The research team, working with seawater collected aboard the research vessel Miguel Oliver during the PELACUS 0324 survey, sampled three stations off the northwest Iberian Peninsula: Northern Galicia, Costa da Morte, and the Ria de Arousa. At each site, they filtered eight liters of seawater through Sterivex cartridge filters and then split the material between two extraction strategies. The first was the standard DNeasy PowerWater Sterivex protocol, a widely used commercial kit designed specifically for enclosed filters. The second added a preliminary bead-beating homogenization step, in which the filter membrane was removed from its cartridge, cut into fragments, and physically shattered with metal beads before following the standard protocol, on the theory that breaking open more cells would release more DNA.

The result was a clear win for simplicity. The standard, hands-off protocol yielded DNA concentrations roughly six to seventeen times higher than the bead-beating variant across the three stations, and it also recovered slightly more amplicon sequence variants, the fine-grained genetic units used to measure biodiversity. Both methods captured a broadly similar cast of marine vertebrates, including sardines, anchovies, sea bream, and even a fin whale at one station, but the extra handling involved in the modified method appears to have cost more than it gained, likely through DNA loss and increased contamination risk when the filter was manipulated outside its sealed unit.

One of the more intriguing questions the study addressed concerns free extracellular DNA, or f-exDNA: genetic material floating loose in seawater, not attached to cells or particles. Some researchers have suggested this fraction could pass straight through filter membranes and be lost. Using controlled seawater samples spiked with common dolphin DNA at known concentrations, the team found that recovery of this free DNA remained low with both extraction methods, generally below one percent, and actually declined as DNA concentrations rose, possibly because binding sites on the filter membrane became saturated. The finding suggests that particle-associated DNA, rather than free-floating strands, is what filters and extraction kits are mostly capturing, though the authors caution that more work is needed to disentangle the different DNA subfractions in natural waters.

The next variable under scrutiny was the polymerase enzyme, the molecular workhorse that copies target DNA during PCR. The team compared a high-fidelity proofreading polymerase, Platinum SuperFi II, which corrects its own replication errors using exonuclease activity, with a cheaper non-proofreading enzyme, Supreme NZYTaq. There is a technical wrinkle here: the same exonuclease activity that makes proofreading enzymes accurate can also chew back mismatches at the ends of primers, potentially altering which taxa amplify successfully. Using artificial mock communities containing DNA from sixteen vertebrate species at equal concentrations, the researchers ran paired statistical tests on the read outputs of both enzymes.

The verdict was taxon-dependent and, in a way, counterintuitive. The proofreading enzyme generated significantly more reads from fish, amplifying bony fish DNA more effectively, while the non-proofreading enzyme produced significantly more cetacean reads, although that effect was weaker and more variable. Both enzymes detected all sixteen species in the mock communities, meaning the cheaper enzyme did not miss anything. In real environmental samples, the picture was messier: the two enzymes produced overlapping but distinct species lists, with seven species shared, six detected only with the proofreading enzyme, and four only with the non-proofreading one, a discrepancy the authors attribute to the inherent stochasticity of the metabarcoding workflow rather than to enzyme choice alone.

For practical purposes, the message is that budget-conscious monitoring programs may not need the expensive high-fidelity option. The non-proofreading enzyme delivered comparable overall species detection in environmental samples and excelled at cetacean detection, making it a cost-effective choice when the goal is tracking marine mammals. The authors do note that polymerases can introduce sequence-composition biases related to guanine-cytosine content, which may distort relative abundance estimates even when presence or absence data remain reliable, so abundance-based ecological interpretations still warrant caution.

The third pillar of the study is MVeM, the Marine Vertebrate eDNA Metabarcoding bioinformatics pipeline, an open-source, adaptable workflow now available on GitHub. After raw reads are trimmed and processed with the DADA2 algorithm to infer sequence variants, taxonomic assignment proceeds through BLAST searches against the NCBI nucleotide database with stringent filters: at least ninety-nine percent sequence identity, sufficient alignment length, and exclusion of generic environmental sample entries so that every assignment traces back to vouchered tissue specimens. The pipeline then layers on several safeguards against false positives.

