HRMS-based suspect screening of pharmaceuticals and their transformation products in multiple environmental compartments: An alternative to target analysis?

The comprehensive monitoring of pharmaceutically active compounds (PhACs) in the environment is challenging given the myriad of substances continuously discharged, the increasing number of new compounds being produced (and released), or the variety of the associated human metabolites and transformation products (TPs). Approaches such as high-resolution mass spectrometry (HRMS)-based suspect analysis have emerged to overcome the drawbacks of classical target analytical methods, e.g., restricted chemical coverage. In this study, we assess the readiness of HRMS-based suspect screening to replace or rather complement target methodologies by comparing the performance of both approaches in terms of i) detection of PhACs in various environmental samples (water, sediments, biofilm, fish plasma, muscle and liver) in a field study; ii) PhACs (semi)quantification and iii) prediction of their environmental risks. Our findings revealed that target strategies alone significantly underestimate the variety of PhACs potentially impacting the environment. However, relying solely on suspect strategies can misjudge the presence and risk of low-level but potentially risky PhACs. Additionally, semiquantitative approaches, despite slightly overestimating concentrations, can provide a realistic overview of PhACs concentrations. Hence, it is recommended to adopt a combined strategy that first evaluates suspected threats and subsequently includes the relevant ones in the established target methodologies.

Efficient removal of toxicity associated to wastewater treatment plant effluents by enhanced Soil Aquifer Treatment.

The regeneration of wastewater has been recognized as an effective strategy to counter water scarcity. Nonetheless, Wastewater Treatment Plant (WWTP) effluents still contain a wide range of contaminants of emerging concern (CECs) even after water depuration. Filtration through Soil Aquifer Treatment (SAT) systems has proven efficient for CECs removal although the attenuation of their associated biological effects still remains poorly understood. To evaluate this, three pilot SAT systems were monitored, two of them enhanced with different reactive barriers. SATs were fed with secondary effluents during two consecutive campaigns. Fifteen water samples were collected from the WWTP effluent, below the barriers and 15 m into the aquifer. The potential attenuation of effluent-associated biological effects by SATs was evaluated through toxicogenomic bioassays using zebrafish eleutheroembryos and human hepatic cells. Transcriptomic analyses revealed a wide range of toxic activities exerted by the WWTP effluents that were reduced by more than 70% by SAT. Similar results were observed when HepG2 hepatic cells were tested for cytotoxic and dioxin-like responses. Toxicity reduction appeared partially determined by the barrier composition and/or SAT managing and correlated with CECs removal. SAT appears as a promising approach to efficiently reduce effluent-associated toxicity contributing to environmental and human health preservation.

Blurred lines: Crossing the boundaries between the chemical exposome and the metabolome.

The aetiology of every human disease lies in a combination of genetic and environmental factors, each contributing in varying proportions. While genomics investigates the former, a comparable holistic paradigm was proposed for environmental exposures in 2005, marking the onset of exposome research. Since then, the exposome definition has broadened to include a wide array of physical, chemical, and psychosocial factors that interact with the human body and potentially alter the epigenome, the transcriptome, the proteome, and the metabolome. The chemical exposome, deeply intertwined with the metabolome, includes all small molecules originating from diet as well as pharmaceuticals, personal care and consumer products, or pollutants in air and water. The set of techniques to interrogate these exposures, primarily mass spectrometry and nuclear magnetic resonance spectroscopy, are also extensively used in metabolomics. Recent advances in untargeted metabolomics using high resolution mass spectrometry have paved the way for the development of methods able to provide in depth characterisation of both the internal chemical exposome and the endogenous metabolome simultaneously. Herein we review the available tools, databases, and workflows currently available for such work, and discuss how these can bridge the gap between the study of the metabolome and the exposome.

Integrating different tools and technologies to advance drinking water quality exposure assessments.

