Contamination Pattern and Risk Assessment of Polar Compounds in Snow Melt: An Integrative Proxy of Road Runoffs.

To assess the contamination and potential risk of snow melt with polar compounds, road and background snow was sampled during a melting event at 23 sites at the city of Leipzig and screened for 489 chemicals using liquid chromatography high-resolution mass spectrometry with target screening. Additionally, six 24 h composite samples were taken from the influent and effluent of the Leipzig wastewater treatment plant (WWTP) during the snow melt event. 207 compounds were at least detected once (concentrations between 0.80 ng/L and 75 µg/L). Consistent patterns of traffic-related compounds dominated the chemical profile (58 compounds in concentrations from 1.3 ng/L to 75 µg/L) and among them were 2-benzothiazole sulfonic acid and 1-cyclohexyl-3-phenylurea from tire wear and denatonium used as a bittern in vehicle fluids. Besides, the analysis unveiled the presence of the rubber additive 6-PPD and its transformation product N-(1.3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6-PPDQ) at concentrations known to cause acute toxicity in sensitive fish species. The analysis also detected 149 other compounds such as food additives, pharmaceuticals, and pesticides. Several biocides were identified as major risk contributors, with a more site-specific occurrence, to acute toxic risks to algae (five samples) and invertebrates (six samples). Ametryn, flumioxazin, and 1,2-cyclohexane dicarboxylic acid diisononyl ester are the main compounds contributing to toxic risk for algae, while etofenprox and bendiocarb are found as the main contributors for crustacean risk. Correlations between concentrations in the WWTP influent and flow rate allowed us to discriminate compounds with snow melt and urban runoff as major sources from other compounds with other dominant sources. Removal rates in the WWTP showed that some traffic-related compounds were largely eliminated (removal rate higher than 80%) during wastewater treatment and among them was 6-PPDQ, while others persisted in the WWTP.

In silico deconjugation of glucuronide conjugates enhances tandem mass spectra library annotation of human samples.

Mass spectral library annotation of liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) data is a reliable approach for fast identification of organic contaminants and toxicants in complex environmental and biological matrices. While determining the exposure of humans or mammals, it is indispensable to include phase I and phase II metabolites (conjugates) along with the parent compounds, but often, tandem mass spectra for these are unavailable. In this study, we present and evaluate a strategy for annotating glucuronide conjugates in LC-HRMS/MS scans by applying a neutral loss search for detection, then truncating the spectra which we refer to as in silico deconjugation, and finally searching these against mass spectral libraries of the aglycones. The workflow was tested on a dataset of in vitro-generated glucuronides of reference standard mixtures and a dataset of 51 authentic urine samples collected from patients with known medication status, acquired on different instrumentations. A total number of 75 different glucuronidated molecular structures were identified by in silico deconjugation and spectral library annotation. We also identified specific molecular structures (sulfonamides, ether bonds, di-glucuronides), which resulted in slightly different fragmentation patterns between the glucuronide and the unconjugated compound. This led to a decreased spectral matching score and in some cases to a false-negative identification. Still, by applying this method, we revealed a reliable annotation of most common glucuronides, leading to a new strategy reducing the need for deconjugation steps or for recording many reference glucuronide spectra for screening approaches.

Improving the Screening Analysis of Pesticide Metabolites in Human Biomonitoring by Combining High-Throughput In Vitro Incubation and Automated LC-HRMS Data Processing.

There is a current need to monitor human exposure to a large number of pesticides and other chemicals of emerging concern (CECs). This requires screening analysis with high confidence for these compounds and their metabolites in complex matrices, which is hampered by the fact that no reference standards are available for most metabolites. We address this challenge by a high-throughput workflow based on incubation of pesticides (or other CECs) with human liver S9, followed by solid-phase extraction, liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis, and automated data processing to generate a database (retention time, precursor m/z, and MS2 spectral library) for the annotation in human samples. The metabolite prioritization consists of statistical comparisons and mass defect and m/z range filtering to obtain a subset of probable phase I metabolites, for which molecular formulas and likely metabolic transformation are retrieved. We tested the workflow on 22 pesticides, for which we could determine 91 metabolite molecular formulas which are only partly covered by the literature and/or predicted by in silico metabolization. Our workflow allows for an efficient generation of metabolite reference information, which can be used directly for annotating LC-HRMS data from human samples. A full structure elucidation of individual metabolites can be limited to those being actually present in human samples.

