Environmental DNA as a Hydrological Tracer: Expanding the Frontiers of Subterranean Ecology and Hydrogeology 

Mike Buchanan 2025

Abstract

Environmental DNA (eDNA) has revolutionised biodiversity monitoring by enabling the detection of species from trace genetic material in environmental samples. More recently, eDNA has been proposed as a novel bio-hydrological tracer, providing opportunities to investigate groundwater connectivity, flow dynamics, and subterranean ecology. This paper critically examines the potential of eDNA in hydrological tracing, situating it alongside classical tracers such as dyes, isotopes, bacterium and salts. Through synthesis of emerging literature, methodological proposals, and case examples, I highlight both the promise and challenges of integrating eDNA into hydrogeological research. I argue that eDNA’s dual ecological and hydrological dimensions could transform our understanding of subterranean connectivity, while acknowledging limitations such as decay rate variability and source attribution complexities. We propose an integrative framework combining eDNA with established tracer methods and hydrological models, supported by experimental designs and conceptual schematics.

1. Introduction

Hydrological tracers have long been fundamental tools in groundwater research, used to reveal subsurface flow paths, residence times, and aquifer connectivity. Conventional tracers, including fluorescent dyes, salts, isotopes and gases, provide reliable measures of water movement but lack ecological context (Smart and Laidlaw, 1977; Käss, 2004). In parallel, environmental DNA (eDNA) has rapidly advanced ecological monitoring, particularly for elusive or rare species (Taberlet et al., 2012; Deiner et al., 2017). Recent work suggests that the transport of eDNA through groundwater and cave systems can be exploited as a biologically informative tracer (Korbel et al., 2024).

This convergence of molecular ecology and hydrogeology represents a conceptual innovation: the use of DNA not only as a marker of species presence but as a tracer of hydrological processes. The present paper explores this intersection, critically reviewing current evidence, outlining methodological pathways, and presenting a framework for future application.

2. Literature Review

2.1 Classical Hydrological Tracers

Dye tracing has been a cornerstone of karst hydrogeology for over a century, providing clear signals of flow connectivity (Field, 2002). Stable isotopes (δ^18O, δ^2H) and chemical tracers further refine assessments of residence times and recharge sources (Kendall and McDonnell, 1998). While effective, these tracers are purely physical or chemical, lacking biological dimension.

Comparative Perspective: Bacterial Tracers vs. eDNA

Historical Use of Bacterial Tracers

• Coliforms and Enterococci have been used as microbial indicators to trace contamination pathways in groundwater, particularly in karst systems (Harvey et al., 1989; Goldscheider et al., 2006).
• Deliberate introduction of non-pathogenic bacterial strains (e.g., Bacillus subtilis spores) has been used in tracer experiments to evaluate subsurface flow and attenuation (Harvey et al., 1989).
• Bacteria function as particulate tracers, transported differently from dissolved dyes or salts, making them useful for studying colloid-facilitated transport.

Advantages of Bacterial Tracers

• Detectable at extremely low concentrations using culture or molecular methods.
• Provide biological realism (actual organisms moving through aquifers).
• Some are resilient (spores), allowing longer-term tracing.

Limitations of Bacterial Tracers

• Survival and growth: Unlike inert tracers, bacteria may multiply or die off, complicating interpretation.
• Public health concerns: Even when using non-pathogenic strains, risk perception and biosafety limit field applications.
• Variable transport: Retention in porous media due to filtration, adsorption, or biofilm interactions makes them less predictable than dyes or isotopes.

eDNA vs. Bacterial Tracers

• Safety: eDNA introduces no living organisms into groundwater, avoiding contamination concerns.
• Transport dynamics: eDNA behaves more like a solute or colloid, showing predictable decay curves, while bacteria interact biologically with the environment.
• Information content: Bacterial tracers provide information on particle transport; eDNA carries ecological identity, linking hydrology with species presence.

Table 1. Comparative features of bacterial tracers and eDNA as hydrological tracers

Aspect	Bacterial Tracers	eDNA Tracers
Safety	Potential biosafety concerns: even non-pathogenic strains raise risk perception	No living organisms introduced; minimal safety concerns
Transport Dynamics	Particulate transport; subject to filtration, adsorption, biofilm interactions	Behaves more like solute/colloid; predictable decay with distance
Detection	Culture-based methods or molecular assays; moderate sensitivity	Sensitive qPCR or metabarcoding; species-level resolution
Information Content	Provides particle/colloid transport insights; limited ecological identity	Ecological identity (species presence) + hydrological inference
Limitations	Survival, growth, die-off complicate interpretation; regulatory hurdles	Decay rate variability; signal may come from distant sources
Typical Applications	Tracing contamination pathways; studying colloid-facilitated transport	Groundwater connectivity studies; linking hydrology and ecology

Integrative Potential

An interesting pathway is the combined study of microbial DNA and eDNA. Microbial community DNA (16S rRNA metabarcoding) could be used alongside macro-organism eDNA to study how microbes and macrofauna move through aquifers together, providing a multi-scale bio-tracing approach.

2.2 Emergence of eDNA Tracing

Early eDNA studies established that genetic material could persist and move through aquatic systems, raising questions about transport and decay dynamics (Shogren et al., 2017). More recently, Carraro et al. (2018) modelled species distributions in river networks using eDNA transport models, demonstrating the potential for hydrological inferences. Korbel et al. (2024) confirmed eDNA persistence in karst conduits, opening new avenues for subsurface hydrology.

