Effect-Directed Analysis Combined with Nontarget Screening to Identify Unmonitored Toxic Substances in the Environment.

Effect-directed analysis (EDA) combined with nontarget screening (NTS) has established a valuable tool for the identification of unmonitored toxic substances in environmental samples. It consists of three main steps: (1) highly potent fraction identification, (2) toxicant candidate selection, and (3) major toxicant identification. Here, we discuss the methodology, current status, limitations, and future challenges of EDA combined with NTS. This method has been applied successfully to various environmental samples, such as sediments, wastewater treatment plant effluents, and biota. We present several case studies and highlight key results. EDA has undergone significant technological advancements in the past 20 years, with the establishment of its key components: target chemical analysis, bioassays, fractionation, NTS, and data processing. However, it has not been incorporated widely into environmental monitoring programs. We provide suggestions for the application of EDA combined with NTS in environmental monitoring programs and management, with the identification of further research needs.

Identification of Mid-Polar and Polar AhR Agonists in Cetaceans from Korean Coastal Waters: Application of Effect-Directed Analysis with Full-Scan Screening.

Major aryl hydrocarbon receptor (AhR) agonists were identified in extracts of blubber, liver, and muscle from six long-beaked common dolphins (Delphinus capensis) and one fin whale (Balaenoptera physalus) collected from Korean coastal waters using effect-directed analysis. Results of the H4IIE-luc bioassay indicated that the polar fractions of blubber and liver extracts from the fin whale exhibited relatively high AhR-mediated potencies. Based on full-scan screening with high-resolution mass spectrometry, 37 AhR agonist candidates, spanning four use categories: pharmaceuticals, pesticides, cosmetics, and natural products, were selected. Among these, five polar AhR agonists were newly identified through toxicological confirmation. Concentrations of polar AhR agonists in cetaceans were tissue-specific, with extracts of blubber and liver containing greater concentrations than muscle extracts. Polar AhR agonists with great log KOA values (>5) were found to biomagnify in the marine food chain potentially. Polar AhR agonists contributed 8.9% of the observed AhR-mediated potencies in blubber and 49% in liver. Rutaecarpine and alantolactone contributed significantly to the total AhR-mediated potencies of blubber, whereas hydrocortisone was a major AhR contributor in the liver of the fin whale. This study is the first to identify the tissue-specific accumulation of polar AhR agonists in blubber and liver extracts of cetaceans.

Characterization of microalgal toxicants in the sediments from an industrial area: Application of advanced effect-directed analysis with multiple endpoint bioassays.

Microalgal toxicants in sediments from an industrialized area (Ulsan Bay) in South Korea were identified using effect-directed analysis (EDA) with full-scan screening analysis (FSA) and microalgal bioassays with multiple endpoints. The growth rate and cell viability of three microalgae (Isochrysis galbana, Dunaliella tertiolecta, and Phaeodactylum tricornutum) were strongly inhibited following exposure to raw organic extracts of sediments from Site D5 (Woehang River). The polar fraction separated using a silica gel column significantly inhibited growth rate, esterase activity, cell membrane intensity, and chlorophyll a autofluorescence. In comparison, non- and mid-polar fractions induced non-toxic or esterase inhibition. Target toxicants, such as polycyclic aromatic hydrocarbons, styrene oligomers, and alkylphenols, were detected at low concentrations (450, 79, and 98 ng g-1 dw, respectively) in the sediment of D5, indicating the presence of unmonitored toxicants. FSA was performed for the polar fraction using LC-QTOFMS, and 31 candidates of toxicants were selected. Toxicological confirmation was conducted for 7 candidates for which standards are available. Out of these, 2-nitrophenol, 3-nitrophenol, and 4-nitrophenol showed significant microalgal toxicity; however, these compounds did not fully explain the induced toxicity. Overall, combining EDA and FSA with multiple endpoint bioassays demonstrated the benefits of characterizing the microalgal toxicants in the environments.

Molecular Characterization of Estrogen Receptor Agonists during Sewage Treatment Processes Using Effect-Directed Analysis Combined with High-Resolution Full-Scan Screening.

