1. Method development and production of an ambient-stable blood certified reference material for total mercury, methylmercury, and trace elementsKoichi Haraguchi, Mineshi Sakamoto, Hiromitsu Nagasaka, Milena Horvat, Ermira Begu, Polona Klemenčič, Adna Alilović Osolin, Masaaki Nakamura, 2026, original scientific article Abstract: Background. Certified reference materials (CRMs) for mercury speciation of biological fluids have hitherto been limited to frozen or cold-storage types, which impedes the participation of laboratories in tropical or remote regions. Existing CRMs often do not fully meet user needs, particularly regarding storage stability and concentration relevance. Results. Using pooled Japanese human blood, we developed a freeze-dried, ambient-stable blood CRM, with certified values of 6.16 μg L−1 for total mercury (THg) and 5.46 μg L−1 for methylmercury (MeHg, as Hg). These concentrations are congruent with median exposure levels in fish-consuming populations (e.g., small island states and riverine Amazonian communities) derived from global biomonitoring data. Homogeneity, stability, and traceability were validated through inter-laboratory comparisons and rigorous uncertainty assessment. Significance. This ambient-stable blood CRM broadens global access to high-quality QA/QC of mercury speciation, especially in regions lacking cold-chain infrastructure. It contributes to capacity building under the Minamata Convention and strengthens interlaboratory comparability. Registration in COMAR and international collaborative deployment are under active development, enhancing the global infrastructure for mercury biomonitoring.
Keywords: passive monitoring, methylmercury, blood samples, mercury detection, human biomonitoring, trace elements
Published in DiRROS: 04.05.2026; Views: 143; Downloads: 108
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2. Sustainable mercury monitoring using graphitic carbon nitride as a 2D binding layer in diffusive gradient thin filmsDmitrii Deev, Raghuraj S. Chouhan, Igor Živković, Ermira Begu, Ana Drinčić, Francisco Ruiz-Zepeda, Andraž Krajnc, Ivan Jerman, Roman Viter, Aleš Lapanje, Milena Horvat, 2026, original scientific article Abstract: Mercury (Hg) is a highly toxic and persistent environmental pollutant whose accurate monitoring remains challenging due to limitations in existing diffusive gradients in thin films (DGT) binding materials, including insufficient selectivity, complex synthesis, and sustainability concerns. In this study, we report the first application of graphitic carbon nitride (GCN) nanosheets as a metal-free and sustainable binding material for Hg²⁺ monitoring in DGT systems, addressing key limitations of current approaches. Few-layer GCN nanosheets were synthesized via thermal polymerization followed by protonation-assisted exfoliation and comprehensively characterized using X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM), atomic force microscopy (AFM), Raman spectroscopy, and scanning electron microscopy (SEM). These analyses confirmed the formation of a structurally stable, nitrogen-rich framework with abundant coordination sites. The GCN–agarose (AG–GCN) composite binding layer demonstrated high mercury binding efficiency (>90%) at environmentally relevant concentrations (2.5–10 ng/mL) and near-neutral pH, attributed to strong coordination between Hg²⁺ ions and electron-donating nitrogen sites within the heptazine structure. Compared to conventional DGT binding phases, the proposed system offers enhanced sustainability, metal-free composition, and strong affinity toward mercury, highlighting its potential for next-generation passive environmental monitoring and advanced nanomaterial-based sensing platforms.
Keywords: passive monitoring, graphitic carbon nitride nanosheets, adsorbent, mercury detection, nanosheets
Published in DiRROS: 04.05.2026; Views: 164; Downloads: 109
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3. Association of trace elements with polycystic ovary syndrome in women - a case-control studyTinkara Srnovršnik, Bojana Pinter, Milena Horvat, Janja Snoj Tratnik, Ingrid Falnoga, Darja Mazej, Ivan Verdenik, Irma Virant-Klun, 2025, original scientific article Abstract: Objectives: There are still limited or lacking data on the association of trace elements (TEs) with polycystic ovary syndrome (PCOS). This case–control study aimed to determine levels of essential TEs (manganese (Mn), copper (Cu), zinc (Zn), selenium (Se), molybdenum (Mo)) and non-essential TEs (arsenic (As), cadmium (Cd), mercury (Hg), lead (Pb)) in urine, whole blood, and serum to investigate a possible association with kidney and liver function, endocrine and metabolic parameters, and environmental and lifestyle sources of potential exposure and provide possible recommendations. Methods: In our case–control study, women with PCOS (n = 35) and healthy controls (n = 35) underwent clinical and ultrasonographic examination, filled in questionnaires targeting general, lifestyle, and environmental information, and provided fasting venous blood samples and first morning urine for biochemical, hormonal, and TE analysis. Multiple linear regression models were used to evaluate the association between TE levels and data obtained through questionnaires. Results: In women with PCOS, lower Mo levels in whole blood (p = 0.024) and serum (p = 0.011) and higher serum Cu levels (p = 0.026) were detected when compared to healthy controls. Results of this study show that amendments in Cu and Mo levels might be related to altered kidney and liver function and disrupted hormonal balance in PCOS women. Cu levels positively correlated with leukocyte count. There was a negative correlation of Mo levels with proteinuria and luteinizing hormone levels. Regarding liver function, Mo negatively correlated with urinary bilirubin levels, and there was a positive association with alanine and aspartate aminotransferase, respectively. Dietary supplement consumption and certain diet habits appeared to be important predictors of exposure to Cu (beef consumption) or Mo (cereal and boiled vegetable consumption) and modify Mo and Cu levels in women. Conclusions: Concentrations of the chemical elements Mo and Cu in biological samples of women appear to be related to PCOS and nutrition. To our knowledge, this is a novel finding for Mo. Additional research is needed to provide more insights into the causality of the PCOS relationship with Mo and Cu in humans.