Chemical Pollution Levels in a River Explain Site-Specific Sensitivities to Micropollutants within a Genetically Homogeneous Population of Freshwater Amphipods.

Anthropogenic micropollutants alter chemical and ecological conditions of freshwater ecosystems and impact aquatic species that live along the pollution gradient of a river. Species sensitivity to micropollutants depends on the site-specific exposure; however, it remains unclear to what degree this sensitivity relates to the species' genetic structure. Here, we explored the relationship between the toxic sensitivity and genetic structure of the amphipod species Gammarus pulex (Linnaeus, 1758) along an organic micropollutant gradient in the Holtemme River in central Germany. We determined the river's site-specific micropollutant patterns and analyzed the genetic structure of G. pulex using nuclear and mitochondrial genetic markers. Furthermore, we examined the exposure sensitivities and bioaccumulation of the commonly detected insecticide imidacloprid in G. pulex from different sites. Our results show that throughout the Holtemme River, G. pulex forms a well-connected and homogeneous population with no observable pollution-related differences in the genetic structure. However, G. pulex from polluted sites responded more sensitively to imidacloprid; survival times for half of the amphipods were up to 54% shorter, the percentage of immobile individuals increased up to 65%, and the modeled imidacloprid depuration rate was lower in comparison to amphipods from non-polluted sites. Altogether, these results suggest that the level of sensitivity of G. pulex amphipods to micropollutants in the river depends on the degree of pollution: amphipods may thrive in food-rich but polluted habitats; yet, their sensitivity is increased when chronically exposed to organic micropollutants.

Modulation of HIV-1 gene expression by binding of a ULM motif in the Rev protein to UHM-containing splicing factors.

The HIV-1 protein Rev is essential for virus replication and ensures the expression of partially spliced and unspliced transcripts. We identified a ULM (UHM ligand motif) motif in the Arginine-Rich Motif (ARM) of the Rev protein. ULMs (UHM ligand motif) mediate protein interactions during spliceosome assembly by binding to UHM (U2AF homology motifs) domains. Using NMR, biophysical methods and crystallography we show that the Rev ULM binds to the UHMs of U2AF65 and SPF45. The highly conserved Trp45 in the Rev ULM is crucial for UHM binding in vitro, for Rev co-precipitation with U2AF65 in human cells and for proper processing of HIV transcripts. Thus, Rev-ULM interactions with UHM splicing factors contribute to the regulation of HIV-1 transcript processing, also at the splicing level. The Rev ULM is an example of viral mimicry of host short linear motifs that enables the virus to interfere with the host molecular machinery.

Symbolic Aggregate Approximation Improves Gap Filling in High-Resolution Mass Spectrometry Data Processing.

Nontargeted mass spectrometry (MS) is widely used in life sciences and environmental chemistry to investigate large sets of samples. A major problem for larger-scale MS studies is data gaps or missing values in aligned data sets. The main causes for these data gaps are the absence of the compound from the sample, issues related to chromatography or mass spectrometry (for example, broad peaks, early eluting peaks, ion suppression, low ionization efficiency), and issues related to software (mainly limitations of peak detection algorithms). While those algorithms are heuristic by necessity and should be used with strict settings to minimize the number of false positive and negative peaks in a data set, gap filling may be used to reduce missing data in single samples remaining after peak detection. In this study, we present a new gap filling algorithm. The method is based on the symbolic aggregation approximation (SAX) algorithm that was developed for the evaluation and classification of time series in data mining studies. We adopted SAX for liquid chromatography high-resolution MS nontarget screening to support the detection of missing peaks in aligned mass spectral data sets. The SAX-based algorithm improves the detection efficiency considerably compared to existing gap filling methods including the Peak Finder algorithm provided in MZmine.

