Application of a novel prioritisation strategy using non-target screening for evaluation of temporal trends (1969-2017) of contaminants of emerging concern (CECs) in archived lynx muscle tissue samples

Most environmental monitoring studies of contaminants of emerging concern (CECs) focus on aquatic species and target specific classes of CECs. Even with wide-scope target screening methods, relevant CECs may be missed. In this study, non-target screening (NTS) was used for tentative identification of potential CECs in muscle tissue of the terrestrial top predator Eurasian lynx (Lynx lynx). Temporal trend analysis was applied as a prioritisation tool for archived samples, using univariate statistical tests (Mann-Kendall and Spearman rank). Pooled lynx muscle tissue collected from 1969 to 2017 was analysed with an eight-point time series using a previously validated screening workflow. Following peak detection, peak alignment, and blank subtraction, 12,941 features were considered for statistical analysis. Prioritisation by time-trend analysis detected 104 and 61 features with statistically significant increasing and decreasing trends, respectively. Following probable molecular formula assignment and elucidation with MetFrag, two compounds with increasing trends, and one with a decreasing trend, were tentatively identified. These results show that, despite low expected concentration levels and high matrix effects in terrestrial species, it is possible to prioritise CECs in archived lynx samples using NTS and univariate statistical approaches

Development of novel methodologies for the identification of unknown compounds in the environment employing non-target screening and high-resolution mass spectrometry

Τα κέντρα επεξεργασίας λυμάτων (ΚΕΛ) δεν απομακρύνουν αποτελεσματικά τους αναδυόμενους ρύπους. Έτσι, οι αναδυόμενοι ρύποι εισάγονται στο υδατικό περιβάλλον και σχηματίζουν πολύπλοκα μίγματα, τα οποία περιέχουν δεκάδες χιλιάδες χημικές ουσίες. Η ανάλυση αυτών των πολύπλοκων δειγμάτων με τεχνικές υψηλής απόδοσης, όπως υγρoχρωματογραφία συζευγμένη με φασματομετρία μαζών υψηλής διακριτικής ικανότητας, παράγουν ένα μεγάλο αριθμό σημάτων. Για την επιτυχή μετάφραση των πολύπλοκων αυτών δεδομένων αλλά και για να επιτευχθεί ένα ολιστικό πρόγραμμα παρακολούθησης του περιβάλλοντος, απαιτείται ολόπλευρη χημική ανάλυση με εφαρμογή στοχευμένης σάρωσης, σάρωσης ύποπτων ενώσεων και μη στοχευμένης σάρωσης. Ο σκοπός της Διατριβής είναι η ανάπτυξη εργαλείων ολόπλευρης χημικής ανάλυσης και η εφαρμογή τους σε σημαντικά Ευρωπαΐκά οικοσυστήματα όπως η λεκάνη απορροής του Δούναβη και η Μάυρη Θάλασσα. Στα πλαίσια του σκοπού αυτού, στο Κεφάλαιο 1 εισάγονται οι αναδυόμενοι ρύποι προτεραιότητας και οι τεχνικές ταυτοποίησης τους, ακολουθούμενοι από λεπτoμερή περιγραφή των στόχων της Διατριβής στο Κεφάλαιο 2. Στο Κεφάλαιο 3 περιγράφεται μια πορεία μη στοχευμένης σάρωσης για την προτεραιοποίηση ουσιών που παρουσιάζουν απότομη μεταβολή στην συγκέντρωση ως προς το χρόνο. Η πορεία αυτή χρησιμοποιήθηκε για την εύρεση φαινομένων απευθείας ρίψης ουσιών στο δίκτυο του ΚΕΛ της Αθήνας. Το Κεφάλαιο 4 περιγράφει την ίδρυση ενός παγκόσμιου δικτύου έγκαιρης προειδοποίησης για την αξιολόγηση της χωρικής και χρονικής κατανομής νέων αναδυόμενων ρύπων. Εξέλιξη αυτού του δικτύου είναι ένα λογισμικό αποθήκευσης δεδομένων LC-HRMS με δυνατότητα εφαρμογής ευρείας αυτόματης σάρωσης ύποπτων ενώσεων, που ενσωματώνει όλα τα έργαλεία που χρησιμοποιούνται στις HRMS μεθόδους (Κεφάλαιο 5). Το λογισμικό χρησιμοποιήθηκε για την ανίχνευση αναδυόμενων ρύπων και χημικών ουσιών της βάσης REACH (i) σε δείγματα από τη Μαύρη θάλασσα (ζωντανοί όργανισμοί, ιζήματα και θαλάσσιο νερό), (ii) σε εξερχόμενα λύματα από τη λεκάνη απορροής του Δούναβη (Κεφάλαιο 6) και (iii) σε εξερχόμενα λύματα που συλλέχθηκαν από τη Γερμανία (Κεφάλαιο 7). Καινοτόμα εργαλεία βιοπαρακολούθησης όπως βιοδοκιμασίες και ανάλυση γονιδίων ανθεκτικών στα αντιβιοτικά συμπλήρωνουν τα αποτελέσματα της ανάλυσης αναδυόμενων ρύπων.Wastewater treatment plants (WWTPs) are unable to remove many contaminants of emerging concern (CECs) efficiently, and therefore introduce them into the aquatic environment, where they form complex chemical mixtures containing typically thousands of individual substances. When analysed by liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS), these complex mixtures produce a high number of signals. To successfully translate these complex data into information required by environmental monitoring programmes, implementation of wide-scope target, suspect and non-target screening using powerful computational tools and related databases is required. The objective of the thesis was to develop novel workflows employing state-of-the-art target, suspect and non-target screening tools and apply them on samples obtained from important European ecosystems such as the Danube River Basin (DRB) and the Black Sea (BS). In this context, an introduction on CECs, workflows and techniques for their identification is presented in Chapter 1, which is followed by the scope of the thesis in Chapter 2. Chapter 3 describes a non-target screening (NTS) workflow capable to prioritise compounds that exhibit large variation in their signal intensity over time (trend-analysis), which was used to detect events of direct disposal or sudden changes in the use of substances in WWTP of Athens. Chapter 4 describes the establishment of a decentralised global emerging contaminant early-warning network to assess the spatial and temporal distribution using suspect screening. A platform to archive LC-HRMS data and apply wide-scope suspect screening of thousands of CECs, that incorporates all recent development in HRMS screening methods, is presented in Chapter 5. The platform was used to screen antibiotics and REACH chemicals in samples from BS (biota, sediment, seawater), various classes of CECs in wastewater from DRB (Chapter 6) and surfactants in wastewater samples collected within the national monitoring campaign in Germany (Chapter 7). Novel biomonitoring tools such as in vitro bioassays and analysis of antibiotic resistant genes (ARGs) supplemented NTS analyses of wastewater samples

