Progress, applications, and challenges in high-throughput effect-directed analysis for toxicity driver identification - is it time for HT-EDA?

The rapid increase in the production and global use of chemicals and their mixtures has raised concerns about their potential impact on human and environmental health. With advances in analytical techniques, in particular, high-resolution mass spectrometry (HRMS), thousands of compounds and transformation products with potential adverse effects can now be detected in environmental samples. However, identifying and prioritizing the toxicity drivers among these compounds remain a significant challenge. Effect-directed analysis (EDA) emerged as an important tool to address this challenge, combining biotesting, sample fractionation, and chemical analysis to unravel toxicity drivers in complex mixtures. Traditional EDA workflows are labor-intensive and time-consuming, hindering large-scale applications. The concept of high-throughput (HT) EDA has recently gained traction as a means of accelerating these workflows. Key features of HT-EDA include the combination of microfractionation and downscaled bioassays, automation of sample preparation and biotesting, and efficient data processing workflows supported by novel computational tools. In addition to microplate-based fractionation, high-performance thin-layer chromatography (HPTLC) offers an interesting alternative to HPLC in HT-EDA. This review provides an updated perspective on the state-of-the-art in HT-EDA, and novel methods/tools that can be incorporated into HT-EDA workflows. It also discusses recent studies on HT-EDA, HT bioassays, and computational prioritization tools, along with considerations regarding HPTLC. By identifying current gaps in HT-EDA and proposing new approaches to overcome them, this review aims to bring HT-EDA a step closer to monitoring applications.

Concentrations of potentially endocrine disrupting chemicals in car cabin air and dust - Effect of temperature and ventilation.

Materials in car cabins contain performance-enhancing semi-volatile organic compounds (SVOCs). As these SVOCs are not chemically bound to the materials, they can emit from the materials at slow rates to the surrounding, causing human exposure. This study aimed at increasing the understanding on abundance of SVOCs in car cabins by studying 18 potential endocrine disrupting chemicals in car cabin air (gas phase and airborne particles) and dust. We also studied how levels of these chemicals varied by temperature inside the car cabin along with ventilation settings, relevant to human exposure. A positive correlation was observed between temperature and SVOC concentration in both the gas and the particle phase, where average gas phase levels at 80 °C were a factor of 18-16,000 higher than average levels at 25 °C, while average particle phase levels were a factor of 4.6-40,000 higher for the studied substances. This study also showed that levels were below the limit of detection for several SVOCs during realistic driving conditions, i.e., with the ventilation activated. To limit human exposure to SVOCs in car cabins, it is recommended to ventilate a warm car before entering and have the ventilation on during driving, as both temperature and ventilation have a significant impact on SVOC levels.

A time-trend guided non-target screening study of organic contaminants in Baltic Sea harbor porpoise (1988-2019), guillemot (1986-2019), and white-tailed sea eagle (1965-2017) using gas chromatography-high-resolution mass spectrometry.

The rate of decline in regulated persistent organic pollutant (POP) concentrations in Baltic Sea biota has leveled off in recent years, with new contaminants frequently being discovered. There is, therefore, a need for comprehensive approaches to study occurrence and temporal trends of a wide range of environmental contaminants, including legacy POPs, contaminants of emerging concern (CECs), and new contaminants. In the current work, non-target screening (NTS) workflows were developed and used for, to the best of our knowledge, the first time-trend directed NTS of biota using gas chromatography-high-resolution mass spectrometry (GC-HRMS). To maximize contaminant coverage, both electron ionization (EI) and electron capture negative ion chemical ionization (ECNI) were used. The EI data were treated using highly automated workflows to find, prioritize, and tentatively identify contaminants with statistically significant temporal trends. The ECNI data were manually processed and reviewed prior to time-trend analysis. Altogether, more than 300 tentatively identified contaminants were found to have significant temporal trends in samples of Baltic guillemot, harbor porpoise, or white-tailed sea eagle. Significant decreases were found for many regulated chemicals, as could be expected, such as PCBs, polychlorinated terphenyls, chlorobenzenes, toxaphenes, DDT, other organochlorine pesticides, and tri- and tetra- bromodiphenyl ethers (BDEs). The rate of decline of legacy POPs agreed well with data reported from targeted analyses. Significant increases were observed for small polycyclic aromatic hydrocarbons, heptaBDEs, CECs, and terpenes and related compounds. The CECs included, among others, one plasticizer tributyl acetylcitrate (ATBC), two antioxidants 2,6-bis(1,1-dimethylethyl)phenol and 2,6-bis(tert-butyl)-4-(4-morpholinyl-methyl)phenol, and two compounds used in polymer production, trimethyl isocyanurate and 2-mercaptobenzothiazole, which had not previously been reported in biota. Their increased concentrations in biota indicate increased use and release. The increase in ATBC may be linked to increased use of it as a substitute for di-2-ethylhexyl phthalate (DEHP), which has been phased out over the last decade.