Contaminants in drinking water are a major contributor to the human exposome and adverse health effects. Assessing drinking water exposure accurately in health studies is challenging, as several of the following study design domains should be addressed as adequately as possible. In this paper, we identify the domains Time, Space, Data Quality, Data Accessibility, economic considerations of Study Size, and Complex Mixtures. We present case studies for three approaches or technologies that address these domains differently in the context of exposure assessment of drinking water quality: regulated contaminants in monitoring databases, high-resolution mass spectrometry (HRMS)-based wide-scope chemical analysis, and effect-based bioassay methods. While none of these approaches address all the domains sufficiently, together they have the potential to carry out exposure assessments that would complement each other and could advance the state-of-science towards more accurate risk analysis. The aim of our study is to give researchers investigating health effects of drinking water quality the impetus to consider how their exposure assessments relate to the above-mentioned domains and whether it would be worthwhile to integrate the advanced technologies presented into planned risk analyses. We highly suggest this three-pronged approach should be further evaluated in health risk analyses, especially epidemiological studies concerning contaminants in drinking water. The state of the knowledge regarding potential benefits of these technologies, especially when applied in tandem, provides more than sufficient evidence to support future research to determine the implications of combining the approaches described in our case studies in terms of protection of public health.

Comprehensive profiling and semi-quantification of exogenous chemicals in human urine using HRMS-based strategies.

Chemicals infiltrate our daily experiences through multiple exposure pathways. Human biomonitoring (HBM) is routinely used to comprehensively understand these chemical interactions. Historically, HBM depended on targeted screening methods limited to a relatively small set of chemicals with triple quadrupole instruments typically. However, recent advances in high-resolution mass spectrometry (HRMS) have facilitated the use of broad-scope target, suspect, and non-target strategies, enhancing chemical exposome characterization within acceptable detection limits. Despite these advancements, establishing robust and efficient sample treatment protocols is still essential for trustworthy broad-range chemical analysis. This study sought to validate a methodology leveraging HRMS-based strategies for accurate profiling of exogenous chemicals and related metabolites in urine samples. We evaluated five extraction protocols, each encompassing various chemical classes, such as pharmaceuticals, plastic additives, personal care products, and pesticides, in terms of their extraction recoveries, linearity, matrix effect, sensitivity, and reproducibility. The most effective protocol was extensively validated and subsequently applied to 10 real human urine samples using wide-scope target analysis encompassing over 2000 chemicals. We successfully identified and semi-quantified a total of 36 chemicals using an ionization efficiency-based model, affirming the methodology's robust performance. Notably, our results dismissed the need for a deconjugation step, a typically labor-intensive and time-consuming process.

Exploring the Occurrence of Organic Contaminants in Human Semen through an Innovative LC-HRMS-Based Methodology Suitable for Target and Nontarget Analysis.

Understanding the potential impact of organic contaminants on male fertility is crucial, yet limited studies have examined these chemicals in semen, with most focusing on urine and blood. To address this gap, we developed and validated a robust LC-HRMS methodology for semen analysis, with a focus on polar and semipolar chemicals. Our methodology enables the quantitative (or semiquantitative) analysis of >2000 chemicals being compatible with suspect and nontarget strategies and providing unprecedented insights into the occurrence and potential bioaccumulation of diverse contaminants in this matrix. We comprehensively analyzed exogenous organic chemicals and associated metabolites in ten semen samples from Spanish participants collected in an area with a large presence of the chemical industry included in the LED-FERTYL Spanish study cohort. This investigation revealed the presence of various contaminants in semen, including plastic additives, PFAS, flame retardants, surfactants, and insecticides. Notably, prevalent plastic additives such as phthalic acid esters and bisphenols were identified, indicating potential health risks. Additionally, we uncovered previously understudied chemicals like the tire additive 2-mercaptobenzothiazole and specific organophosphate flame retardants. This study showcases the potential of our methodology as a valuable tool for large-scale cohort studies, providing insights into the association between contaminant exposure and the risk of male fertility impairments.

What is in the fish? Collaborative trial in suspect and non-target screening of organic micropollutants using LC- and GC-HRMS.