Among those safeguards are a customizable geographic ban list that automatically screens out species implausible for the study region, a filter against nuclear mitochondrial DNA segments, or NUMTs, which are mitochondrial fragments lurking in the nuclear genome that can masquerade as genuine detections, and a Lowest Common Ancestor fallback that assigns ambiguous matches to a genus or family rather than gambling on a species call. Contamination control is handled with an unusual touch of machine learning: instead of a fixed read threshold, an unsupervised classification method flags potential contaminants sample by sample, accounting for variable sequencing depth while still preserving genuinely abundant biological signals that might appear as faint traces in control blanks.

Importantly, the authors are candid that their environmental comparisons rest on limited replication, so the extraction and enzyme trends should be read as exploratory rather than statistically definitive. Even so, the study lands at a moment when eDNA is moving from research curiosity to policy tool, feeding into fisheries assessments and conservation planning. By showing that a simple, minimally invasive extraction, a thoughtfully chosen polymerase, and a rigorous, transparent bioinformatics framework can together deliver reliable marine vertebrate inventories, the work offers exactly what the field has been asking for: a standardized, accessible recipe for reading the ocean’s genetic fingerprint, one water sample at a time.

Subject of Research: Optimization of environmental DNA metabarcoding methods for monitoring marine vertebrate biodiversity

Article Title: Evaluating eDNA metabarcoding methods for marine vertebrate monitoring

Article References: Afonso, L., Álvarez-González, M., Pascoal, F., Sánchez-Barreiro, F., Rojo, V., Saavedra, C., Costa, J., Covelo, P., Pierce, G. J., Correia, A. M., Magalhães, C., & Suarez-Bregua, P. (2026). Evaluating eDNA metabarcoding methods for marine vertebrate monitoring. Metabarcoding and Metagenomics, 10, Article e191426. https://doi.org/10.3897/mbmg.10.191426

Image Credits: AI Generated

DOI: 10.3897/mbmg.10.191426

Keywords: environmental DNA, eDNA metabarcoding, marine vertebrates, biodiversity monitoring, DNA extraction, polymerase, bioinformatics pipeline, MVeM, Sterivex filters, cetaceans, contamination control, marine conservation

The scale of the problem begins with definition. Unlike microplastics, which are relatively easy to see, count, and chemically identify, nanoplastics occupy a size range where detection becomes genuinely difficult. Particles below roughly one micrometre scatter light weakly, aggregate unpredictably in environmental waters, and defy many of the spectroscopic techniques that work well for larger fragments. Researchers have proposed various size thresholds, sometimes linking the ability to cross biological barriers to specific size classes, but there is still no universally agreed definition of what constitutes a nanoplastic. This ambiguity matters because it makes comparisons between studies unreliable and complicates any attempt to set regulatory limits. The review emphasises that environmental nanoplastics are not uniform spheres of polystyrene but heterogeneous, weathered, chemically transformed particles whose properties differ dramatically from the pristine model materials used in most laboratory experiments.

Perhaps the most provocative section of the review concerns the evidence for tissue translocation, the process by which particles move from the gut or gills into internal organs and, potentially, the bloodstream. Experimental studies using model nanoplastics suggest that these particles can indeed reach internal tissues in organisms ranging from scallops and zebrafish to earthworms, lettuce, and snails. Studies of zebrafish have documented size-dependent accumulation and depuration dynamics, with smaller particles distributing more widely across tissues. Work on scallops at environmentally realistic concentrations has traced whole-body distribution and clearance. Research on plants has shown root uptake and movement into edible tissues, raising questions about food safety, and mass-based measurements have even tracked polystyrene nanoplastics moving up a lettuce-to-snail food chain. Yet the authors issue a sobering caveat: quantitative confirmation that intact particles, rather than dissolved tracer or leached dye, actually cross into tissues remains limited across the literature.

That caveat strikes at the heart of a methodological controversy that has been brewing for years. Many uptake studies rely on fluorescently labelled particles, visualising bright signals inside organisms and interpreting them as internalised plastic. But fluorescence is a notoriously indirect proxy. Dyes can leach from particle surfaces and accumulate in tissues independently of the particles themselves. Biological tissues autofluoresce, producing false signals. Particles adhering to external surfaces can be mistaken for internal burden. Without adequate controls and independent chemical confirmation, a glowing image of a fish larva tells us surprisingly little about where plastic actually is. The review highlights that these limitations, documented repeatedly in the literature, mean that some of the most widely cited evidence for nanoplastic translocation may overstate the case. This does not mean translocation does not happen; it means the field cannot yet quantify it with confidence.