Endocrine-disrupting potential was evaluated during the sewage treatment process using in vitro bioassays. Aryl hydrocarbon receptor (AhR)-, androgen receptor (AR)-, glucocorticoid receptor (GR)-, and estrogen receptor (ER)-mediated activities were assessed over five steps of the treatment process. Bioassays of organic extracts showed that AhR, AR, and GR potencies tended to decrease through the sewage treatment process, whereas ER potencies did not significantly decrease. Bioassays on reverse-phase high-performance liquid chromatography fractions showed that F5 (log KOW 2.5-3.0) had great ER potencies. Full-scan screening of these fractions detected two novel ER agonists, arenobufagin and loratadine, which are used pharmaceuticals. These compounds accounted for 3.3-25% of the total ER potencies and 4% of the ER potencies in the final effluent. The well-known ER agonists, estrone and 17ß-estradiol, accounted for 60 and 17% of the ER potencies in F5 of the influent and primary treatment, respectively. Fourier transform ion cyclotron resonance mass spectrometry analysis showed that various molecules were generated during the treatment process, especially CHO and CHOS (C: carbon, H: hydrogen, O: oxygen, and S: sulfur). This study documented that widely used pharmaceuticals are introduced into the aquatic environments without being removed during the sewage treatment process.

Identification of AhR agonists in sediments of the Bohai and Yellow Seas using advanced effect-directed analysis and in silico prediction.

Novel aryl hydrocarbon receptor (AhR) agonists were identified in coastal sediments in the Yellow and Bohai Seas by use of a combination of effect-directed analysis (EDA) and in silico prediction. A total of 125 sediments were screened for AhR-mediated potencies using H4IIE-luc bioassay. Great potencies were observed in organic extracts, mid-polar fraction (F2), and subfractions of F2 (F2.6-F2.9) of sediments collected from Nantong, Qinhuangdao, and Yancheng. Less than 15% AhR potencies could be explained by detected dioxin-like PAHs. Full-scan screening analysis was conducted for the more potent fractions using GC-QTOFMS to investigate the presence of unmonitored AhR agonists. A five-step prioritization strategy was applied; 92 candidate compounds satisfied all criteria. Among these chemicals, thirteen were evaluated for AhR efficacy. Six compounds; benz[b]anthracene, 6-methylchrysene, 2-methylbenz[a]anthracene, 1-methylbenz[a]anthracene, 1,12-dimethylbenzo[c]phenanthrene, and indeno[1,2,3-cd]fluoranthene, exhibited significant AhR-mediated efficacies. 1,12-dimethylbenzo[c]phenanthrene and indeno[1,2,3-cd]fluoranthene were identified as novel AhR agonists. Potency balance analysis showed that the six newly identified AhR agonists explained 0.4-100% of the total AhR-mediated potencies determined. Overall, combining EDA and in silico prediction applied in this study demonstrated the benefits of assessing the potential toxic effects of previously unidentified AhR agonists in sediments from the coasts of China and Korea.

Spatiotemporal distribution characteristics of yessotoxins and pectenotoxins in phytoplankton and shellfish collected from the southern coast of South Korea.

The distribution characteristics of lipophilic marine biotoxins (LMTs), such as yessotoxins (YTXs) and pectenotoxins (PTXs) in phytoplankton, mussels, and commercial seafood were determined for the southern coast of South Korea. Gonyaulax spinifera and Dinophysis acuminata, which are the causative microalgae of YTXs and PTXs, were recorded during summer. Homo-YTX and PTX-2 were predominantly detected in phytoplankton (max: 5.7 µg g-1 ww), whereas only YTXs were detected in mussels (max: 1.1 µg g-1 ww). LMT concentrations in mussels were positively correlated with those in phytoplankton. However, there was a 1-month time gap in maximum LMT concentrations between mussels and phytoplankton. Homo-YTX was detected in commercial seafood, including red scallop and comb pen shell. However, homo-YTX concentrations in shellfish were below the recommended value of the European Food Safety Authority (3.75 mg YTX equivalents kg-1); thus, the consumption of this seafood was not considered to be a significant risk for human health.

Identification of novel polar aryl hydrocarbon receptor agonists accumulated in liver of black-tailed gulls in Korea using advanced effect-directed analysis.