A collaborative trial involving 16 participants from nine European countries was conducted within the NORMAN network in efforts to harmonise suspect and non-target screening of environmental contaminants in whole fish samples of bream (Abramis brama). Participants were provided with freeze-dried, homogenised fish samples from a contaminated and a reference site, extracts (spiked and non-spiked) and reference sample preparation protocols for liquid chromatography (LC) and gas chromatography (GC) coupled to high resolution mass spectrometry (HRMS). Participants extracted fish samples using their in-house sample preparation method and/or the protocol provided. Participants correctly identified 9-69 % of spiked compounds using LC-HRMS and 20-60 % of spiked compounds using GC-HRMS. From the contaminated site, suspect screening with participants' own suspect lists led to putative identification of on average ∼145 and ∼20 unique features per participant using LC-HRMS and GC-HRMS, respectively, while non-target screening identified on average ∼42 and ∼56 unique features per participant using LC-HRMS and GC-HRMS, respectively. Within the same sub-group of sample preparation method, only a few features were identified by at least two participants in suspect screening (16 features using LC-HRMS, 0 features using GC-HRMS) and non-target screening (0 features using LC-HRMS, 2 features using GC-HRMS). The compounds identified had log octanol/water partition coefficient (KOW) values from -9.9 to 16 and mass-to-charge ratios (m/z) of 68 to 761 (LC-HRMS and GC-HRMS). A significant linear trend was found between log KOW and m/z for the GC-HRMS data. Overall, these findings indicate that differences in screening results are mainly due to the data analysis workflows used by different participants. Further work is needed to harmonise the results obtained when applying suspect and non-target screening approaches to environmental biota samples.

MR dynamic-susceptibility-contrast perfusion metrics in the presurgical discrimination of adult solitary intra-axial cerebellar tumors.

OBJECTIVES: Adult solitary intra-axial cerebellar tumors are uncommon. Their presurgical differentiation based on neuroimaging is crucial, since management differs substantially. Comprehensive full assessment of MR dynamic-susceptibility-contrast perfusion-weighted imaging (DSC-PWI) may reveal key differences between entities. This study aims to provide new insights on perfusion patterns of these tumors and to explore the potential of DSC-PWI in their presurgical discrimination. METHODS: Adult patients with a solitary cerebellar tumor on presurgical MR and confirmed histological diagnosis of metastasis, medulloblastoma, hemangioblastoma, or pilocytic astrocytoma were retrospectively retrieved (2008-2023). Volumetric segmentation of tumors and normal-appearing white matter (for normalization) was semi-automatically performed on CE-T1WI and coregistered with DSC-PWI. Mean normalized values per patient tumor-mask of relative cerebral blood volume (rCBV), percentage of signal recovery (PSR), peak height (PH), and normalized time-intensity curves (nTIC) were extracted. Statistical comparisons were done. Then, the dataset was split into training (75%) and test (25%) cohorts and a classifier was created considering nTIC, rCBV, PSR, and PH in the model. RESULTS: Sixty-eight patients (31 metastases, 13 medulloblastomas, 13 hemangioblastomas, and 11 pilocytic astrocytomas) were included. Relevant differences between tumor types' nTICs were demonstrated. Hemangioblastoma showed the highest rCBV and PH, pilocytic astrocytoma the highest PSR. All parameters showed significant differences on the Kruskal-Wallis tests (p < 0.001). The classifier yielded an accuracy of 98% (47/48) in the training and 85% (17/20) in the test sets. CONCLUSIONS: Intra-axial cerebellar tumors in adults have singular and significantly different DSC-PWI signatures. The combination of perfusion metrics through data-analysis rendered excellent accuracies in discriminating these entities. CLINICAL RELEVANCE STATEMENT: In this study, the authors constructed a classifier for the non-invasive imaging presurgical diagnosis of adult intra-axial cerebellar tumors. The resultant tool can be a support for decision-making in the clinical practice and enables optimal personalized patient management. KEY POINTS: • Adult intra-axial cerebellar tumors exhibit specific, singular, and statistically significant different MR dynamic-susceptibility-contrast perfusion-weighted imaging (DSC-PWI) signatures. • Data-analysis, applied to MR DSC-PWI, could provide added value in the presurgical diagnosis of solitary cerebellar metastasis, medulloblastoma, hemangioblastoma, and pilocytic astrocytoma. • A classifier based on DSC-PWI metrics yields excellent accuracy rates and could be used as a support tool for radiologic diagnosis with clinician-friendly displays.

Tumoral and normal brain tissue extraction protocol for wide-scope screening of organic pollutants.