Chemoenzymatic Total Synthesis of Sorbicatechol Structural Analogues and Evaluation of Their Antiviral Potential.

Sorbicillinoids are fungal polyketides characterized by highly complex and diverse molecular structures, with considerable stereochemical intricacy combined with a high degree of oxygenation. Many sorbicillinoids possess promising biological activities. An interesting member of this natural product family is sorbicatechol A, which is reported to have antiviral activity, particularly against influenza A virus (H1N1). Through a straightforward, one-pot chemoenzymatic approach with recently developed oxidoreductase SorbC, the characteristic bicyclo[2.2.2]octane core of sorbicatechol is structurally diversified by variation of its natural 2-methoxyphenol substituent. This facilitates the preparation of a focused library of structural analogues bearing substituted aromatic systems, alkanes, heterocycles, and ethers. Fast access to this structural diversity provides an opportunity to explore the antiviral potential of the sorbicatechol family.

Discovery of the Streptoketides by Direct Cloning and Rapid Heterologous Expression of a Cryptic PKS II Gene Cluster from Streptomyces sp. Tü 6314.

Genome sequencing and bioinformatic analysis have identified numerous cryptic gene clusters that have the potential to produce novel natural products. Within this work, we identified a cryptic type II PKS gene cluster (skt) from Streptomyces sp. Tü 6314. Facilitated by linear plus linear homologous recombination-mediated recombineering (LLHR), we directly cloned the skt gene cluster using the Streptomyces site-specific integration vector pSET152. Direct cloning allowed for rapid heterologous expression in Streptomyces coelicolor, leading to the identification and structural characterization of six polyketides (three known compounds and new streptoketides), four of which exhibit anti-HIV activities. Our study shows that the pSET152 vector can be directly used for LLHR, expanding the Rec/ET direct cloning toolbox and providing the possibility for rapid heterologous expression of gene clusters from Streptomyces.

Application of the Sea Urchin Embryo Test in Toxicity Evaluation and Effect-Directed Analysis of Wastewater Treatment Plant Effluents.

Sea urchin embryo assay was used to assess general toxicity at four wastewater treatment plant effluents of Biscay (Gorliz, Mungia, Gernika, and Galindo), and within the tested range, all the extracts showed embryo growth inhibition and skeleton malformation activities with EC50 values, in relative enrichment factor units, between 1.1-16.8 and 1.1-8.8, respectively. To identify the causative compounds, effect-directed analysis was successfully applied for the first time using a sea urchin embryo test to the secondary treatment of the Galindo effluent. To this end, two subsequent fractionation steps were performed using C18 (21 fractions) and aminopropyl columns (15 fractions). By this fractionation, the number of features detected by LC-HRMS in the raw sample was drastically reduced from 1500 to 9, and among them, two pesticides (mexacarbate, 17 ng/L, and fenpropidin, 23 ng/L), two antidepressants (amitriptyline, 304 ng/L, and paroxetine, 26 ng/L), and two anthelmintic agents (mebendazole, 65 ng/L, and albendazole, 48 ng/L) could be identified in the two toxic fractions. The artificial mixture of the identified six compounds could explain 79% of the observed effect, with albendazole and paroxetine as the predominant contributors (49% and 49%, respectively) affecting the sea urchin embryogenesis activity.

Assessing the Mixture Effects in In Vitro Bioassays of Chemicals Occurring in Small Agricultural Streams during Rain Events.

Rain events may impact the chemical pollution burden in rivers. Forty-four small streams in Germany were profiled during several rain events for the presence of 395 chemicals and five types of mixture effects in in vitro bioassays (cytotoxicity; activation of the estrogen, aryl hydrocarbon, and peroxisome proliferator-activated receptors; and oxidative stress response). While these streams were selected to cover a wide range of agricultural impacts, in addition to the expected pesticides, wastewater-derived chemicals and chemicals typical for street runoff were detected. The unexpectedly high estrogenic effects in many samples indicated the impact by wastewater or overflow of combined sewer systems. The 128 water samples exhibited a high diversity of chemical and effect patterns, even for different rain events at the same site. The detected 290 chemicals explained only a small fraction (<8%) of the measured effects. The experimental effects of the designed mixtures of detected chemicals that were expected to dominate the mixture effects of detected chemicals were consistent with predictions for concentration addition within a factor of two for 94% of the mixtures. Overall, the burden of chemicals and effects was much higher than that previously detected in surface water during dry weather, with the effects often exceeding proposed effect-based trigger values.

