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/.

A quest to identify suitable organic tracers for estimating children's dust ingestion rates.

Chemical exposure via dust ingestion is of great interest to researchers and regulators because children are exposed to dust through their daily activities, and as a result, to the many chemicals contained within dust. Our goal was to develop a workflow to identify and rank organic chemicals that could be used as tracers to calculate children's dust ingestion rates. We proposed a set of criteria for a chemical to be considered a promising tracer. The best tracers must be (1) ubiquitous in dust, (2) unique to dust, (3) detectable as biomarkers in accessible biological samples, and (4) have available or obtainable ADME information for biomarker-based exposure reconstruction. To identify compounds meeting these four criteria, we developed a workflow that encompasses non-targeted analysis approaches, literature and database searching, and multimedia modeling. We then implemented an ad hoc grading system and ranked candidate chemicals based on fulfillment of our criteria (using one small, publicly available dataset to show proof of concept). Initially, five chemicals (1,3-diphenylguanidine, leucine, piperine, 6:2/8:2 fluorotelomer phosphate diester, 6:2 fluorotelomer phosphate diester) appeared to satisfy many of our criteria. However, a rigorous manual investigation raised many questions about the applicability of these chemicals as tracers. Based on the results of this initial pilot study, no individual compounds can be unequivocally considered suitable tracers for calculating dust ingestion rates. Future work must therefore consider larger datasets, generated from broader measurement studies and literature searches, as well as refinements to selection criteria, to identify robust and defensible tracer compounds.

Measured concentrations of consumer product chemicals in California house dust: Implications for sources, exposure, and toxicity potential.

Household dust is a reservoir of various consumer product chemicals. Thus, characterizing comprehensive chemical profiles of house dust may help improve our understanding of residential chemical exposure. We have previously developed a method for detecting a broad spectrum of chemicals in dust by applying a combination of target, suspect screening, and non-target methods with mass spectrometry preceded by liquid chromatography and gas chromatography. Building upon a previous study that detected 271 compounds in 38 dust samples, we presented concentrations of 144 compounds that were confirmed and quantified by standards in the same set of samples. Ten compounds were measured with median concentrations greater than 10 000 ng/g of dust: cis-hexadec-6-enoic acid, squalene, cholesterol, vitamin E, bis(2-ethylhexyl) phthalate, dioctyl terephthalate, linoleic acid, tricaprylin, tris(1-chloroisopropyl) phosphate, and oxybenzone. We also reviewed in vitro toxicity screening data to identify compounds that were not previously detected in indoor dust but have potential for adverse health effects. Among 119 newly detected compounds, 13 had endocrine-disrupting potential and 7 had neurotoxic potential. Toxicity screening data were not available for eight biocides, which may adversely affect health. Our results strive to provide more comprehensive chemical profiles of house dust and identified information gaps for future health studies.

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.

Wastewater alters feeding rate but not vitellogenin level of Gammarus fossarum (Amphipoda).

Wastewater treatment plant (WWTP) effluents release complex mixtures of organic and inorganic micropollutants, including endocrine disrupting compounds, into receiving water bodies. These substances may cause adverse effects in aquatic communities as well as in ecosystem functions they provide. The aim of this study was to determine the potential impact of secondary treated wastewater released into a small Swiss stream on leaf litter decomposition based on feeding rates of the amphipod shredder Gammarus fossarum measured in situ. Additionally, endocrine disrupting effects downstream of the WWTP were investigated by measuring vitellogenin (vg) induction in male gammarids exposed in situ, as well as estrogen receptor activation using the Yeast Estrogen Screen (YES) involving passive sampler and grab water sample extracts. Extracts were also analysed for 424 organic micropollutants and selected transformation products. Gammarid feeding rate was significantly reduced 100, 200 and 400 m downstream of the WWTP effluent relative to the upstream site. While YES results showed significantly elevated estrogenicity at downstream sites, vg production in male gammarids was not induced. A laboratory experiment, in which gammarids were exposed to WWTP effluent, supported this observation. These results, hence, suggest that treated wastewater released into aquatic ecosystems impairs the ecosystem function of leaf litter decomposition. Vg levels in male gammarids measured by UPLC-MS/MS did, however, not alter.

Exposure Assessment For Air-To-Skin Uptake of Semivolatile Organic Compounds (SVOCs) Indoors.