Bacterial communities as indicators of environmental pollution by POPs in marine sediments.

Decades of intensive discharge from industrial activities into coastal systems has resulted in the accumulation of a variety of persistent organic pollutants (POPs) in marine waters and sediments, having detrimental impacts on aquatic ecosystems and the resident biota. POPs are among the most hazardous chemicals originating from industrial activities due to their biotoxicity and resistance to environmental degradation. Bacterial communities are known to break down many of these aromatic compounds, and different members of naturally occurring bacterial consortia have been described to work in syntrophic association to thrive in heavily contaminated waters and sediments, making them potential candidates as bioindicators of environmental pollution. In this study environmental, sampling was combined with chemical analysis of pollutants and high-resolution sequencing of bacterial communities using Next Generation Sequencing molecular biology tools. The aim of the present study was to describe the bacterial communities from marine sediments containing high loads of POPs and to identify relevant members of the resident microbial communities that may act as bioindicators of contamination. Marine sediments were collected from a coastal bay area of the Baltic Sea historically influenced by intense industrial activity, including metal smelting, oil processing, and pulp and paper production. Different types of POPs were detected at high concentrations. Fiberbank sediments, resulting from historic paper industry activity, were found to harbour a clearly distinct bacterial community including a number of bacterial taxa capable of cellulolytic and dechlorination activities. Our findings indicate that specific members of the bacterial communities thrive under increasing levels of POPs in marine sediments, and that the abundances of certain taxa correlate with specific POPs (or groups), which could potentially be employed in monitoring, status assessment and environmental management purposes.

Corrigendum: Effects of Organic Pollutants on Bacterial Communities Under Future Climate Change Scenarios.

[This corrects the article DOI: 10.3389/fmicb.2018.02926.].

Determination of picomolar concentrations of thiol compounds in natural waters and biological samples by tandem mass spectrometry with online preconcentration and isotope-labeling derivatization.

We present a sensitive, selective and robust method for the determination of 14 thiol compounds in aqueous samples. Thiols were derivatized with ω-bromoacetonylquinolinium bromide (BQB) and its deuterium labeled equivalent D7-ω-bromoacetonylquinolinium bromide (D7). Derivatized thiols were preconcentrated by online solid-phase extraction (SPE) followed by liquid chromatography separation and electrospray ionization tandem mass spectrometry determination (SPE/LC-ESI-MS/MS). The robustness of the method was validated for wide ranges in pH, salinity, and concentrations of sulfide and dissolved organic carbon (DOC) to cover contrasting natural water types. The limits of detection (LODs) for the thiols were 3.1-66 pM. Between 6 and 14 of the thiols were detected in different natural sample types at variable concentrations: boreal wetland porewater (0.7-51 nM), estuarine sediment porewater (50 pM-11 nM), coastal sea water (60 pM-16 nM), and sulfate reducing bacterium cultures (80 pM-4 nM). MS/MS fragmentation of the compounds produces two pairs of common product ions, m/z 130.2/137.1 and 218.1/225.1, which enables scanning for unknown thiols in precursor ion scan mode. Using this approach, we identified cysteine, mercaptoacetic acid, N-acetyl-L-cysteine and sulfurothioic S-acid in boreal wetland porewater. The performance of the developed method sets a new state of the art for the determination of thiol compounds in environmental and biological samples.

The strength in numbers: comprehensive characterization of house dust using complementary mass spectrometric techniques.