Biological evaluation of molecules of the azaBINOL class as antiviral agents: Inhibition of HIV-1 RNase H activity by 7-isopropoxy-8-(naphth-1-yl)quinoline.

Inspired by bioactive biaryl-containing natural products found in plants and the marine environment, a series of synthetic compounds belonging to the azaBINOL chiral ligand family was evaluated for antiviral activity against HIV-1. Testing of 39 unique azaBINOLs and two BINOLs in a single-round infectivity assay resulted in the identification of three promising antiviral compounds, including 7-isopropoxy-8-(naphth-1-yl)quinoline (azaBINOL B#24), which exhibited low-micromolar activity without associated cytotoxicity. The active compounds and several close structural analogues were further tested against three different HIV-1 envelope pseudotyped viruses as well as in a full-virus replication system (EASY-HIT). The in vitro studies indicated that azaBINOL B#24 acts on early stages of viral replication before viral assembly and budding. Next we explored B#24's activity against HIV-1 reverse transcriptase (RT) and individually tested for polymerase and RNase H activity. The azaBINOL B#24 inhibits RNase H activity and binds directly to the HIV-1 RT enzyme. Additionally, we observe additive inhibitory activity against pseudotyped viruses when B#24 is dosed in competition with the clinically used non-nucleoside reverse transcriptase inhibitor (NNRTI) efavirenz. When tested against a multi-drug resistant HIV-1 isolate with drug resistance associated mutations in regions encoding for HIV-1 RT and protease, B#24 only exhibits a 5.1-fold net decrease in IC50 value, while efavirenz' activity decreases by 7.6-fold. These results indicate that azaBINOL B#24 is a potentially viable, novel lead for the development of new HIV-1 RNase H inhibitors. Furthermore, this study demonstrates that the survey of libraries of synthetic compounds, designed purely with the goal of facilitating chemical synthesis in mind, may yield unexpected and selective drug leads for the development of new antiviral agents.

Isolation and characterization of eleven novel microsatellite markers for fine-scale population genetic analyses of Gammarus pulex (Crustacea: Amphipoda).

The freshwater amphipod species Gammarus pulex (Linnaeus, 1758) is widespread across Europe and Asia and is able to live in a broad range of environmental conditions. Yet, it is of great interest to which degree it is able to tolerate and adapt to the current rapid anthropogenic environmental changes affecting its habitat, such as pollution, changes in river morphology, and invasions of alien gammarid species. Microsatellite primers for genetic population studies with G. pulex have been developed but due to the existence of several genetically different lineages within the species, the application of these primers is not always successful. In order to investigate the impacts of anthropogenic environmental changes on the spatio-genetic patterns of G. pulex lineage E in streams in the Saale river catchment in Germany, we designed eleven novel polymorphic microsatellites for this lineage using a high-throughput sequencing approach. These microsatellites enabled highly specific characterization of three closely related populations. The results show genetically distinct populations reflected by both a principal coordinates analysis and an analysis of molecular variance. Several of the newly designed microsatellite primers also enabled successful cross-amplification of the respective microsatellites in specimens of G. pulex lineage C, while only two microsatellites were amplified successfully and showed polymorphisms for all of the analyzed specimens of G. fossarum Koch, 1836. The microsatellites identified here are suitable for future assessments of micro-evolutionary dynamics of G. pulex from central Germany.

Non-targeted mercapturic acid screening in urine using LC-MS/MS with matrix effect compensation by postcolumn infusion of internal standard (PCI-IS).