The analytical toolbox available to researchers is expanding, but each technique carries trade-offs. Pyrolysis gas chromatography coupled with mass spectrometry can identify polymer type and provide mass-based quantification, but it destroys the sample and cannot report particle numbers or sizes. Raman and infrared spectroscopy offer polymer specificity but struggle at the nanoscale, where diffraction limits and weak signals intervene. Single-particle inductively coupled plasma mass spectrometry has recently enabled quantitative tracking of nanoplastic uptake and distribution in zebrafish, representing a promising advance for metal-doped or metal-bearing particles. Spectro-microscopic methods combining chemical identification with nanoscale imaging are emerging as powerful hybrid approaches. Yet interlaboratory comparisons of microplastic analysis have revealed alarming variability between laboratories, underscoring the urgent need for harmonised protocols, certified reference materials, and rigorous contamination control. Airborne deposition, plastic-containing labware, and reagent contamination can all inflate measured concentrations, and studies lacking proper blanks are difficult to interpret.

Even where exposure can be demonstrated, the biological consequences remain hard to pin down. Across taxa, the review finds a recurring set of reported effects: oxidative stress, immune and inflammatory responses, genotoxicity, and metabolic disturbance. Zebrafish larvae exposed to polystyrene nanoplastics show disrupted glucose metabolism and altered cortisol levels, with possible links to behavioural changes. Studies report neurotoxicity, endocrine disruption, reproductive effects, and DNA damage in cell cultures and organisms. Human-relevant findings add urgency: plastic particles have been detected in human blood and in placental tissue, and in vitro work on human blood cells and lymphocytes shows oxidative and genotoxic responses. But here lies the interpretative trap. These biomarkers are famously non-specific. Oxidative stress rises in response to heat, toxins, pathogens, poor nutrition, and countless other stressors. A fish with elevated antioxidant enzyme activity may be responding to nanoplastics, or to any of a dozen other pressures in its environment.

This lack of specificity is precisely why the authors argue that single biomarkers cannot serve as stand-alone indicators of nanoplastic exposure or effect. Instead, they advocate a tiered conceptual framework for future biomonitoring built on three integrated layers. The first layer demands polymer-specific particle confirmation, ensuring that what is measured is genuinely plastic of a known type rather than an artefact. The second layer calls for assessment of internal burden using complementary metrics, combining mass-based, number-based, and size-based measurements, because each captures a different dimension of exposure and no single metric tells the whole story. The third layer interprets these measurements through mechanistically linked multi-biomarker panels, where combinations of responses along coherent biological pathways, such as adverse outcome pathways linking molecular initiating events to apical outcomes, provide the contextual weight that isolated markers lack.

The framework also draws a crucial conceptual line between three things that are often conflated: environmental occurrence, internal exposure, and biological effect. A particle present in water or sediment says nothing about whether organisms have taken it up. A particle in the gut says nothing about whether it has crossed a barrier. And a stressed biomarker profile says nothing, on its own, about the cause. Distinguishing these layers, the authors contend, is essential for building monitoring programmes that produce defensible conclusions. They further stress the need for contamination control at every step, complementary analytical methods rather than reliance on any single technique, environmentally relevant exposure conditions in experimental work, and transparent reporting of uncertainty. Model particles that are spherical, monodisperse, and freshly manufactured may behave nothing like the ragged, aged, protein-coated particles found in a river, and extrapolating from the former to the latter carries risks that risk assessors must acknowledge.

The bottom line of the review is deliberately unglamorous but important: nanoplastics are not yet ready for routine biomonitoring. That conclusion may disappoint those hoping for a simple metric to track this emerging threat, but it reflects scientific honesty about the state of the evidence. The path forward, the authors argue, lies in coordinated analytical validation and mechanism-based interpretation, which together could eventually support the inclusion of nanoplastics in environmental monitoring and risk assessment. Given the trajectory of plastic production and fragmentation, the particles are not going away. What is needed now is the methodological rigour to measure them reliably, the mechanistic understanding to interpret what their presence means for living systems, and the patience to build monitoring frameworks on foundations solid enough to bear regulatory weight. Until then, the invisible plastic inside wildlife remains a compelling hypothesis awaiting proof.

