Fronzi et al. (2026) Toward quantitative DNA tracer tests: development and validation of a novel capture device for groundwater flow characterization in karst and carbonate aquifers

Identification

• Journal: Journal of Hydrology
• Year: 2026
• Date: 2026-03-20
• Authors: Davide Fronzi, Vesna Milanović, Stefano Palpacelli, Alberto Tazioli, Lucia Aquilanti
• DOI: 10.1016/j.jhydrol.2026.135324

Research Groups

• Department of Science and Matter Engineering, Environment and Urban Planning (SIMAU), Marche Polytechnic University, Ancona, Italy
• Department of Agricultural, Food and Environmental Sciences (D3A), Marche Polytechnic University, Ancona, Italy
• Independent Geologist, Italy

Short Summary

This study develops and validates a novel passive device for selectively capturing biotinylated synthetic DNA tracers, enabling time-integrated, semi-quantitative assessment of groundwater flow. Laboratory and field tests demonstrate that the device produces breakthrough curves consistent with conventional tracers, facilitating broader application of DNA tracers in complex hydrogeological systems.

Objective

• To develop and validate a novel passive device that selectively captures biotinylated synthetic DNA tracers, enabling time-integrated, semi-quantitative assessment of groundwater flow and pollutant migration, thereby overcoming limitations of manual sampling in prolonged tracer tests.

Study Configuration

• Spatial Scale: Laboratory column tests (column height 46 cm, internal diameter 4.8 cm, internal volume 832.40 cm³); Field-scale experiment in a natural stream (approximately 200 m reach).
• Temporal Scale: Column test: contact time for traps 55–60 min, breakthrough curves (BTCs) monitored up to 8 pore volumes. Field test: tracer tests conducted over approximately 3 hours 45 minutes, each device deployed for 5 min, BTCs monitored for approximately 50 min.

Methodology and Data

• Models used: Not applicable (no explicit hydrological or transport models were used for simulation; the study focused on device development and tracer behavior benchmarking).
• Data sources:
  • Biotinylated synthetic DNA tracer (72-nucleotide single-stranded DNA).
  • Streptavidin-functionalized superparamagnetic beads (1.0 µm diameter, ~500 pmol/mg binding capacity) encapsulated in 0.6% (w/v) agarose hydrogel pearls.
  • Conventional tracers: Potassium chloride (KCl), Sodium chloride (NaCl), Fluorescein.
  • Measurements: Electric conductivity (Diver CTD Eijkelkamp, WTW-type conductivity probe), Fluorescein concentration (PME fluorometric probe Cyclops-7 Logger), Quantitative Polymerase Chain Reaction (qPCR) for DNA quantification (CFX Connect Real-Time System).
  • Column characteristics: Height, diameter, volume, soil bulk density, pore volume, porosity.

Main Results

• qPCR performance: Achieved 100.2% amplification efficiency and R² = 0.997 for biotinylated DNA tracer quantification, with a sensitivity of less than 10 DNA molecules per reaction and linearity across eight orders of magnitude (8.8 to 8.8 × 10⁷ molecules per reaction).
• Column test: Breakthrough curves (BTCs) from both manual sampling and the passive device closely matched conservative tracer (KCl) responses. The passive device produced slightly broader BTCs with a less steep descending limb, consistent with its time-integrated capture mechanism. Peak DNA concentration from manual samples was ~3.87 × 10⁹ molecules/mL, and from traps was ~2.1 × 10⁶ molecules/trap, both peaking at ~3 pore volumes.
• Field test: Conservative tracers (NaCl, Fluorescein) showed rapid concentration increases around 8 min post-injection, peaking between 13 and 14 min, and returning to baseline by approximately 50 min. DNA concentrations from manual sampling peaked around 15 min at ~1.5 × 10⁸ molecules/mL. The DNA trap signal displayed a broader and bimodal distribution, with an initial peak at ~18 min (~7.8 × 10⁴ molecules/trap) and a secondary peak after 28 min (~2.7 × 10⁴ molecules/trap).
• Tracer behavior: No evidence of specific retention or irreversible interaction between the synthetic DNA tracer and the calcium-carbonate material was observed in either experiment.
• Capture efficiency: The number of DNA molecules recovered by the device was substantially lower than the injected mass (e.g., 10⁶ recovered vs. 10¹⁴ injected in lab; 10⁴ recovered vs. 10¹⁵ injected in field), indicating a semi-quantitative, time-integrative sampling approach rather than complete mass recovery.

Contributions

• Developed and validated a novel passive device for selective, time-integrated capture of biotinylated synthetic DNA tracers, overcoming limitations of manual sampling in hydrological studies.
• Demonstrated the device's ability to produce semi-quantitative BTCs consistent with conventional tracers in both laboratory and field settings, particularly relevant for karst and carbonate aquifers.
• Provided a reproducible and operationally efficient method for tracing hydrological pathways, facilitating broader and more quantitative application of DNA tracers in groundwater investigations.
• Confirmed the conservative transport behavior of biotinylated DNA tracers in calcium-carbonate environments, comparable to conventional tracers.
• Introduced a system with high specificity, environmental safety, low cost, and operational simplicity, making it suitable for remote or ecologically sensitive environments.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Citation

@article{Fronzi2026Toward,
  author = {Fronzi, Davide and Milanović, Vesna and Palpacelli, Stefano and Tazioli, Alberto and Aquilanti, Lucia},
  title = {Toward quantitative DNA tracer tests: development and validation of a novel capture device for groundwater flow characterization in karst and carbonate aquifers},
  journal = {Journal of Hydrology},
  year = {2026},
  doi = {10.1016/j.jhydrol.2026.135324},
  url = {https://doi.org/10.1016/j.jhydrol.2026.135324}
}