While the targeted analysis of mercapturic acid (MA) metabolites in human urine is used to assess exposure to selected chemicals, this compound class has only rarely been addressed in non-target screening utilizing diagnostic neutral loss liquid chromatography tandem mass spectrometry (LC-MS/MS). Additionally, this type of analysis is severely affected by matrix effects (MEs) causing poor comparability of samples and distortion of signal intensities. However, MEs have been neglected in urinary MA non-target screening so far. Therefore, we developed a non-target screening method relying on neutral loss scanning for MAs using post column infusion of an isotope-labelled standard. For signal correction, we synthesized a structural analogue to MAs, N-acetyl-S-methyl-homocysteine-D3, lacking the characteristic neutral loss of the MAs. For method development, 16 structurally different model MA compounds and 20 spiked urine samples were used. Twelve out of the 16 model compounds could be analysed by the developed method. We found severe matrix effects (largely signal suppression) for the spiked model compounds, with only 34% of all peaks' intensities changing by less than a factor of two. This could be compensated by the post column internal standard infusion with now 68% of all peaks' intensities changing by less than a factor of two. For three compounds, an over-compensation was observed resulting in an increase of signal of up to a factor of 16. In the 20 urine samples, altogether 558 native MAs (between 74 and 175 per sample) could be detected after ME compensation. These results indicate that a large number of so far uncharacterized MAs are present in urine, which yield a potential for biomarker discovery and pattern characterisation. Graphical Abstract.

Synthetic AAV/CRISPR vectors for blocking HIV-1 expression in persistently infected astrocytes.

Astrocytes, the most abundant cells in the mammalian brain, perform key functions and are involved in several neurodegenerative diseases. The human immunodeficiency virus (HIV) can persist in astrocytes, contributing to the HIV burden and neurological dysfunctions in infected individuals. While a comprehensive approach to HIV cure must include the targeting of HIV-1 in astrocytes, dedicated tools for this purpose are still lacking. Here we report a novel Adeno-associated virus-based vector (AAV9P1) with a synthetic surface peptide for transduction of astrocytes. Analysis of AAV9P1 transduction efficiencies with single brain cell populations, including primary human brain cells, as well as human brain organoids demonstrated that AAV9P1 targeted terminally differentiated human astrocytes much more efficiently than neurons. We then investigated whether AAV9P1 can be used to deliver HIV-inhibitory genes to astrocytes. To this end we generated AAV9P1 vectors containing genes for HIV-1 proviral editing by CRISPR/Cas9. Latently HIV-1 infected astrocytes transduced with these vectors showed significantly diminished reactivation of proviruses, compared with untransduced cultures. Sequence analysis identified mutations/deletions in key HIV-1 transcriptional control regions. We conclude that AAV9P1 is a promising tool for gene delivery to astrocytes and may facilitate inactivation/destruction of persisting HIV-1 proviruses in astrocyte reservoirs.

Pesticide Body Burden of the Crustacean Gammarus pulex as a Measure of Toxic Pressure in Agricultural Streams.

Risk assessments of toxicants in aquatic environments are typically based on the evaluation of concentrations in water or sediment. However, concentrations in water are highly variable, while the body burden may provide a better time-integrated measure of pesticide exposure and potential effects in aquatic organisms. Here, we quantified pesticide body burdens in a dominant invertebrate species from agricultural streams, Gammarus pulex, compared them with pesticide concentrations in water samples, and linked the pesticide contamination with observed ecological effects on macroinvertebrate communities. In total, 19 of 61 targeted analytes were found in the organisms, ranging from 0.037 to 93.94 ng g-1 (wet weight). Neonicotinoids caused the highest toxic pressure among the pesticides detected in G. pulex. Using linear solvation energy relationships (LSERs), we derived equivalent pesticide concentrations in streamwater based on the body burden. These equivalent concentrations correlated with the concentrations in water samples collected after runoff (65% of variance explained). Pesticide pressure significantly affected the aquatic macroinvertebrate community structure, expressed as SPEARpesticides, and caused, on average, 3-fold increased insecticide tolerance in G. pulex as a result of adaptation. The toxic pressure derived from body burden and from water samples similarly explained the change in community structure (68% and 64%). However, the increased tolerance of G. pulex to pesticides was better explained by the toxicity derived from body burden (70%) than by the toxicity from water samples (53%). We conclude that the internal body burden of macroinvertebrates is suitable to assess the overall pesticide exposure and effects in agricultural streams.