Subject of Research: Analytical challenges, tissue translocation, and biomarkers of nanoplastic contamination in environmental biota

Article Title: Nanoplastics in environmental biota: analytical challenges, tissue translocation, and biomarkers for biomonitoring

Article References: Curcic, M., Hadzic, B., & Lazarevic, Z. (2026). Nanoplastics in environmental biota: analytical challenges, tissue translocation, and biomarkers for biomonitoring. Environmental Monitoring and Assessment, 198(10), Article 1040. https://doi.org/10.1007/s10661-026-15891-0

Image Credits: AI Generated

DOI: 10.1007/s10661-026-15891-0

Keywords: nanoplastics, biomonitoring, microplastics, tissue translocation, biomarkers, oxidative stress, ecotoxicology, analytical chemistry, environmental monitoring, zebrafish, genotoxicity, contamination control

The research team, led by Maryam Sharafi Farzad and colleagues at the University of Copenhagen, set out to validate the Precision ID mtDNA Whole Genome Panel from Thermo Fisher Scientific specifically for telogen hair analysis and to implement it in routine forensic casework. Mitochondrial DNA offers a crucial advantage in this context: cells contain many more copies of mitochondrial DNA than nuclear DNA, and the molecule is far more resistant to degradation. Although mtDNA is maternally inherited and therefore lacks the discriminatory power to identify a single individual, it remains highly valuable for maternal lineage analysis, missing-person investigations, disaster victim identification, and cases where only degraded biological material is available. Traditionally, mtDNA typing relied on Sanger sequencing of the control region alone, a labor-intensive and costly approach with limited discriminatory power. Massively parallel sequencing technologies such as the Precision ID panel now allow entire mitochondrial genomes to be sequenced quickly and affordably, dramatically increasing both sensitivity and information content.

To establish the sensitivity of the workflow, the researchers prepared dilution series from NIST Standard Reference Material DNA and six reference samples, spanning an extraordinary range from 3 to 3,600 mitochondrial DNA copies per microliter. The results were striking. Complete mitochondrial genome sequences were consistently recovered in 99 percent of samples across the dilution series at DNA input levels as low as 47 mtDNA copies, demonstrating remarkable robustness under low-template conditions. Full profiles were obtained in half of the samples containing only 23 copies, and even some samples at 12 copies produced partial results. Statistical modeling using log-log linear regression revealed that library yield scaled nearly proportionally with mtDNA input in the NIST series, with a doubling of input DNA producing an approximately 2.16-fold increase in library output. Read depth increased linearly up to roughly 560 to 1,235 copies per reaction before plateauing due to saturation, underscoring the importance of accurate library quantification and normalization.

The heart of the study involved 100 volunteers who each provided 20 shed hairs collected from their own clothing, with 98 of them also contributing buccal swab samples for comparison. Microscopic examination identified the club-shaped roots characteristic of telogen hairs, and one hair per donor was selected for analysis. Quantification revealed that 98 of the 100 hair extracts met or exceeded the manufacturer-recommended input of 3,000 mtDNA copies, and both small and large mtDNA quantification targets were detected in every single sample. In contrast, 25 hairs contained no detectable nuclear DNA at all, and most of the remainder showed concentrations below 6.8 picograms per microliter with extensive degradation. The researchers also found that reducing the post-extraction volume from 50 to 30 microliters significantly increased both mtDNA copy number and sequencing read depth, a simple procedural change that boosted sensitivity without introducing contamination. Notably, donor variables such as age, gender, hair dye use, and washing habits showed no statistically significant association with mtDNA quantity or quality.

Comparing the mitochondrial genomes sequenced from 91 paired hair and buccal samples revealed fascinating subtleties of mitochondrial biology. Initial sequence concordance between hairs and their corresponding buccal references was 93.4 percent. However, when the team applied the interpretation guidelines of the International Society for Forensic Genetics and excluded low-confidence discrepancies associated with high strand bias, heteroplasmic variants, and sequencing artifacts, concordance rose to a perfect 100 percent. This finding is critically important for forensic practice: apparent mismatches between a hair and a reference sample frequently reflect technical limitations and tissue-specific heteroplasmy rather than true genetic differences. Heteroplasmy, the coexistence of multiple mitochondrial variants within an individual, arises from bottleneck effects and drift during tissue development, and previous research has shown that it can drift notably in hair. According to ISFG guidelines, a single mismatch or heteroplasmic difference should never be interpreted as evidence of exclusion, and this study provides empirical weight for that principle in the context of whole mitochondrial genome sequencing.