Keywords: essential elements, non-essential elements, polycystic ovary syndrome, endocrine disruptors, hepatic damage, trace elements
Published in DiRROS: 16.04.2026; Views: 120; Downloads: 86
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4. The Exposure and Health Examination Survey mother-child(ren) cohort profile : applying the exposome to the comprehension of child’s health and development joining singletons and twins dataMohamed-Yassir Errahmani, Sara Maio, Sandra Baldacci, Neža Palir, Darja Mazej, Janja Snoj Tratnik, Ingrid Falnoga, Milena Horvat, Isabella Annesi-Maesano, 2025, original scientific article Abstract: he Exposure and Health Examination Survey (EXHES) cohort aims to elucidate the impact of environmental exposures (the external exposome) and their biological markers (the internal exposome) on childhood health conditions, asthma and allergies, obesity, and cognitive development in particular. Utilizing singletons and twins helped differentiate environmental effects from genetic influences due to the shared genetic background in twins. The EXHES cohort includes 2356 mother-child pairs across 10 European countries, comprising 1945 singletons and 411 twins, with data collected during the crucial first 1000 days of life. Data were gathered through epidemiological questionnaires and biomarkers, including blood, urine, hair, and breast milk from mothers, and cord blood, placenta, and cord tissues from children. Findings confirm that twin pregnancies are linked with increased risks of pregnancy complications, preterm birth, cesarean delivery, low birthweight, maternal health problems during pregnancy and a lower risk of macrosomia. Moreover, mothers of twins were more likely to have asthma, while higher maternal education was associated with a lower likelihood of twin births. The EXHES cohort provides a robust framework to be adopted in other studies for comparing singletons and twins to better understand how the exposome affects early child development and health outcomes. This approach offers new insights into the interplay between environmental and biological factors in shaping long-term health.
Keywords: environmental exposure, biomarkers
Published in DiRROS: 18.02.2026; Views: 373; Downloads: 204
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5. Human biomonitoring in support of the Minamata Convention : a case of phasing out dental amalgamVanja Usenik, Adna Alilović Osolin, Janja Snoj Tratnik, Marta Jagodic Hudobivnik, Darja Mazej, David Kocman, Davor Kontić, Milena Horvat, 2026, original scientific article Abstract: This study analysed urinary mercury (U-Hg) concentrations in 1412 Slovenian children across four human biomonitoring campaigns conducted between 2007 and 2024. Median U-Hg levels declined from 0.76 ng mL−1 (0.72 µg g−1 creatinine) in the 2007 PHIME cohort to 0.22 ng mL−1 (0.21 µg g−1 creatinine) in the 2018–2024 SLO-HBM-II cohort, paralleling a decrease in the prevalence (from 65 to 3%) and the average number of dental amalgam fillings in children. Multilevel mixed-effects models showed a consistent temporal decline in U-Hg that persisted after adjustment for demographic and environmental covariates. In contrast, the inclusion of the amalgam number substantially attenuated the time trend, indicating that reduced amalgam use likely contributed to the observed decrease. As amalgam prevalence fell, other sources of exposure, such as fish consumption, became relatively more prominent predictors of U-Hg, while children living in historically Hg-contaminated areas showed persistently higher levels. Although Slovenia had already phased down dental amalgam in children before ratifying the Minamata Convention, these long-term biomonitoring data illustrate how changes in exposure sources are reflected in internal Hg levels. The study demonstrates the value of repeated national HBM programmes for identifying dominant exposure pathways, investigating their evolution over time, and providing evidence relevant to effectiveness-evaluation frameworks under Article 22 of the Minamata Convention.