Untargeted analysis of a composite house dust sample has been performed as part of a collaborative effort to evaluate the progress in the field of suspect and nontarget screening and build an extensive database of organic indoor environment contaminants. Twenty-one participants reported results that were curated by the organizers of the collaborative trial. In total, nearly 2350 compounds were identified (18%) or tentatively identified (25% at confidence level 2 and 58% at confidence level 3), making the collaborative trial a success. However, a relatively small share (37%) of all compounds were reported by more than one participant, which shows that there is plenty of room for improvement in the field of suspect and nontarget screening. An even a smaller share (5%) of the total number of compounds were detected using both liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS). Thus, the two MS techniques are highly complementary. Most of the compounds were detected using LC with electrospray ionization (ESI) MS and comprehensive 2D GC (GC×GC) with atmospheric pressure chemical ionization (APCI) and electron ionization (EI), respectively. Collectively, the three techniques accounted for more than 75% of the reported compounds. Glycols, pharmaceuticals, pesticides, and various biogenic compounds dominated among the compounds reported by LC-MS participants, while hydrocarbons, hydrocarbon derivatives, and chlorinated paraffins and chlorinated biphenyls were primarily reported by GC-MS participants. Plastics additives, flavor and fragrances, and personal care products were reported by both LC-MS and GC-MS participants. It was concluded that the use of multiple analytical techniques was required for a comprehensive characterization of house dust contaminants. Further, several recommendations are given for improved suspect and nontarget screening of house dust and other indoor environment samples, including the use of open-source data processing tools. One of the tools allowed provisional identification of almost 500 compounds that had not been reported by participants.

Comprehensive assessment of organic contaminant removal from on-site sewage treatment facility effluent by char-fortified filter beds.

To remove organic contaminants from wastewater using cost-efficient and currently existing methods, our study investigated char-fortified filter beds for on-site sewage treatment facilities (OSSFs) in a long-term field setting. OSSFs are commonly used in rural and semi-urban areas worldwide to treat wastewater when municipal wastewater treatment is not economically feasible. First, we screened for organic contaminants with gas chromatography and liquid chromatography mass spectrometry-based targeted and untargeted analysis and then we developed quantitative structure-property relationship models to search for key molecular features responsible for the removal of organic contaminants. We identified 74 compounds (24 confirmed by reference standards) including plasticizers, UV stabilizers, fragrances, pesticides, surfactant and polymer impurities, pharmaceuticals and their metabolites, and many biogenic compounds. Sand filters that are used as a secondary step after the septic tank in OSSFs could remove hydrophobic contaminants. The addition of biochar significantly increased the removal of these and a few hydrophilic compounds (Wilcoxon signed-rank test, α = 0.05). Besides hydrophobicity-driven sorption, biodegradation was suggested to be the most important removal pathway in this long-term field application. However, further improvements are necessary to remove very hydrophilic contaminants as they were not removed with sand and biochar-fortified sand.

Effects of Organic Pollutants on Bacterial Communities Under Future Climate Change Scenarios.

Coastal ecosystems are highly dynamic and can be strongly influenced by climate change, anthropogenic activities (e.g., pollution), and a combination of the two pressures. As a result of climate change, the northern hemisphere is predicted to undergo an increased precipitation regime, leading in turn to higher terrestrial runoff and increased river inflow. This increased runoff will transfer terrestrial dissolved organic matter (tDOM) and anthropogenic contaminants to coastal waters. Such changes can directly influence the resident biology, particularly at the base of the food web, and can influence the partitioning of contaminants and thus their potential impact on the food web. Bacteria have been shown to respond to high tDOM concentration and organic pollutants loads, and could represent the entry of some pollutants into coastal food webs. We carried out a mesocosm experiment to determine the effects of: (1) increased tDOM concentration, (2) organic pollutant exposure, and (3) the combined effect of these two factors, on pelagic bacterial communities. This study showed significant responses in bacterial community composition under the three environmental perturbations tested. The addition of tDOM increased bacterial activity and diversity, while the addition of organic pollutants led to an overall reduction of these parameters, particularly under concurrent elevated tDOM concentration. Furthermore, we identified 33 bacterial taxa contributing to the significant differences observed in community composition, as well as 35 bacterial taxa which responded differently to extended exposure to organic pollutants. These findings point to the potential impact of organic pollutants under future climate change conditions on the basal coastal ecosystem, as well as to the potential utility of natural bacterial communities as efficient indicators of environmental disturbance.

Multicriteria approach to select polyaromatic river mutagen candidates.