 2.3 DNA Transport and Decay

DNA persistence is mediated by degradation due to microbial activity, enzymatic breakdown, UV exposure and hydrochemical conditions (Collins et al., 2018). Transport can extend kilometres downstream, but signal strength decays with distance and turbulence. These features align eDNA with classical tracer decay curves but add ecological complexity.

3. Methodological Framework

3.1 Source–Sink Identification

Targeted sampling upstream and downstream of recharge points can reveal flow connectivity. Species-specific assays (e.g., qPCR) allow precise detection of DNA sources.

3.2 Dual-Tracer Experiments

Pairing eDNA with dyes or isotopes enables cross-validation. For instance, simultaneous dye injection and eDNA sampling at discharge points can strengthen inference of connectivity.

3.3 Decay and Persistence Experiments

Introducing controlled DNA fragments or tissues into cave pools allows quantification of decay rates under varying physicochemical conditions.

3.4 Metabarcoding for Community-Level Insights

Instead of single taxa, high-throughput sequencing of eDNA allows entire communities to be traced, offering ecosystem-wide connectivity insights.

4. Case Studies

• Karst systems: Korbel et al. (2024) detected invertebrate DNA moving through conduits, validating connectivity hypotheses.
• River networks: Carraro et al. (2018) applied eDNA modelling to infer distribution and abundance patterns influenced by hydrological transport.
• Porous substrates: Shogren et al. (2017) demonstrated eDNA movement through sediments, analogous to solute transport models.

5. Challenges and Limitations

• Decay variability: eDNA degrades at rates highly dependent on environmental conditions, requiring site-specific calibration.
• Non-point sources: Recharge and organic matter inputs confound signal interpretation.
• Transport vs. presence: Detection does not guarantee local occupancy but may reflect transport from upstream.
• Quantification difficulties: DNA concentrations are not yet dependable proxies for biomass or flow velocity.

6. Integrative Conceptual Framework

Figure 1 – A schematic comparing the transport of a classical dye tracer (blue arrows) vs. eDNA (green arrows, showing decay over distance) in a karst aquifer.

 

Figure 2 – A conceptual framework diagram showing how classical tracers, eDNA approaches, and hydrological models integrate to produce combined insights into groundwater connectivity and subterranean ecology.

  7. Discussion

The dual role of eDNA as both ecological signal and hydrological tracer presents a change in basic assumptions in groundwater research. While limitations remain, particularly in calibration and interpretation, the method has potential to provide bio-hydrological insights unavailable through conventional tracers. The integration of molecular tools with hydrogeological modelling could open new research frontiers in subterranean connectivity, biodiversity conservation, and aquifer management.

8. Conclusion

The use of eDNA as a hydrological tracer is still in its infancy, but early studies highlight significant promise. By complementing classical tracers with biological information, eDNA can provide deeper insights into groundwater systems. Standardisation of methods, controlled experiments, and interdisciplinary collaboration will be essential to realise its full potential.

References

Carraro, L., Hartikainen, H., Jokela, J. & Bertuzzo, E. (2018) Estimating species distribution and abundance in river networks using eDNA. Proceedings of the National Academy of Sciences, 115(46), 11724–11729.

Collins, R.A., Wangensteen, O.S., Sims, D.W., Genner, M.J. & Mariani, S. (2018) Persistence of environmental DNA in marine systems. Communications Biology, 1(1), 185.

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Field, M.S. (2002) The QTRACER2 program for tracer-breakthrough curve analysis for tracer tests in karstic aquifers and other hydrologic systems. U.S. Environmental Protection Agency Report.

Käss, W. (2004) Tracing Technique in Geohydrology. Rotterdam: Balkema.

Kendall, C. & McDonnell, J.J. (1998) Isotope Tracers in Catchment Hydrology. Amsterdam: Elsevier.

Harvey, R.W., George, L.H., Smith, R.L. & LeBlanc, D.R. (1989) Transport of microspheres and indigenous bacteria through a sandy aquifer: Results of natural- and forced-gradient tracer experiments. Environmental Science & Technology, 23(1), 51–56.

Goldscheider, N., Hunkeler, D. & Rossi, P. (2006) Review: Microbial biogeography in karst aquifers: Review and research perspectives. Hydrogeology Journal, 14(3), 347–360.

Korbel, K.L., Hose, G.C., Carini, G. & Seymour, J.R. (2024) Detection, movement and persistence of invertebrate eDNA in subterranean systems. Molecular Ecology Resources, 24(2), 211–225.

Shogren, A.J., Tank, J.L., Andruszkiewicz, E.A., Olds, B. & Jerde, C.L. (2017) Modelling the transport of environmental DNA through a porous substrate. Freshwater Biology, 62(1), 97–107.

Smart, P.L. & Laidlaw, I.M.S. (1977) An evaluation of some fluorescent dyes for water tracing. Water Resources Research, 13(1), 15–33.

Taberlet, P., Coissac, E., Hajibabaei, M. & Rieseberg, L.H. (2012) Environmental DNA. Molecular Ecology, 21(8), 1789–1793.