Little is known about the presence of organic pollutants in human brain (and even less in brain tumors). In this regard, it is necessary to develop new analytical protocols capable of identifying a wide range of exogenous chemicals in this type of samples (by combining target, suspect and non-target strategies). These methodologies should be robust and simple. This is particularly challenging for solid samples, as reliable extraction and clean-up techniques should be combined to obtain an optimal result. Hence, the present study focuses on the development of an analytical methodology that allows the screening of a wide range of organic chemicals in brain and brain tumor samples. This protocol was based on a solid-liquid extraction based on bead beating, solid-phase extraction clean-up with multi-layer mixed-mode cartridges, reconstitution and LC-HRMS analysis. To evaluate the performance of the extraction methodology, a set of 66 chemicals (e.g., pharmaceuticals, biocides, or plasticizers, among others) with a wide range of physicochemical properties was employed. Quality control parameters (i.e., linear range, sensitivity, matrix effect (ME%), and recoveries (R%)) were calculated and satisfactory results were obtained for them (e.g., R% within 60-120% for 32 chemicals, or ME% higher than 50% (signal suppression) for 79% of the chemicals).

Target analysis and suspect screening of UV filters, parabens and other chemicals used in personal care products in human cord blood: Prenatal exposure by mother-fetus transfer.

Prenatal exposure to certain organic chemicals like pesticides and phenols has been lifelong associated with birth outcomes and health disorders. Many personal care product (PCP) ingredients have similar properties or structures to those chemicals. Previous studies have documented the occurrence of UV filters (UVFs) and paraben preservatives (PBs) in the placenta, but observational studies concerning PCPs chemicals and foetal exposure are particularly scarce. Thus, this work aimed to assess the presence of a wide range of PCPs chemicals using target and suspect screening in the umbilical cord blood of new born babies to evaluate their potential transfer to the fetus. To do so, we analysed 69 umbilical cord blood plasma samples from a mother-child cohort from Barcelona (Spain). We quantified 8 benzophenone-type UVFs and their metabolites, and 4 PBs using validated analytical methodologies based on target screening using liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Then, we screened for additional 3246 substances using high-resolution mass spectrometry (HRMS) and advanced suspect analysis strategies. Six UVFs and three parabens were detected in the plasma with frequencies between 1.4% and 17.4% and concentrations up to 53.3 ng/mL (benzophenone-2). Thirteen additional chemicals were tentatively identified in the suspect screening, and ten were further confirmed with the corresponding standards. Among them, we found the organic solvent N-methyl-2-pyrrolidone, the chelating agent 8-hydroxyquinoline, and the antioxidant 2,2'-methylenebis(4-methyl-6-tert-butylphenol), which have been demonstrated to display reproductive toxicity. UVFs and PBs presence in the umbilical cord blood demonstrates mother-fetus transfer through the placental barrier and prenatal exposure to these PCPs chemicals, which may lead to adverse effects in the early stages of fetal development. Considering the small cohort used in this study, the reported results should be interpreted as a preliminary reference for the background umbilical cord transfer levels of the target PCPs chemicals. Further research is needed to determine the long-term consequences of prenatal exposure to PCPs chemicals.

Chemicals of emerging concern in coastal aquifers: Assessment along the land-ocean interface.

Submarine Groundwater Discharge (SGD) is recognized as a relevant source of pollutants to the sea, but little is known about its relevance as a source of chemicals of emerging concern (CECs). Here, both the presence and distribution of a wide range of CECs have been evaluated in the most comprehensive manner to date, in a well-characterized Mediterranean coastal aquifer near Barcelona (Spain). Samples from coastal groundwater and seawater allowed for the unique spatial characterization of the pollutants present in the land-ocean interface, an outstanding research gap that required attention. The main goals were (1) to determine CECs in the aquifer, so as to evaluate the SGD as a relevant source of marine pollution, and (2) to identify new tracers to improve our understanding of SGD dynamics. To this end, 92 CECs were located in the aquifer by using wide-scope analytical target methodologies (>2000 chemicals). Among them, the perfluoroalkyl and polyfluoroalkyl substances (PFAS), along with the pharmaceuticals carbamazepine and topiramate, were revealed to be good markers for tracing anthropogenic contamination in ground- and seawater, in concrete situations (e.g., highly contaminated sites). Additionally, non-target analysis expanded the number of potential tracers, making it a promising tool for identifying both the source and the fate of pollutants.

Occurrence and fate of contaminants of emerging concern and their transformation products after uptake by pak choi (Brassica rapa subsp. chinensis).