The identification of unknown compounds remains one of the most challenging tasks to link observed toxic effects in complex environmental mixtures to responsible toxicants in effect-directed analysis (EDA). Here, a workflow is presented based on nontarget liquid chromatography-high resolution mass spectrometry (LC-HRMS) starting with molecular formulas determined in a previous study. A compound database search (ChemSpider) was performed to retrieve candidates for each formula. Subsequently, the number of candidates was reduced by applying MS-, physical-chemical, and chromatography-based selection criteria including HRMS/MS fragmentation and plausibility, ionization efficiency with different ion sources and detection modes, acid/base behavior, octanol/water partitioning, retention time prediction and finally toxic effects (mutagenicity caused by aromatic amines). The workflow strongly decreased the number of possible candidates and resulted in the tentative identification of possible mutagens and the positive identification of the nonmutagen benzyl(diphenyl) phosphine oxide in a mutagenic fraction. The positive identification of mutagens was hampered by a lack of commercially available standards. The workflow is an innovative and promising approach and forms an excellent basis for possible further advancements.

Integrated biological-chemical approach for the isolation and selection of polyaromatic mutagens in surface waters.

Many environmental mutagens, including polyaromatic compounds are present in surface waters, often in complex mixtures and at low concentrations. The present study provides and applies a novel, integrated approach to isolate polyaromatic mutagens in river water using a sample from the River Elbe. The sample was taken downstream of industrial discharges using blue rayon (BR) as a passive sampler that selectively adsorbs polyaromatic compounds and was subjected to effect-directed fractionation in order to characterise the compounds causing the detected effect(s). The procedure relies on three complementary fractionation steps, the Ames fluctuation assay with strains TA98, YG1024 and YG1041 with and without S9 activation and analytical screening. Several mutagenic fractions were isolated by combining mutagenicity testing with fractionation. The enhanced mutagenicity in the nitroreductase and/or O-acetyltransferase overexpressing strains YG1024 and YG1041 strains suggested amino- and/or nitro-compounds causing mutagenicity in several fractions. Analytical screening of mutagenic fractions with LC-HRMS/MS provided a list of molecular formulas typically containing one to ten nitrogen and at least two oxygen atoms supporting the presence of amino and nitro-compounds in the mutagenic fractions.

Consensus structure elucidation combining GC/EI-MS, structure generation, and calculated properties.

This article explores consensus structure elucidation on the basis of GC/EI-MS, structure generation, and calculated properties for unknown compounds. Candidate structures were generated using the molecular formula and substructure information obtained from GC/EI-MS spectra. Calculated properties were then used to score candidates according to a consensus approach, rather than filtering or exclusion. Two mass spectral match calculations (MOLGEN-MS and MetFrag), retention behavior (Lee retention index/boiling point correlation, NIST Kovat's retention index), octanol-water partitioning behavior (log K(ow)), and finally steric energy calculations were used to select candidates. A simple consensus scoring function was developed and tested on two unknown spectra detected in a mutagenic subfraction of a water sample from the Elbe River using GC/EI-MS. The top candidates proposed using the consensus scoring technique were purchased and confirmed analytically using GC/EI-MS and LC/MS/MS. Although the compounds identified were not responsible for the sample mutagenicity, the structure-generation-based identification for GC/EI-MS using calculated properties and consensus scoring was demonstrated to be applicable to real-world unknowns and suggests that the development of a similar strategy for multidimensional high-resolution MS could improve the outcomes of environmental and metabolomics studies.

Passive dosing: an approach to control mutagen exposure in the Ames fluctuation test.

One of the major challenges for mutagenicity assessment of environmental samples and individual compounds for example in the Ames fluctuation test (AFT) is the establishment and control of a well defined exposure concentration. Thus, a combination of passive dosing with silicone O-rings (SRs) together with an analytical confirmation of the freely dissolved concentration (FDC) is presented. FDCs are often determined with a combination of solid phase micro-extraction (SPME) with gas chromatography (GC). For compounds with poor performance in GC, a high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of bi-distilled water dosed with identically loaded SRs is suggested to avoid interference of the bacterial culture. The approach was tested for six amino-, nitro-, and keto-substituted polycyclic aromatic compounds with a logK(OW) range of 2.5-5.1 without metabolic activation. The method provided reliable concentration-effect relationships and freely dissolved 50% effect concentrations (DEC(50)) 3-33 times lower than nominal effect concentrations (NEC(50)) derived in parallel solvent-dosed AFT. Partition coefficients and NEC(50)/DEC(50) ratios were well correlated with lipophilicity.