Bioluminescence Inhibition Research Articles

A bioluminescence-based bioassay for hazard assessment of food-grade silver foil (E174) and its validation by atomic absorption spectroscopy

E174 (elemental silver) is a globally used food additive to increase its aesthetic value. Quality E174 manufacturing demands skilled personnel and purity of silver, making the process expensive. In this regard, cases wherein food commodities are coated with spurious E174 are rising. The contaminants used for the cheap production of E174 are comparatively inexpensive metals such as aluminum (Al), and copper (Cu) that increase the malleability, and ductility of the end product, but are hazardous to living organisms at higher concentrations. Bioluminescent bacteria (BB) having an in vivo luminescence of intensity (I0) undergo bioluminescence inhibition (BLI) on acute exposure to a hazardous material; emitting a lowered luminescence intensity (I) that is measured using a luminometer and expressed in terms of relative luminescence unit (RLU).In the present research, the effect of E174 samples procured from nine different vendors was observed on calcium alginate immobilized bioluminescent bacteria (biophotonic bead, BB’) in terms of % residual luminescence (% RL) expressed as [(I/I0)*100]. Further, the E174 samples (c, e, f, g, h, and i) were analyzed for the metal contaminants (Al, and Cu) using atomic absorption spectroscopy (AAS). The E174 samples were tested at 500 ppm, 0.5 ppm, and 5 ppb for their effect on BB’ in terms of %RL. A comparison of % RL with AAS strongly suggests the role of hazardous metal contaminants in mediating BLI response. Therefore, BB’ may be perceived as a rapid, sensitive, and inexpensive biosensing tool for the quality assessment of E174.

Disposable Face Masks into aquatic media: chemical and biological testing of the released compounds during the leaching process

The present study investigated the fate, and the biological effects posed by the presence of Disposable Face Masks (DFMs) into fresh- and saltwater media, using both chemical and biological testing. To this end, slightly fragmented DFMs were maintained in tanks with artificial sea water (ASW) or dH2O (DFMASW and DFMdH2O, respectively) for a period of 20 days (under continuous agitation, oxygen supply, and light/dark ration 1:1) to simulate both fresh- and saltwater natural conditions. Thereafter, DFMs leaching substances were determined, before proceeding to biological testing with the use of the marine bacterium Aliivibrio fischeri (Bioluminescence Inhibition assay), the fresh- and saltwater algal species Chlorococcum sp. and Tetraselmis suecica (algal bioassays), as well as the fairy shrimp Thamnocephalus platyurus, the water flea Daphnia magna, and the rotifer Brachionus calyciflorus (acute toxicity screening tests, in terms of microbiotest). According to the results, once into aquatic media (DFMASW and DFMdH2O) DFMs are subjected to degradation, leading to the release of organic, inorganic, and polymeric compounds (PP microfibers). Considering that possible interactions of the leaching substances could differentially affect the aquatic biota, the present study showed that DFMs leaching substances could be harmful to the fairy shrimp T. platyurus, and the water flea D. magna, with slight to non-toxic effects to be observed in case of Chlorococcum sp. and Tetraselmis suecica, the marine bacterium Aliivibrio fischeri, and the rotifer B. calyciflorus. The present findings showed that the DFMs improper disposal into fresh- and/or saltwater media, followed by their degradation and leaching processes, could lead to the release of substances of great environmental concern, thus promoting awareness about their proper handling and management, as well as the long-term monitoring of their environmental risk.

Parameters Optimization for Improving Bioluminescence Inhibition Assay Using Vibrio fischeri Bacteria to Detect Lipopolysaccharide Toxicity in Aquatic Environments.

Bioluminescence inhibition of Vibrio fischeri is a widely used method for toxicity testing in aquatic environments. Certain complex biological contaminants, such as lipopolysaccharide (LPS), can interfere with metabolic pathways during toxicity assays. The standard 15-minute Vibrio fischeri bioluminescence assay has limitations when evaluating and screening water toxicity against complex and emerging chemicals like LPS. To accurately determine the effects of such substances, it is crucial to use a bioassay that encompasses a sufficient cell cycle period. This study tested LPS at varying incubation times (ranging from 60s to 60min) and concentrations (1-1*10- 12 mg/ml) to identify the appropriate incubation time for bioluminescence inhibition and toxicity testing. The results indicated that bioluminescence inhibition begins within 60s and reaches maximum inhibition at 60min. However, at 30 and 45min, the bacterial response to different concentrations of LPS varied, with some concentrations causing increased bioluminescence. The EC50 values at different times (60s, 15, 30, 45, and 60min) were found to be 0.0012, 0.0063, 4.07e + 54, 3.85e-8, and 3.34e-9mg/ml respectively. This study highlights the importance of considering incubation time when using bioluminescence inhibition to detect acute toxicity in aquatic ecosystems. A longer incubation time may enhance the method's sensitivity and improve its ability to detect low levels of toxins, such as LPS, in water resources.

Emission Factors, Chemical Composition and Ecotoxicity of PM10 from Road Dust Resuspension in a Small Inland City

Road dust resuspension in urban environments can contribute to high human exposure to metal(loid)s, polycyclic aromatic hydrocarbons, and other potentially toxic organic compounds. However, for many regions, information on loadings, emission factors and chemical profiles is lacking to accurately apply emission inventories and source apportionment models. In the present study, PM10 samples were collected with an in situ road dust sampler from eleven representative streets of Bragança, an inland city of the Iberian Peninsula, and were analysed for organic and elemental carbon by a thermal-optical technique, elemental composition by ICP-MS and ICP-OES, and ecotoxicity by a luminescence inhibition bioassay with Allivibrio fischeri. A global emission factor of 5.36 ± 2.35 mg veh−1 km−1 was obtained but in suburban areas the values reached twice the average. Total carbon accounted for 14.9 ± 6.8% of the PM10 mass, while element oxides represented the largest share (28.6 ± 18.7%). Very high enrichments were found for typical traffic-related elements such as Cu, Zn, S, Pb and Ni. The geochemical index Igeo further confirmed that road dust of the study region is extremely contaminated by elements mainly originated from tyre and brake wear. Although the total non-carcinogenic and carcinogenic risks associated with metal exposure were found to be low for both children and adults, the bioluminescence inhibition assay showed (eco)toxic responses for all samples, indicating that road dust resuspension may pose a significant human health and ecological threat.

