though large volume methods more reliably detected low-concentration targets. Large volume samples were less stable during storage. Sample matrix and losses during recovery and storage help to explain the similar sensitivity of large volume and small volume methods in surface water. Microbial target, concentration, sample matrix, and study goals inform method selection.