Screening of Pesticide and Biocide Patterns As Risk Drivers in Sediments of Major European River Mouths: Ubiquitous or River Basin-Specific Contamination?

Pesticides and biocides (PaB) are ubiquitously present in aquatic ecosystems due to their widespread application and have been detected in rivers at concentrations that may cause distress to aquatic life. Many of these compounds accumulate in sediments acting as long-term source for aquatic ecosystems. However, data on sediment contamination with current-use PaB in Europe are scarce. Thus, in this study, we elucidated PaB patterns and associated risks in sediments of seven major European rivers focusing on their last stretch as an integrative sink of particles transported by these rivers. Sediments were extracted with pressurized liquid extraction (PLE) using a broad-spectrum method recovering many compound classes with a wide range of physicochemical properties. Altogether 126 compounds were analyzed and 81 of them were detected with LC-HRMS and GC-NCI-MS/MS at least in one of the sediments. The highest number of compounds was detected (59) in River Elbe sediments close to Cuxhaven with outstanding concentrations ranging from 0.8 to 1691 mg/g organic carbon. Multivariate analysis identified a cluster with 3 ubiquitous compounds (cyhalothrin, carbendazim, fenpropimorph) and three clusters of chemicals with higher variability within and between rivers. Risk assessment indicates an acute toxic risk to benthic crustaceans at all investigated sites with the pyrethroids tefluthrin and cyfluthrin together with the fungicide carbendazim as the main drivers. Risks to algae were driven at most sites almost exclusively by photosynthesis inhibitors with estuary-specific herbicide mixtures, while in the rivers Po and Gironde cell division inhibitors played an important role at some sites. Mixtures of specific concern have been defined and suggested for integration in future monitoring programs.

Identification of Unknown Antiandrogenic Compounds in Surface Waters by Effect-Directed Analysis (EDA) Using a Parallel Fractionation Approach.

Among all the nuclear-receptor mediated endocrine disruptive effects, antiandrogenicity is frequently observed in aquatic environments and may pose a risk to aquatic organisms. Linking these effects to responsible chemicals is challenging and a great share of antiandrogenic activity detected in the environment has not been explained yet. To identify drivers of this effect at a hot spot of antiandrogenicity in the German river Holtemme, we applied effect-directed analysis (EDA) including a parallel fractionation approach, a downscaled luciferase reporter gene cell-based anti-AR-CALUX assay and LC-HRMS/MS nontarget screening. We identified and confirmed the highly potent antiandrogen 4-methyl-7-diethylaminocoumarin (C47) and two derivatives in the active fractions. The relative potency of C47 to the reference compound flutamide was over 5.2, whereas the derivatives were less potent. C47 was detected at a concentration of 13.7 µg/L, equal to 71.4 µg flutamide equivalents per liter (FEq/L) in the nonconcentrated water extract that was posing an antiandrogenic activity equal to 45.5 (±13.7 SD) FEq/L. Thus, C47 was quantitatively confirmed as the major cause of the measured effect in vitro. Finally, the antiandrogenic activity of C47 and one derivate was confirmed in vivo in spiggin-gfp Medaka. An endocrine disrupting effect of C47 was observed already at the concentration equal to the concentration in the nonconcentrated water extract, underlining the high risk posed by this compound to the aquatic ecosystem. This is of some concern since C47 is used in a number of consumer products indicating environmental as well as human exposure.

Performance of combined fragmentation and retention prediction for the identification of organic micropollutants by LC-HRMS.