Non-target and suspect screening strategies for electrodialytic soil remediation evaluation: Assessing changes in the molecular fingerprints and per- and polyfluoroalkyl substances (PFASs)

Contamination of soils with organic pollutants is an increasing global problem, so novel soil remediation techniques are urgently needed. One such technique is electrokinetic remediation, in which an electric field is applied over the soil to extract contaminants. Previous evaluations of the technique have been limited to a few specific compounds. In this study, we integrated the latest advances in high-resolution mass spectrometry (HRMS) to identify molecular fingerprints, and used the results to improve the mechanistic understanding necessary for successful remediation. A laboratory-scale 0.38 mA cm(-2) electrodialytic treatment was applied for 21 days to a contaminated soil from a firefighter training facility in Sweden. Non-target analysis allowed generic evaluation of changes in the soil organic fraction by tentatively determining the elemental composition of compounds present. The results showed that smaller oxygen-rich molecules were significantly transported to the anode by electromigration, while larger hydrogen-saturated molecules were transported to the cathode by electroosmotic flow. Wide suspect screening with >3000 per- and polyfluoroalkyl substances (PFASs) tentatively identified seven new PFASs in the test soil, including perfluoroheptanesulfonic acid (PFHpS), and PFASs with butoxy, ethoxy, ethanol, and ethylcyclohexanesulfonate functional groups

PEAKTRAMS: An automated computational approach for the simultaneous detection of features in reverse phase and hilic hrms screening