Recovery and reuse of nutrients is a major challenge in agriculture. A new process contributing to a circular economy is the anaerobic digestion of food waste, which is a sustainable way of recycling nutrients as the digestate can be used as fertiliser in agriculture and horticulture. However, the digestate may be polluted with contaminants of emerging concern (CECs) that can be circulated back into the food chain, posing a risk to the environment and human health. In this work, the nutrient solution was spiked with 18 selected CECs frequently detected in food waste biogas facilities, and subsequent uptake and fate of these CECs were evaluated in pak choi grown in two different nutrient solutions (mineral and organic). All spiked compounds except two (propylparaben, fenbendazole) were taken up by pak choi plants, with perfluorobutanoic acid (PFBA) and sertraline displaying the highest concentrations (270 and 190 µg/kg fresh weight, respectively). There were no statistically significant differences in uptake between mineral and organic nutrient solutions. Uptake of per- and polyfluoroalkyl substances (PFAS) was negatively correlated with perfluorocarbon chain length and dependent on the functional group (r = -0.73). Sixteen transformation products (TPs) were tentatively identified using suspect screening, most of which were Phase II or even Phase III metabolites. Six of these TPs were identified for the first time in plant metabolism and their metabolic pathways were considered.

The NORMAN Suspect List Exchange (NORMAN-SLE): facilitating European and worldwide collaboration on suspect screening in high resolution mass spectrometry.

Background: The NORMAN Association (https://www.norman-network.com/) initiated the NORMAN Suspect List Exchange (NORMAN-SLE; https://www.norman-network.com/nds/SLE/) in 2015, following the NORMAN collaborative trial on non-target screening of environmental water samples by mass spectrometry. Since then, this exchange of information on chemicals that are expected to occur in the environment, along with the accompanying expert knowledge and references, has become a valuable knowledge base for "suspect screening" lists. The NORMAN-SLE now serves as a FAIR (Findable, Accessible, Interoperable, Reusable) chemical information resource worldwide. Results: The NORMAN-SLE contains 99 separate suspect list collections (as of May 2022) from over 70 contributors around the world, totalling over 100,000 unique substances. The substance classes include per- and polyfluoroalkyl substances (PFAS), pharmaceuticals, pesticides, natural toxins, high production volume substances covered under the European REACH regulation (EC: 1272/2008), priority contaminants of emerging concern (CECs) and regulatory lists from NORMAN partners. Several lists focus on transformation products (TPs) and complex features detected in the environment with various levels of provenance and structural information. Each list is available for separate download. The merged, curated collection is also available as the NORMAN Substance Database (NORMAN SusDat). Both the NORMAN-SLE and NORMAN SusDat are integrated within the NORMAN Database System (NDS). The individual NORMAN-SLE lists receive digital object identifiers (DOIs) and traceable versioning via a Zenodo community (https://zenodo.org/communities/norman-sle), with a total of > 40,000 unique views, > 50,000 unique downloads and 40 citations (May 2022). NORMAN-SLE content is progressively integrated into large open chemical databases such as PubChem (https://pubchem.ncbi.nlm.nih.gov/) and the US EPA's CompTox Chemicals Dashboard (https://comptox.epa.gov/dashboard/), enabling further access to these lists, along with the additional functionality and calculated properties these resources offer. PubChem has also integrated significant annotation content from the NORMAN-SLE, including a classification browser (https://pubchem.ncbi.nlm.nih.gov/classification/#hid=101). Conclusions: The NORMAN-SLE offers a specialized service for hosting suspect screening lists of relevance for the environmental community in an open, FAIR manner that allows integration with other major chemical resources. These efforts foster the exchange of information between scientists and regulators, supporting the paradigm shift to the "one substance, one assessment" approach. New submissions are welcome via the contacts provided on the NORMAN-SLE website (https://www.norman-network.com/nds/SLE/). Supplementary Information: The online version contains supplementary material available at 10.1186/s12302-022-00680-6.

Are preserved coastal water bodies in Spanish Mediterranean basin impacted by human activity? Water quality evaluation using chemical and biological analyses.