Keywords: concentrations in blood
Published in DiRROS: 10.02.2026; Views: 513; Downloads: 168
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6. Optimisation of a sample preparation method for the determination of multi-elemental compositions in human hair by triple quadrupole ICP-MS analysisAgneta Annika Runkel, Marta Jagodic Hudobivnik, Igor Živković, Polona Klemenčič, Darja Mazej, Milena Horvat, 2026, original scientific article Abstract: Monitoring toxic elements has a long tradition in Slovenia due to historical mining. More recently, attention has shifted to essential elements, since both deficiencies and excesses can harm health. Regular monitoring of (non-)essential elements supports risk assessment and policymaking. While urine and blood are common biomonitoring matrices, hair offers a non-invasive alternative that reflects exposure over several months, though standardised methodologies for hair analysis remain limited. This study aimed to develop and validate a sensitive and robust analytical method for the determination of 29 elements in human hair, addressing key challenges in sample preparation and contamination control. We developed a sensitive and robust method for the determination of 29 elements (Ag, Al, As, Ba, Ca, Cd, Co, Cr, Cu, Fe, Hg, K, Mg, Mn, Mo, Na, Ni, P, Pb, Rb, S, Sb, Se, Sn, Sr, Ti, U, V, and Zn) in 3 cm segments of human hair that involves a washing procedure with acetone and Milli-Q water, microwave digestion with 65% HNO3, and analysis with Triple Quadrupole Inductively Coupled Plasma Mass Spectrometry (ICP-MS/MS). Evaluation of preparation steps revealed stainless-steel scissors as a major contamination source. Glass digestion vessels were unsuitable for several elements due to high detection limits and relative standard deviations. The optimised method reduced analytical variability and improved sensitivity compared to published protocols. This validated method enables reproducible multi-elemental analysis in hair, highlights overlooked contamination risks, and is now applied in human biomonitoring studies to strengthen exposure assessment and standardisation efforts.
Keywords: determination of elements, optimization, human biomonitoring
Published in DiRROS: 27.01.2026; Views: 322; Downloads: 208
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7. Quantification of phthalate and DINCH metabolites in human urine and maternal breast milk : assessing maternal body burden and infant exposureAgneta Annika Runkel, Žan Rekar, Neja Kosirnik, Darja Mazej, Milena Horvat, Janja Snoj Tratnik, Tina Kosjek, 2026, original scientific article Abstract: Based on toxicological evidence, human exposure to phthalates (PHs) and diisononylcyclohexane-1,2-dicarboxylate (DINCH) may contribute to adverse health effects, especially during vulnerable developmental stages. To support the exposure assessment for this group of endocrine disruptors, we developed and validated a method for the analysis of 14 PH and 3 DINCH metabolites in human urine and maternal milk, applied the method in a pilot study, and identified crucial obstacles in the path of establishing maternal milk as a routine matrix in human biomonitoring. Urine and milk samples were extracted with solid-phase extraction (SPE) and QuEChERS salts, respectively, and analysed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The method accuracy was confirmed for urine samples via a certified standard reference material and the G-EQUAS intercomparison programme. We identified a need for sampling protocols, reference materials, and external method verification schemes in order to establish maternal milk as a routine matrix. Finally, the method was tested for its applicability in a pilot biomonitoring study on 30 paired urine and milk samples from lactating mothers, with medians ranging from Keywords: phthalate metabolites, DINCH, human biomonitoring
Published in DiRROS: 14.01.2026; Views: 319; Downloads: 205
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8. Green synthesis of a magnetite/graphitic carbon nitride 2D nanocomposite for efficient ▫$Hg^{2+}$▫ remediationRaghuraj S. Chouhan, Jan Gačnik, Igor Živković, Sreekanth Vijayakumaran Nair, Nigel Willy Van de Velde, Alenka Vesel, Primož Šket, Sonu Gandhi, Ivan Jerman, Milena Horvat, 2023, original scientific article Abstract: Novel nanoadsorbents demonstrate the potential to efficiently eliminate harmful substances, such as Hg2+, from the environment while preserving ecological balance. However, the search for environmentally-friendly nanomaterials as ideal adsorbents, as well as the development of suitable synthesis protocols, remains a challenge. This study presents an effective thermal-sonication technique for producing unique de novo tris-s-triazine carbon nitride nanosheets decorated with magnetite nanoparticles (M-g-CN). The structural integrity and chemical properties of the M-g-CN nanocomposite were extensively characterized using a battery of analytical instruments, including FTIR, SEM, TEM, XRD, XPS, AFM, Raman, and NMR. The obtained data, along with the analysis results, are discussed in detail. The novel synthesis method yields a high percentage (97.7%) of stable, highly selective, and reusable M-g-CN (40 mg mL−1). The resulting M-g-CN effectively binds Hg2+, with binding efficiencies of 96.0%, 97.5%, 98.2%, and 99.4% for Hg2+ concentrations of 50 pg mL−1 in marine, stream, precipitation, and ultra-pure matrices, respectively. Also, the magnetite-decorated particles can be easily retrieved using an applied magnetic field. This greener and scalable synthesis method does not require harsh chemicals, making it cost-effective, eco-friendly, and non-toxic compared to other technologies such as carbon filters, ion exchange resins, chemical precipitation, membrane filtration, electrochemical methods, and biological remediation. Overall, the synthesized M-g-CN exhibits wide-ranging potential applications, particularly as a green adsorbent in passive samplers or materials for remediation purposes.