Reproducibility testing further strengthened the case for implementation. The researchers amplified and sequenced duplicate libraries from 43 telogen hair samples and 46 buccal samples, finding over 99 percent concordance of the software-classified true variants between replicates. The handful of discrepancies involved shifts between point and length heteroplasmy classifications, insertion-length differences at homopolymeric C-tracts, and low-frequency artifacts at known hotspots, none of which affected haplotype interpretation. Haplogroup assignments remained identical across all replicates and dilution levels, confirming that reduced template input did not compromise phylogenetic classification. The data analysis itself was also highly repeatable, with two independent runs of the Converge analysis software producing identical results across every measured parameter, from variant frequencies and read depths to strand bias metrics and variant state classifications.

Contamination control, a perennial concern in low-copy-number DNA work, was rigorously assessed. Of 16 negative controls processed during the study, none produced a complete mitochondrial DNA sequence. Six controls showed low-level mtDNA signal consisting of only two to eight variants, predominantly common polymorphisms, and none of these partial profiles corresponded to any of the analyzed samples, the NIST control, or laboratory personnel. Several variants in the controls were traced to primer binding regions, indicating primer variants rather than true biological signal. All work was conducted in a dedicated laboratory following ISFG and SWGDAM guidelines under the ISO 17025 validation framework, and every extraction, quantification, and sequencing batch included negative controls, providing a template for laboratories considering adoption of the method.

Perhaps most compelling for investigators is the performance of the validated workflow on real evidence. The team sequenced 11 archived DNA extracts from hairs collected in 2010 from concluded criminal cases, samples that had previously been deemed unsuitable for STR profiling because they contained no detectable or less than 7 picograms per microliter of nuclear DNA. Remarkably, these aged forensic samples yielded complete mitochondrial genome sequences with an average read depth of 1,037-fold, an exceptionally high coverage figure. Across the three fully reported casework samples, 102 variants were identified, 98 of which were confirmed as true variants with high confidence scores, and no nuclear mitochondrial DNA segments, or NUMTs, were detected to confound the analysis. The success demonstrates that even DNA extracts stored for over a decade can return informative whole mitochondrial genomes, offering hope for cold case reviews.

The study is not without caveats that laboratories must respect. A few amplicons, particularly those covering mitochondrial regions 8622 to 8796, 1317 to 1491, and 1948 to 2111, consistently underperformed across sample types, mirroring poor performance reported in earlier evaluations of the panel. Length heteroplasmy artifacts at homopolymeric stretches such as positions 309 and 11038 appeared with variant strand bias exceeding 0.7, requiring careful filtering. At very low template levels, below roughly 47 copies, stochastic effects increased the frequency of unlikely and unclear variant calls. The authors emphasize that reliable results from low-copy samples demand increased read allocation, replicate sequencing, manufacturer-recommended run thresholds, and stringent filtering of negative controls and NUMTs. The laboratory will continue using an input of 3,000 mtDNA copies for casework despite evidence that lower inputs can suffice, retaining a conservative margin for evidentiary work.

Taken together, the findings position whole mitochondrial genome sequencing of telogen hairs as a validated, sensitive, and contamination-controlled method ready for routine forensic implementation. With more than 85 percent of hair evidence in some laboratories currently unanalyzable by STR profiling, the ability to recover complete mitochondrial genomes from shed hairs with high read depth and near-perfect concordance when properly interpreted represents a meaningful expansion of the forensic toolkit. As massively parallel sequencing continues its migration from research laboratories into accredited casework units, hairs that once sat silently in evidence storage may soon speak, narrowing gaps in investigations, strengthening maternal lineage identifications, and giving cold cases a second chance at resolution.

Subject of Research: Validation of whole mitochondrial genome sequencing of shed telogen hairs for forensic casework

Article Title: Precision ID mtDNA Whole Genome Panel and sequencing of telogen hairs – perspectives for validation and implementation in casework

Article References: Sharafi Farzad, M., Claessens, F., Petersen, C. B., Pereira, V., Morling, N., & Mogensen, H. S. (2026). Precision ID mtDNA Whole Genome Panel and sequencing of telogen hairs – perspectives for validation and implementation in casework. International Journal of Legal Medicine. https://doi.org/10.1007/s00414-026-03992-x

Image Credits: AI Generated

DOI: 10.1007/s00414-026-03992-x

Keywords: mitochondrial DNA, telogen hairs, massively parallel sequencing, Precision ID mtDNA Whole Genome Panel, forensic casework, mtDNA heteroplasmy, low-template DNA, STR profiling, profile concordance, contamination control, ISFG guidelines, hair shaft evidence