Ring Test Reveals Hidden Flaws in eDNA Labs Hunting Whales and Sharks

Environmental DNA has become one of the most powerful tools in modern ecology. By collecting a few liters of seawater, filtering it, and reading the genetic traces left behind by passing animals, researchers can detect whales, dolphins, and sharks without ever seeing them. The method is non-invasive, scalable, and increasingly attractive for monitoring rare and protected species across vast ocean basins. Yet a new study published in Metabarcoding and Metagenomics shows just how fragile the chain of laboratory steps behind those detections can be, and why multinational eDNA projects ignore standardization at their peril.

An international team working under the eWHALE project, a collaborative initiative spanning the North-East Atlantic and the Mediterranean Sea, set out to answer a deceptively simple question: if four laboratories extract DNA from the very same water samples, do they get the same results? The answer, revealed through a rigorous inter-laboratory comparison known as a ring test, was not always. Despite similar total DNA concentrations across all participating facilities, one laboratory’s extraction protocol performed significantly worse at detecting the target species, and the discrepancy was only caught because the partners had agreed to test themselves against each other before the real monitoring work began.

The ring test involved four institutions: the University of Innsbruck in Austria, INRAE in France, University College Cork in Ireland, and the Institute of Marine Research in Norway. In summer 2023, each partner contributed water samples collected near marine megafauna in their home waters. Innsbruck supplied samples gathered near sperm whales in the Mediterranean and around the Azores, INRAE contributed samples taken near porbeagle sharks off Brittany, Cork collected water in the Shannon Estuary where bottlenose dolphins surface in groups, and the Norwegian institute sampled near basking sharks by the Lofoten Islands. In total, 39 eDNA samples entered the comparison.

The experimental design was elegant in its simplicity. Each laboratory first lysed its own filters, breaking open cells and releasing DNA into a storage buffer. Then, 250-microliter aliquots of every lysate were shipped to all four partners, where each facility extracted DNA using its own preferred protocol. The extracts were shipped back to the laboratory of origin for analysis. This exchange meant that every original sample could be measured four times over, once per extraction method, allowing researchers to isolate the effect of the extraction chemistry itself from the noise of field sampling.

The extraction methods themselves reflected the everyday reality of eDNA research. Innsbruck used a Qiagen BioSprint 96 Workstation, an automated platform that relies on paramagnetic beads to capture DNA. INRAE employed a Macherey-Nagel NucleoSpin Tissue Kit with repeated heated elutions, while Cork and the Norwegian institute both used the widely recommended Qiagen DNeasy Blood and Tissue Kit, albeit with different modifications, including vacuum-assisted filtration in Norway and an extended 56-degree incubation of lysates in Ireland. Each of these small choices, the study found, could ripple forward into the final检测结果, though the differences were often invisible when measuring total DNA alone.

To evaluate detection performance, the team developed three novel species-specific quantitative PCR assays, one each for sperm whale, porbeagle shark, and basking shark, using mitochondrial genes and a software pipeline called assayID that ranks candidate primer and probe designs by specificity and sensitivity. These TaqMan assays, alongside an existing SYBR Green assay for dolphin DNA developed at Cork, were validated in silico, in vitro, and finally against field samples, reaching Level 3 of the widely accepted five-level validation framework proposed by Thalinger and colleagues. The assays proved remarkably sensitive, with limits of detection as low as 0.00001 nanograms per microliter for dolphins, but sensitivity alone could not rescue poor extracts.

The statistical verdict was clear. Across 468 qPCR reactions, 115 amplified their target species. Total DNA concentrations measured by Qubit fluorometer did not differ significantly between laboratories, yet detection probability did. Extracts produced by the Norwegian laboratory amplified target species DNA significantly less often than those from the other three partners, in both an all-inclusive dataset and a conservative dataset restricted to amplifications at or above each assay’s limit of detection. When the team examined the protocols side by side, two subtle differences emerged between the Norwegian and Irish workflows, which otherwise used nearly identical kits: the extra hour of lysate incubation in Ireland may have improved DNA binding to silica membranes, while the vacuum system used in Norway may have compromised binding efficiency or left residual ethanol in the extracts.

Species identity mattered as much as laboratory identity. Sperm whale DNA was detected in 43 reactions and dolphin DNA in 56, while porbeagle shark yielded only 13 detections and basking shark a mere three, all vanishing entirely from the conservative dataset. The researchers attribute this pattern partly to biology: marine mammals slough skin and defecate at the surface between feeding dives, seeding the water with DNA, whereas sharks shed far less genetic material. Sampling design compounded the problem, as sperm whale samples were drawn from 10-liter volumes while shark samples came from only 5 liters, and some basking shark samples were likely collected too far from the animals themselves. A significant interaction between laboratory and species further suggested that extraction efficiency depends on the environmental character of each sample, meaning no single protocol is universally superior.

The study also offers a cautionary lesson about the limits of detection. Nearly 80 percent of the positive amplifications fell below the assays’ formal limits of detection, and excluding those low-level signals reduced overall detection rates by roughly 20 percent. Interpreting such borderline amplifications requires care, the authors argue, because treating them as false positives risks discarding real detections of genuinely rare species, while trusting them uncritically invites statistical noise. For cross-laboratory comparisons in particular, the conservative approach provides the fairest benchmark, since DNA extracted from the same environmental sample should theoretically be identical regardless of who processed it.

The practical outcome of the ring test was immediate: the Norwegian laboratory modified its extraction protocol for the remainder of the project, and the eWHALE partners now possess a documented, quantified understanding of how their methods compare. Three newly published qPCR assays are available for other eDNA practitioners targeting sperm whale, porbeagle shark, and basking shark. More broadly, the study stands as a template for what multinational eDNA monitoring should look like from the start, with validation exercises built in before any ecological conclusions are drawn. As eDNA moves from an academic curiosity toward routine conservation decision-making, the authors argue, this kind of inter-laboratory rigor is not optional overhead but the very foundation upon which comparable, trustworthy biodiversity data must rest.

Subject of Research: Inter-laboratory comparison of environmental DNA extraction protocols for detecting marine megafauna using novel species-specific qPCR assays

Article Title: Inter-laboratory ring test for environmental DNA extraction protocols: implications for marine megafauna detection using three novel qPCR assays

Article References: Rodriguez, L. K., De Bonis, L., McKee, J., McKenna, J. A., Urvois, T., Barbaccia, E., Dillane, E., Lanfredi, C., Hjellnes, H., Jung, A., Villa, E., Azzellino, A., Westgaard, J.-I., Quéméré, E., & Thalinger, B. (2025). Inter-laboratory ring test for environmental DNA extraction protocols: implications for marine megafauna detection using three novel qPCR assays. Metabarcoding and Metagenomics, 9, Article e128235. https://doi.org/10.3897/mbmg.9.128235

Image Credits: AI Generated

DOI: 10.3897/mbmg.9.128235

Keywords: environmental DNA, eDNA, ring test, marine megafauna, qPCR, sperm whale, porbeagle shark, basking shark, dolphins, DNA extraction, laboratory standardization, eWHALE project