Comparison of chromatograms obtained in reserved phase (RP) liquid chromatography and hydrophilic interaction liquid chromatography (HILIC) can provide valuable information for the identification and confirmation of suspect and non-target compounds. The plausibility of the obtained chromatographic retention times (RTs) in both modes as well as the ψomparison of the MS/MS spectra are strong points to be considered. This work presents the development of a novel automatic approach for the identification of common peaks between RP and HILIC chromatograms. The core of the program is written in R-project while a simple and user friendly graphical user interface (GUI) was built in JAVA. The first step consists of the introduction of the target chromatograms of the same sample (one obtained by RP and one by HILIC) plus the corresponding blank chromatograms. Blank subtraction was performed first using an algorithm to find in each scan the common m/z features (with a given mass accuracy). This algorithm also considers the RTs (a tolerance interval is applied), so the subtraction takes place even with slight drifts in the RTs between target and blank chromatograms. After blank subtraction, two different lists are obtained with the detected peaks in both RP and HILIC modes. Subsequently, m/z values are compared and matches are listed. The developed workflow was validated with solvent standards and with spiked wastewater samples with a mixture of compounds with a wide range of physicochemical properties. Successful results were obtained for 26 out of the 27 evaluated substances, allowing the recording of the corresponding RTs in both RP and HILIC mode

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/)

The role of natural science collections in the biomonitoring of environmental contaminants in apex predators in support of the EU's zero pollution ambition

The chemical industry is the leading sector in the EU in terms of added value. However, contaminants pose a major threat and significant costs to the environment and human health. While EU legislation and international conventions aim to reduce this threat, regulators struggle to assess and manage chemical risks, given the vast number of substances involved and the lack of data on exposure and hazards. The European Green Deal sets a ‘zero pollution ambition for a toxic free environment’ by 2050 and the EU Chemicals Strategy calls for increased monitoring of chemicals in the environment. Monitoring of contaminants in biota can, inter alia: provide regulators with early warning of bioaccumulation problems with chemicals of emerging concern; trigger risk assessment of persistent, bioaccumulative and toxic substances; enable risk assessment of chemical mixtures in biota; enable risk assessment of mixtures; and enable assessment of the effectiveness of risk management measures and of chemicals regulations overall. A number of these purposes are to be addressed under the recently launched European Partnership for Risk Assessment of Chemicals (PARC). Apex predators are of particular value to biomonitoring. Securing sufficient data at European scale implies large-scale, long-term monitoring and a steady supply of large numbers of fresh apex predator tissue samples from across Europe. Natural science collections are very well-placed to supply these. Pan-European monitoring requires effective coordination among field organisations, collections and analytical laboratories for the flow of required specimens, processing and storage of specimens and tissue samples, contaminant analyses delivering pan-European data sets, and provision of specimen and population contextual data. Collections are well-placed to coordinate this. The COST Action European Raptor Biomonitoring Facility provides a well-developed model showing how this can work, integrating a European Raptor Biomonitoring Scheme, Specimen Bank and Sampling Programme. Simultaneously, the EU-funded LIFE APEX has demonstrated a range of regulatory applications using cutting-edge analytical techniques. PARC plans to make best use of such sampling and biomonitoring programmes. Collections are poised to play a critical role in supporting PARC objectives and thereby contribute to delivery of the EU’s zero-pollution ambition

Pioneering an effect-based early warning system for hazardous chemicals in the environment

Existing regulatory frameworks often prove inadequate in promptly identifying contaminants of emerging concern (CECs) and determining their impacts on biological systems at an early stage. The establishment of Early Warning Systems (EWSs) for CECs is becoming increasingly relevant for policy-making, aiming to proactively detect chemical hazards and implement effective mitigation measures. Effect-based methodologies, including bioassays and effect-directed analysis (EDA), offer valuable input to EWSs by pinpointing the relevant toxicity drivers and prioritizing the associated risks. This review evaluates the analytical techniques currently available to assess biological effects, and provides a structured plan for their systematic integration into an EWS for hazardous chemicals in the environment. Key scientific advancements in effect-based approaches and EDA are discussed, underscoring their potential for early detection and management of chemical hazards. Additionally, critical challenges such as data integration and regulatory alignment are addressed, emphasizing the need for continuous improvement of the EWS and the incorporation of analytical advancements to safeguard environmental and public health from emerging chemical threats.Pioneering an effect-based early warning system for hazardous chemicals in the environmentpublishedVersio