Although bioaccumulation of persistent organic pollutants in seabirds has been examined, few studies have been conducted to identify previously unidentified substances. Here, aryl hydrocarbon receptor (AhR) agonists were identified in livers of black-tailed gulls from South Korea using effect-directed analysis combined with full-scan screening analysis. Significant AhR-mediated potencies were observed in the polar fractions of liver extracts using H4IIE-luc bioassay. Eight known polar AhR agonists accounted for 11-20% of the total AhR-mediated potencies in the polar fractions; hydrocortisone and rutaecarpine were the major contributors. Twenty-two AhR agonist candidates in the polar fractions were identified using liquid chromatography-quadrupole time-of-flight mass spectrometry during a six-step selection process. Of these, [10]-gingerol, angelicin, corticosterone, eupatilin, etofenprox, oxadixyl, and tretinoin were identified as novel AhR agonists. The contribution to potencies increased with inclusion of novel AhR agonists (27-52%); corticosterone and [10]-gingerol contributed significantly. Quantitative structure-activity relationship suggested that the novel AhR agonists have other potential toxic effects, including carcinogenicity and mutagenicity. Polar AhR agonists have been used for pharmaceuticals and pesticides. Some novel AhR agonists have log KOW > 2 and log KOA ≥ 6, which indicates that these compounds can be biomagnified in air-breathing organisms, such as seabirds.

Effect-directed identification of novel aryl hydrocarbon receptor-active aromatic compounds in coastal sediments collected from a highly industrialized area.

In this study, we identified major aryl hydrocarbon receptor (AhR) agonists in the sediments from Yeongil Bay (n = 6) using effect-directed analysis. Using the H4IIE-luc bioassays, great AhR-mediated potencies were found in aromatic fractions (F2) of sediment organic extracts from silica gel column chromatography and sub-fractions (F2.6-F2.8) from reverse phase-HPLC. Full-scan mass spectrometric analysis using GC-QTOFMS was conducted to identify novel AhR agonists in highly potent fractions, such as F2.6-F2.8 of S1 (Gumu Creek). Selection criteria for AhR-active compounds consisted of three steps, including matching factor of NIST library (≥70), aromatic structures, and the number of aromatic rings (≥4). Fifty-nine compounds were selected as tentative AhR agonist candidates, with the AhR-mediated activity being assessed for six compounds for which standard materials were available commercially. Of these compounds, 20-methylcholanthrene, 7-methylbenz[a]anthracene, 10-methylbenz[a]pyrene, and 7,12-dimethylbenz[a]anthracene exhibited significant AhR-mediated potency. Relative potency values of these compounds were determined relative to benzo[a]pyrene to be 3.2, 1.4, 1.2, and 0.2, respectively. EPA positive matrix factorization modeling indicated that the sedimentary AhR-active aromatic compounds primarily originated from coal combustion and vehicle emissions. Potency balance analysis indicated that four novel AhR agonists explained 0.007% to 1.7% of bioassay-derived AhR-mediated potencies in samples.

Novel polar AhR-active chemicals detected in sediments of an industrial area using effect-directed analysis based on in vitro bioassays with full-scan high resolution mass spectrometric screening.

Studies investigating aryl hydrocarbon receptor (AhR)-active compounds in the environment typically focus on non- and mid-polar substances, such as PAHs; while, information on polar AhR agonists remains limited. Here, we identified polar AhR agonists in sediments collected from the inland creeks of an industrialized area (Lake Sihwa, Korea) using effect-directed analysis combined with full-scan screening analysis (FSA; using LC-QTOFMS). Strong AhR-mediated potencies were observed for the polar and latter fractions of RP-HPLC (F3.5-F3.8) from sediment organic extracts in the H4IIE-luc in vitro bioassays. FSA was performed on the corresponding fractions. Twenty-eight tentative AhR agonists were chosen using a five-step process. Toxicological confirmation using bioassay revealed that canrenone, rutaecarpine, ciprofloxacin, mepanipyrim, genistein, protopine, hydrocortisone, and medroxyprogesterone were significantly active. The relative potencies of these AhR-active compounds compared to that of benzo[a]pyrene ranged from 0.00002 to 2.0. Potency balance analysis showed that polar AhR agonists explained, on average, ~6% of total AhR-mediated potencies in samples. Some novel polar AhR agonists also exhibited endocrine-disrupting potentials capable of binding to estrogen and glucocorticoid receptors, as identified by QSAR modeling. In conclusion, the focused studies on distributions, sources, fate, and ecotoxicological effects of novel polar AhR agonists in the environment are necessary.