Methodological advances and future directions of microalgal bioassays for evaluation of potential toxicity in environmental samples: A review.

Microalgal bioassays are widely applied to evaluate the potential toxicity of various persistent toxic substances in environmental samples due to multiple advantages, including high sensitivity, short test duration, and cost-effectiveness. Microalgal bioassay is gradually developing in method, and the scope of application to environmental samples is also expanding. Here, we reviewed the published literature on microalgal bioassays for environmental assessments, focusing on types of samples, sample preparation methods, and endpoints, and highlighted key scientific advancements. Bibliographic analysis was performed with the keywords 'microalgae' and 'toxicity' or 'bioassay', and 'microalgal toxicity'; 89 research articles were selected and reviewed. Traditionally, most studies implementing microalgal bioassays focused on water samples (44%) with passive samplers (38%). Studies using the direct exposure method (41%) of injecting microalgae into sampled water mainly evaluated toxic effects by growth inhibition (63%). Recently, various automated sampling techniques, in situ bioanalytical methods with multiple endpoints, and targeted and non-targeted chemical analyses have been applied. More research is needed to identify causative toxicants affecting microalgae and to quantify the cause-effect relationships. This study provides the first comprehensive overview of recent advances in microalgal bioassays performed with environmental samples, suggesting future research directions based on current understanding and limitations.

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.

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.

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.

Identification of potential toxicants in sediments from an industrialized area in Pohang, South Korea: Application of a cell viability assay of microalgae using flow cytometry.

Potential toxicants in sediments collected from an industrialized bay of Korea were identified by use of effect-directed analysis (EDA). Three marine microalgal bioassays (Dunaliella tertiolecta, Isochrysis galbana, and Phaeodactylum tricornutum) with diverse endpoints were employed. Initial screening of raw organic extracts of sediments indicated large variations among locations and species in a traditional endpoint "inhibition of growth". After fractionation, inhibition of growths increased significantly, particularly in some fractions containing aromatics with log KOW 5-6 (F2.6). While viabilities of cells were adversely affected in more fractions, including F2.6-F2.7 (log KOW 5-7) and F3.5-F3.6 (log KOW 4-6). Among the several endpoints of viability, esterase activity seemed to be more sensitive, followed by integrity of cell membranes, chlorophyll a, cell size, and intracellular complexity. Instrumental analyses indicated that toxicities to microalgae observed in F2.7 could not be fully explained by target PAHs. Full-scan screening analysis using GC-QTOFMS identified 58 compounds in F2.7 with matching scores ≥90%. Based on toxic potencies for these compounds predicted by ECOSAR, several causative agents, including 1-phenylpyrene, dibenz[a,c]anthracene, and picene were suggested. Overall, viability of microalgae provided sensitive and high-resolution toxicity screening of samples into integrative assessment of sediment.

Multiple Bioassays and Targeted and Nontargeted Analyses to Characterize Potential Toxicological Effects Associated with Sediments of Masan Bay: Focusing on AhR-Mediated Potency.

An enhanced, multiple lines of evidence approach was applied to assess potential toxicological effects associated with polluted sediments. Two in vitro bioassays (H4IIE-luc and Vibrio fischeri) and three in vivo bioassays (microalgae: Isochrysis galbana and Phaeodactylum tricornutum; zebrafish embryo: Danio rerio) were applied. To identify causative chemicals in samples, targeted analyses (polycyclic aromatic hydrocarbons (PAHs), styrene oligomers (SOs), and alkylphenols) and nontargeted full-scan screening analyses (FSA; GC- and LC-QTOFMS) were performed. First, great AhR-mediated potencies were observed in midpolar and polar fractions of sediment extracts, but known and previously characterized AhR agonists, including PAHs and SOs could not fully explain the total potencies of samples. Enoxolone was identified as a novel AhR agonist in a highly potent sediment fraction by use of FSA. Enoxolone has a relative potency of 0.13 compared to benzo[a]pyrene (1.0) in the H4IIE-luc bioassay. Nonylphenols associated with membrane damage that influenced the viability of the microalgae were also observed. Finally, inhibitions of bioluminescence of V. fischeri and lethality of D. rerio embryos were strongly related to nonpolar compounds. Overall, the present work addressed assay- and end point-specific variations and sensitivities for potential toxicities of mixture samples, warranting a significant utility of the "multiple lines of evidence" approach in ecological risk assessment.