Keywords: mercury, elimination, nanoadsorbents, nanocomposites
Published in DiRROS: 05.01.2026; Views: 450; Downloads: 216
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9. Recent progress and advancement in detecting Methylmercury using a battery of biosensors and biomolecular-based techniques : An updated overviewAllwin Mabes Raj, Raghuraj S. Chouhan, Aljoša Košak, Milena Horvat, Aleksandra Lobnik, Tomaž Rijavec, Aleš Lapanje, 2025, original scientific article Abstract: Methylmercury (MeHg) represents the most toxic form of mercury, owing to its ability to permeate both the blood-brain and placental barriers, leading to bioaccumulation in organisms. In the marine food web, MeHg concentrations can reach levels millions of times higher than those found in the surrounding environment, posing significant ecological and human health risks. This review provides a comprehensive overviews and critical evaluation of the available biosensor detection platforms for the detection of MeHg, with a focus on their performance based on key parameters such as (i) sensitivity, (ii) selectivity, (iii) response time, and (iv) adaptability to diverse environmental matrices. We examine recent advancements in MeHg biosensing technologies, emphasizing innovative approaches that surpass current methodologies regarding detection limits, reversibility, response time, and operational stability. Furthermore, we present an in-depth discussion on future directions for the development of in situ MeHg detection platforms, with potential applications in both biomedical and environmental monitoring. The review concludes by outlining the challenges and opportunities for advancing MeHg sensing technologies to enhance real-time detection in aqueous environments.
Published in DiRROS: 15.12.2025; Views: 1032; Downloads: 178
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10. Nontargeted urinary profiling strategy for endocrine-disrupting chemicals in women with ovarian malignanciesHelena Plešnik, Žan Rekar, Stefanela Stevanović, Irma Virant-Klun, Senka Imamović-Kumalić, Mateja Sladič, Darja Mazej, Janja Snoj Tratnik, Milena Horvat, Tina Kosjek, 2025, original scientific article Abstract: Endocrine-disrupting chemicals (EDCs), includingknown and unknown parent compounds, their metabolites, andtransformation products, are pervasive in daily life, posingincreasing risks to human health and the environment. Thisstudy employed a high-resolution mass spectrometry-basednontargeted screening approach, integrating polar (HILIC) andreversed-phase separations to expand the chemical space coverageand, supported by open-science tools and resources, evaluatedurinary chemical profiles to assess internal EDC exposure. Among106 annotated biomarkers of exposure, six exhibited significantlyhigher normalized intensities in patients with ovarian malignanciescompared to healthy controls (p < 0.05). This suggests theirgreater exposure to phthalates (diethylhexyl phthalate and diethylphthalate), pesticides (metolachlor metabolite and 4-nitrophenol), a UV filter (benzophenone-1), and an industrial byproduct (4-methyl-2-nitrophenol). These compounds may interfere with hormonal regulation, potentially contributing to cancer development.While these findings highlight potential differences in internal EDC exposure, the study primarily demonstrates the applicability ofnontargeted urinary profiling for chemical exposure assessment. By providing new insights into EDCs burden and its pathologicalimplications, this work contributes to advancing next-generation chemical risk assessment within the European Partnership for theAssessment of Risks from Chemicals initiative and supports the development of preventive strategies to mitigate environmentalcancer risks
Keywords: ovarian cancer, biomarkers, exposure, nontargeted screening, high-resolution mass spectrometry, analytical coverage, reversed-phase, HILIC
Published in DiRROS: 02.12.2025; Views: 632; Downloads: 317
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