In nontarget screening, structure elucidation of small molecules from high resolution mass spectrometry (HRMS) data is challenging, particularly the selection of the most likely candidate structure among the many retrieved from compound databases. Several fragmentation and retention prediction methods have been developed to improve this candidate selection. In order to evaluate their performance, we compared two in silico fragmenters (MetFrag and CFM-ID) and two retention time prediction models (based on the chromatographic hydrophobicity index (CHI) and on log D). A set of 78 known organic micropollutants was analyzed by liquid chromatography coupled to a LTQ Orbitrap HRMS with electrospray ionization (ESI) in positive and negative mode using two fragmentation techniques with different collision energies. Both fragmenters (MetFrag and CFM-ID) performed well for most compounds, with average ranking the correct candidate structure within the top 25% and 22 to 37% for ESI+ and ESI- mode, respectively. The rank of the correct candidate structure slightly improved when MetFrag and CFM-ID were combined. For unknown compounds detected in both ESI+ and ESI-, generally positive mode mass spectra were better for further structure elucidation. Both retention prediction models performed reasonably well for more hydrophobic compounds but not for early eluting hydrophilic substances. The log D prediction showed a better accuracy than the CHI model. Although the two fragmentation prediction methods are more diagnostic and sensitive for candidate selection, the inclusion of retention prediction by calculating a consensus score with optimized weighting can improve the ranking of correct candidates as compared to the individual methods. Graphical abstract Consensus workflow for combining fragmentation and retention prediction in LC-HRMS-based micropollutant identification.

A sediment extraction and cleanup method for wide-scope multitarget screening by liquid chromatography-high-resolution mass spectrometry.

Previous studies on organic sediment contaminants focused mainly on a limited number of highly hydrophobic micropollutants accessible to gas chromatography using nonpolar, aprotic extraction solvents. The development of liquid chromatography-high-resolution mass spectrometry (LC-HRMS) permits the spectrum of analysis to be expanded to a wider range of more polar and ionic compounds present in sediments and allows target, suspect, and nontarget screening to be conducted with high sensitivity and selectivity. In this study, we propose a comprehensive multitarget extraction and sample preparation method for characterization of sediment pollution covering a broad range of physicochemical properties that is suitable for LC-HRMS screening analysis. We optimized pressurized liquid extraction, cleanup, and sample dilution for a target list of 310 compounds. Finally, the method was tested on sediment samples from a small river and its tributaries. The results show that the combination of 100 °C for ethyl acetate-acetone (50:50, neutral extract) followed by 80 °C for acetone-formic acid (100:1, acidic extract) and methanol-10 mM sodium tetraborate in water (90:10, basic extract) offered the best extraction recoveries for 287 of 310 compounds. At a spiking level of 1 µg mL-1, we obtained satisfactory cleanup recoveries for the neutral extract-(93 ± 23)%-and for the combined acidic/basic extracts-(42 ± 16)%-after solvent exchange. Among the 69 compounds detected in environmental samples, we successfully quantified several pharmaceuticals and polar pesticides.

Mutagenicity in Surface Waters: Synergistic Effects of Carboline Alkaloids and Aromatic Amines.

For decades, mutagenicity has been observed in many surface waters with a possible link to the presence of aromatic amines. River Rhine is a well-known example of this phenomenon but responsible compound(s) are still unknown. To identify the mutagenic compounds, we applied effect-directed analysis (EDA) utilizing novel analytical and biological approaches to a water sample extract from the lower Rhine. We could identify 21 environmental contaminants including two weakly mutagenic aromatic amines, and the known alkaloid comutagen norharman along with two related ß-carboline alkaloids, carboline, and 5-carboline, which were reported the first time in surface waters. Results of mixture tests showed a strong synergism of the identified aromatic amines not only with norharman, but also with carboline and 5-carboline. Additionally, other nitrogen-containing compounds also contributed to the mutagenicity when aromatic amines were present. Thus, comutagenicity of ß-carboline alkaloids with aromatic amines is shown to occur in surface waters. These results strongly suggest that surface water mutagenicity is highly complex and driven by synergistic mechanisms of a complex compound mixture (of which many are yet unidentified) rather than by single compounds. Therefore, mixture effects should be considered not only from mutagens alone, but also including possible comutagens and nonmutagenic compounds.