Semivolatile organic compounds (SVOCs) are ubiquitous in the indoor environment and a priority for exposure assessment because of the environmental health concerns that they pose. Direct air-to-skin dermal uptake has been shown to be comparable to the inhalation intake for compounds with certain chemical properties. In this study, we aim to further understand the transport of these types of chemicals through the skin, specifically through the stratum corneum (SC). Our assessment is based on collecting three sequential forehead skin wipes, each hypothesized to remove pollutants from successively deeper skin layers, and using these wipe analyses to determine the skin concentration profiles. The removal of SVOCs with repeated wipes reveals the concentration profiles with depth and provides a way to characterize penetration efficiency and potential transfer to blood circulation. We used a diffusion model applied to surface skin to simulate concentration profiles of SVOCs and compared them with the measured values. We found that two phthalates, dimethyl and diethyl phthalates, penetrate deeper into skin with similar exposure compared to other phthalates and targeted SVOCs, an observation supported by the model results as well. We also report the presence of statistically significant declining patterns with skin depth for most SVOCs, indicating that their diffusion through the SC is relevant and eventually can reach the blood vessels in the vascularized dermis. Finally, using a nontarget approach, we identified skin oxidation products, linked to respiratory irritation symptoms, formed from the reaction between ozone and squalene.

Household Dust as a Repository of Chemical Accumulation: New Insights from a Comprehensive High-Resolution Mass Spectrometric Study.

Chemical exposure in household dust poses potential risks to human health but has been studied incompletely thus far. Most analytical studies have focused on one or several compound classes, with analysis performed by either liquid or gas chromatography coupled with mass spectrometry (LC-MS or GC-MS). However, a comprehensive investigation of individual dust samples is missing. The present study comprehensively characterizes chemicals in dust by applying a combination of target, suspect, and nontarget screening approaches using both LC and GC with quadrupole time-of-flight (Q/TOF) MS. First, the extraction method was optimized to streamline detection of LC-Q/TOF and GC-Q/TOF amenable compounds and was successfully validated with over 100 target compounds. Nontarget screening with GC-Q/TOF was done by spectral deconvolution followed by a library search. Suspect screening by LC-Q/TOF was carried out with an accurate mass spectral library. Finally, LC-Q/TOF nontarget screening was carried out by extracting molecular features, acquiring tandem mass spectrometric (MS/MS) spectra, and performing compound identification by use of in silico fragmentation software tools. In total, 271 chemicals could be detected in 38 dust samples, 163 of which could be unambiguously confirmed by a reference standard. Many of them, such as the plastic leachable 7,9-di- tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione (CAS 82304-66-3) and three organofluorine compounds, are of emerging concern and their presence in dust has been underestimated. Advantages and drawbacks of the different approaches and analytical instruments are critically discussed.

Using Mutations for Pesticide Resistance to Identify the Cause of Toxicity in Environmental Samples.

Traditional Toxicity Identification Evaluations (TIE) are applied to identify causal agents in complex environmental samples showing toxicity and rely upon physical or chemical manipulation of samples. However, mutations conferring toxicant resistance provide the opportunity for a novel biologically based TIE. Populations within the Hyalella azteca complex from pesticide-affected waterways were 2 and 3 orders of magnitude more resistant to the pyrethroid cyfluthrin and the organophosphate chlorpyrifos, respectively, than laboratory-cultured H. azteca widely used for toxicity testing. Three resistant populations, as well as laboratory-cultured, nonresistant H. azteca, were exposed to urban and agricultural runoff. Every sample causing death or paralysis in the nonresistant individuals had no effect on pyrethroid-resistant individuals, providing strong evidence that a pyrethroid was the responsible toxicant. The lack of toxicity to chlorpyrifos-sensitive, but pyrethroid-resistant, individuals suggested chlorpyrifos was not a likely toxicant, a hypothesis supported by chemical analysis. Since these mutations that confer resistance to pesticides are highly specific, toxicity to wild-type, but not resistant animals, provides powerful evidence of causality. It may be possible to identify strains resistant to even a wider variety of toxicants, further extending the potential use of this biologically based TIE technique beyond the pyrethroid and organophosphate-resistant strains currently available.

LC- and GC-QTOF-MS as Complementary Tools for a Comprehensive Micropollutant Analysis in Aquatic Systems.

Efficient strategies are required to implement comprehensive suspect screening methods using high-resolution mass spectrometry within environmental monitoring campaigns. In this study, both liquid and gas chromatography time-of-flight mass spectrometry (LC-QTOF-MS and GC-QTOF-MS) were used to screen for >5000 target and suspect compounds in the Sacramento-San Joaquin River Delta in Northern California. LC-QTOF-MS data were acquired in All-Ions fragmentation mode in both positive and negative electrospray ionization (ESI). LC suspects were identified using two accurate mass LC-QTOF-MS/MS libraries containing pesticides, pharmaceuticals, and other environmental contaminants and a custom exact mass database with predicted transformation products (TPs). The additional fragment information from the All-Ions acquisition improved the confirmation of the compound identity, with a low false positive rate (9%). Overall, 25 targets, 73 suspects, and 5 TPs were detected. GC-QTOF-MS extracts were run in negative chemical ionization (NCI) for 21 targets (mainly pyrethroids) at sub-ng/L levels. For suspect screening, extracts were rerun in electron ionization (EI) mode with a retention time locked method using a GC-QTOF-MS pesticide library (containing exact mass fragments and retention times). Sixteen targets and 42 suspects were detected, of which 12 and 17, respectively, were not identified by LC-ESI-QTOF-MS. The results highlight the importance of analyzing water samples using multiple separation techniques and in multiple ionization modes to obtain a comprehensive chemical contaminant profile. The investigated river delta experiences significant pesticide inputs, leading to environmentally critical concentrations during rain events.