The Spanish Mediterranean basin is particularly susceptible to climate change and human activities, making it vulnerable to the influence of anthropogenic contaminants. Therefore, conducting comprehensive and exhaustive water quality assessment in relevant water bodies of this basin is pivotal. In this work, surface water samples from coastal lagoons or estuaries were collected across the Spanish Mediterranean coastline and subjected to target and suspect screening of 1,585 organic micropollutants by liquid chromatography coupled to ion mobility separation and high resolution mass spectrometry. In total, 91 organic micropollutants could be confirmed and 5 were tentatively identified, with pharmaceuticals and pesticides being the most prevalent groups of chemicals. Chemical analysis data was compared with data on bioanalysis of those samples (recurrent aryl hydrocarbon receptor (AhR) activation, and estrogenic receptor (ER) inhibition in wetland samples affected by wastewater streams). The number of identified organic contaminants containing aromatic rings could explain the AhR activation observed. For the ER antagonistic effects, predictions on estrogenic inhibition potency for the detected compounds were used to explain the activities observed. The integration of chemical analysis with bioanalytical observations allowed a comprehensive overview of the quality of the water bodies under study.

Development and Application of Liquid Chromatographic Retention Time Indices in HRMS-Based Suspect and Nontarget Screening.

There is an increasing need for comparable and harmonized retention times (tR) in liquid chromatography (LC) among different laboratories, to provide supplementary evidence for the identity of compounds in high-resolution mass spectrometry (HRMS)-based suspect and nontarget screening investigations. In this study, a rigorously tested, flexible, and less system-dependent unified retention time index (RTI) approach for LC is presented, based on the calibration of the elution pattern. Two sets of 18 calibrants were selected for each of ESI+ and ESI-based on the maximum overlap with the retention times and chemical similarity indices from a total set of 2123 compounds. The resulting calibration set, with RTI set to range between 1 and 1000, was proposed as the most appropriate RTI system after rigorous evaluation, coordinated by the NORMAN network. The validation of the proposed RTI system was done externally on different instrumentation and LC conditions. The RTI can also be used to check the reproducibility and quality of LC conditions. Two quantitative structure-retention relationship (QSRR)-based models were built based on the developed RTI systems, which assist in the removal of false-positive annotations. The applicability domains of the QSRR models allowed completing the identification process with higher confidence for substances within the domain, while indicating those substances for which results should be treated with caution. The proposed RTI system was used to improve confidence in suspect and nontarget screening and increase the comparability between laboratories as demonstrated for two examples. All RTI-related calculations can be performed online at http://rti.chem.uoa.gr/.

Development of a sensitive analytical method for the simultaneous analysis of Benzophenone-type UV filters and paraben preservatives in umbilical cord blood.

UV filters and parabens are compounds used in large quantities in modern societies and have become ubiquitous in the environment. They are considered compounds of emerging concern due to the unwanted effects they cause in the environment and their bioaccumulation potential in humans. Considering their endocrine disrupting activity and their so far unknown effects in newborns, a continuous monitoring of these substances is required. In this work, we developed and validated a new sensitive methodology for the analysis of 8 UV filters and metabolites, and 4 parabens in umbilical cord blood samples. The method consisted of a liquid-liquid extraction and phase separation by freezing. Then, the organic extract was further analyzed at alkaline pH using liquid chromatography coupled to tandem-mass spectrometry (LC-MS/MS) using a QqLIT hybrid mass spectrometer as analyzer. The low limits of detection achieved (0.01-0.42 ng/mL) allowed the reliable simultaneous quantification of UV filters and parabens in this complex biological matrix.•Simple, fast and sensitive analysis of UV filters and parabens in cord blood samples.•First simultaneous analysis of UV filters and parabens in cord blood.•Allows the evaluation of perinatal transfer of UV filters and parabens from the mother to the fetus.

Inter-laboratory mass spectrometry dataset based on passive sampling of drinking water for non-target analysis.

Non-target analysis (NTA) employing high-resolution mass spectrometry is a commonly applied approach for the detection of novel chemicals of emerging concern in complex environmental samples. NTA typically results in large and information-rich datasets that require computer aided (ideally automated) strategies for their processing and interpretation. Such strategies do however raise the challenge of reproducibility between and within different processing workflows. An effective strategy to mitigate such problems is the implementation of inter-laboratory studies (ILS) with the aim to evaluate different workflows and agree on harmonized/standardized quality control procedures. Here we present the data generated during such an ILS. This study was organized through the Norman Network and included 21 participants from 11 countries. A set of samples based on the passive sampling of drinking water pre and post treatment was shipped to all the participating laboratories for analysis, using one pre-defined method and one locally (i.e. in-house) developed method. The data generated represents a valuable resource (i.e. benchmark) for future developments of algorithms and workflows for NTA experiments.