Evaluation of in-situ calibration of Chemcatcher passive samplers for 322 micropollutants in agricultural and urban affected rivers.

In a large field study, the in-situ calibration of the Chemcatcher(®) passive sampler - styrenedivinylbenzene (SDB) covered by a polyether sulfone (PES) membrane - was evaluated for 322 polar organic micropollutants. Five rivers with different agricultural and urban influences were monitored from March to July 2012 with two methods i) two-week time-proportional composite water samples and ii) two-week passive sampler deployment. All substances - from different substance classes with logKow -3 to 5, and neutral, anionic, cationic, and zwitterionic species - were analyzed by liquid-chromatography high-resolution tandem mass spectrometry. This study showed that SDB passive samplers are well-suited for the qualitative screening of polar micropollutants because the number of detected substances was similar (204 for SDB samples vs. 207 for composite water samples), limits of quantification were comparable (median: 1.3 ng/L vs. 1.6 ng/L), and the handling in the field and laboratory is fast and easy. The determination of in-situ calibrated sampling rates (field Rs) was possible for 88 compounds where the R(2) from the regression (water concentration vs. sampled mass on SDB disk) was >0.75. Substances with moderately fluctuating river concentrations such as pharmaceuticals showed much better correlations than substances with highly fluctuating concentrations such as pesticides (R(2) > 0.75 for 93% and 60% of the investigated substances, respectively). Flow velocity (0.05-0.8 m/s) and temperature (5-20 °C) did not have an evident effect on the field Rs. It was observed that ionic species had significantly lower field Rs than neutral species. Due to the complexity of the different transport processes, a correlation between determined field Rs and logDow could only predict Rs with large uncertainties. We conclude that only substances with relatively constant river concentrations can be quantified accurately in the field by passive sampling if substance-specific Rs are determined. For that purpose, the proposed in-situ calibration is a very robust method and the substance specific Rs can be used in future monitoring studies in rivers with similar environmental conditions (i.e., flow velocity, temperature, pH).

Picogram per liter detections of pyrethroids and organophosphates in surface waters using passive sampling.

Pyrethroids and organophosphates are among the most toxic insecticides for aquatic organisms, leading to annual-average environmental quality standards (AA-EQS) in the picogram per liter range in surface waters. For monitoring purposes, it is therefore crucial to develop very sensitive analytical methods. Until now, it is very difficult to reach detection limits at or below given AA-EQSs. Here, we present a passive sampling method using silicone rubber (SR) sheets for the sampling of ten pyrethroids and two organophosphates in surface waters. An analytical method was developed, optimized and validated for the extraction of the insecticides from the SR sheets by accelerated solvent extraction followed by clean-up on C18 and silica gel and detection with GC-MS/MS in positive ionization mode. Good precision (<20%) and absolute recovery (>50%) was observed for all substances, accuracy was between 66% and 139%. Limits of detection between 6 and 200 pg/L were achieved for all substances in surface waters using average sampling rates for PCBs and PAHs. The lack of substance-specific sampling rates and missing performance reference compounds led to an uncertainty in the concentration estimation of factor three in both directions. In a large field study, comprising 40 environmental samples from nine Swiss rivers, eight out of 12 substances were detected (most frequently: chlorpyrifos, cypermethrin). Most of the estimated organophosphate concentrations were between 0.1 and 1 ng/L, most pyrethroid detections below 0.1 ng/L. Four substances (chlorpyrifos-methyl, cypermethrin, deltamethrin and lambda-cyhalothrin) showed exceedances of their respective AA-EQS in multiple samples, also when the uncertainties in the concentration estimation were considered. As pyrethroid and organophosphate detection by SR passive sampling is very practicable and allows sensitive analysis, it has the potential to become a new tool in the monitoring of non-polar pesticides.

How a complete pesticide screening changes the assessment of surface water quality.

A comprehensive assessment of pesticides in surface waters is challenging due to the large number of potential contaminants. Most scientific studies and routine monitoring programs include only 15-40 pesticides, which leads to error-prone interpretations. In the present study, an extensive analytical screening was carried out using liquid chromatography-high-resolution mass spectrometry, covering 86% of all polar organic pesticides sold in Switzerland and applied to agricultural or urban land (in total 249 compounds), plus 134 transformation products; each of which could be quantified in the low ng/L range. Five medium-sized rivers, containing large areas of diverse crops and urban settlements within the respective catchments, were sampled between March and July 2012. More than 100 parent compounds and 40 transformation products were detected in total, between 30 and 50 parent compounds in each two-week composite sample in concentrations up to 1500 ng/L. The sum of pesticide concentrations was above 1000 ng/L in 78% of samples. The chronic environmental quality standard was exceeded for 19 single substances; using a mixture toxicity approach, exceedances occurred over the whole measurement period in all rivers. With scenario calculations including only 30-40 frequently measured pesticides, the number of detected substances and the mixture toxicity would be underestimated on average by a factor of 2. Thus, selecting a subset of substances to assess the surface water quality may be sufficient, but a comprehensive screening yields substantially more confidence.

Exploring the Behaviour of Emerging Contaminants in the Water Cycle using the Capabilities of High Resolution Mass Spectrometry.

To characterize a broad range of organic contaminants and their transformation products (TPs) as well as their loads, input pathways and fate in the water cycle, the Department of Environmental Chemistry (Uchem) at Eawag applies and develops high-performance liquid chromatography (LC) methods combined with high-resolution tandem mass spectrometry (HRMS/MS). In this article, the background and state-of-the-art of LC-HRMS/MS for detection of i) known targets, ii) suspected compounds like TPs, and iii) unknown emerging compounds are introduced briefly. Examples for each approach are taken from recent research projects conducted within the department. These include the detection of trace organic contaminants and their TPs in wastewater, pesticides and their TPs in surface water, identification of new TPs in laboratory degradation studies and ozonation experiments and finally the screening for unknown compounds in the catchment of the river Rhine.

Alleviating the reference standard dilemma using a systematic exact mass suspect screening approach with liquid chromatography-high resolution mass spectrometry.

In this study, the efficiency of a suspect screening strategy using liquid chromatography-high resolution mass spectrometry (LC-HRMS) without the prior purchase of reference standards was systematically optimized and evaluated for assessing the exposure of rarely investigated pesticides and their transformation products (TPs) in 76 surface water samples. Water-soluble and readily ionizable (electrospray ionization) substances, 185 in total, were selected from a list of all insecticides and fungicides registered in Switzerland and their major TPs. Initially, a solid phase extraction-LC-HRMS method was established using 45 known, persistent, and high sales volume pesticides. Seventy percent of these target substances had limit of quantitation (LOQ) < 5 ng L(-1). This compound set was then used to develop and optimize a HRMS suspect screening method using only the exact mass as a priori information. Thresholds for blank subtraction, peak area, peak shape, signal-to-noise, and isotopic pattern were applied to automatically filter the initially picked peaks. The success rate was 70%; false negatives mainly resulted from low intense peaks. The optimized approach was applied to the remaining 140 substances. Nineteen additional substances were detected in environmental samples, two TPs for the first time in the environment. Sixteen substances were confirmed with reference standards purchased subsequently, while three TP standards could be obtained from industry or other laboratories. Overall, this screening approach was fast and very successful and can easily be expanded to other micropollutant classes for which reference standards are not readily accessible such as TPs of household chemicals.

Multi-level approach for the integrated assessment of polar organic micropollutants in an international lake catchment: the example of Lake Constance.

Polar organic micropollutants (MPs) can have ecotoxicological effects on aquatic ecosystems and their occurrence in drinking water is a threat to public health. An extensive exposure assessment of MPs in large river and lake catchments is a necessary but challenging proposition for researchers and regulators. To get a complete picture of MP exposure in a large catchment, we employed a novel integrated strategy including MP measurement in the international catchment of Lake Constance and mass-flux modeling. A comprehensive screening of 252 MPs in the lake water by high-resolution mass spectrometry was used to identify the most commonly present MPs for the study site. It was found that the wastewater borne MPs diclofenac, carbamazepine, sulfamethoxazole, acesulfame, sucralose, benzotriazole, and methylbenzotriazole accounted for the most frequent and prominent findings. The concentration pattern of these compounds in the catchment was calculated based on regionalized inputs from wastewater treatment plants (WWTPs) and substance specific elimination rates. In 52, 8, and 3 of the 112 investigated river locations the concentration exceeded the predicted no-effect levels for diclofenac, sulfamethoxazole and carbamazepine, respectively. By coupling the catchment and lake model the effect of future trends in usage as well as possible mitigation options were evaluated for the tributaries and the lake. The upgrade of the major WWTPs in the catchment with a postozonation step would lead to a load reduction between 32% and 52% for all substances except for sucralose (10%).