Biotransformation of diclofenac by Stenotrophomonas humi strain DIC_5 and toxicological examination of the resulting metabolites

The widely used non-steroidal anti-inflammatory drug, diclofenac, detected in increasing concentrations in freshwater ecosystems, is among the most pressing environmental problems today. In this study, the bacterial isolate Stenotrophomonas humi strain DIC_5 was capable of degrading diclofenac. It eliminated 75.1% of diclofenac at an initial concentration of 1.5 mg/L after 8 days in the presence of glucose (3.0 g/L). During the process, nitro-diclofenac was identified as a resulting metabolite, whose concentration increased significantly in the bacterial medium from the 7th day of the experiment, while the concentration of diclofenac decreased correspondingly. The ecotoxicological tests on Aliivibrio fischeri and zebrafish embryos showed that the bacterial metabolites without diclofenac have a higher toxicity (up to 35.5% bacterial bioluminescence inhibition and 36.7% embryo mortality) than the diclofenac degradation residues (28% and 26.7%, respectively). Based on these results, neither diclofenac nor its degradation products exhibit toxic effects on the test organisms. Conversely, the toxic effect caused by the bacteria was reduced in the presence of diclofenac. Our work highlights the importance of using biotic controls in biotransformation trials, especially when the foreign material is applied in intermediate or environmentally relevant concentration ranges.Key points• Biotransformation of diclofenac by bacteria isolated from a bacterial biofilm.• Biotransformation of diclofenac led to the formation of nitro-diclofenac.• Microorganisms are alternatives for reducing the concentration of diclofenac in water.

Application of a laccase-catalyzed oxidative coupling for the removal and detoxification of typical flotation reagent salicylhydroxamic acid

Residual organic flotation reagents in mineral processing wastewater (MPW) can pose environmental hazards and hinder the sustainable development of the mining industry. Laccase as a versatile biocatalyst can eliminate various recalcitrant contaminants from the environment, but its potential applications for the removal and detoxification of organic flotation reagents have until now been overlooked. Herein, the influence of superparamagnetic immobilized laccase (SPM-IMLac) on the enzymatic removal of salicylhydroxamic acid (SHA), a typical mineral flotation reagent, as well as the mechanism of this process have been explored for the first time. The results indicated that SPM-IMLac can effectively remove SHA over a broad pH range and this process follows the pseudo-first-order reaction kinetics. A methodology which combined high resolution mass spectrometry (HRMS) and a solid phase extraction (SPE) technique revealed that SPM-IMLac could catalyze a single-electron oxidation of SHA to generate self/cross-oligomers via radical-driven C-C and/or C-O-C covalent coupling pathways. The Vibrio fischeri bioluminescence inhibition assay and a total organic carbon (TOC) analysis corroborated that the SHA transformation was accompanied by detoxification and by a reduction in TOC of the reaction solution, the extent of which correlated with the extent of the oxidative coupling. Moreover, SPM-IMLac could be easily recovered and reused, while keeping its high removal efficiency of SHA in the actual MPW matrices. This study not only provides fascinating insights into the transformation mechanisms and environmental fate of SHA mediated by SPM-IMLac, but it also exploits a viable and sustainable approach for the remediation of organic flotation reagents-contaminated MPW.

Pyrrolidinium-Based Ionic Liquids as Advanced Non-Aqueous Electrolytes for Safer Next Generation Lithium Batteries

In the current context of increasing energy demand, ionic liquids (ILs) are presented as possible candidates to replace conventional electrolytes and to develop more efficient energy storage devices. The IL 1-Methyl-1-propylpyrrolidinium bis(trifluoromethanesulfonyl)imide has been selected for this work, due to the good thermal and chemical stabilities and good electrochemical performance of the pyrrolidinium cation based ILs. Binary mixtures of this IL and lithium salt with the same anion, [TFSI], have been prepared with the aim of assessing them, as possible electrolytes for lithium batteries. These mixtures were thermally and electrochemically characterised through DSC and dielectric spectroscopy studies. The ionic conductivity decreases as the salt concentration increases, finding values ranging between 0.4 S/m and 0.1 S/m at room temperature. Additionally, a wide liquid range was found for the mixtures, which would reduce or even eliminate some of the most common problems of current electrolytes, such as their crystallisation at low temperatures and flammability. Finally, the toxicity of pure IL and the intermediate salt concentration was also evaluated in terms of the bioluminescence inhibition of the Alivibrio Fischeri bacteria, observing that, although the toxicity increases with the salt addition, both samples can be classified as practically harmless.

Application of the luminous bacterium Photobacterium phosphoreum for toxicity monitoring of selenite and its reduction to selenium(0) nanoparticles

Luminous marine bacteria are traditionally used as a bioassay due to the convenience and high rate of registering the intensity of their physiological function – luminescence. This study aimed to develop the application of Photobacterium phosphoreum in traditional and novel fields – toxicity monitoring and biotechnology. We demonstrated (1) effects of selenite ions on bioluminescence, and (2) biotransformation of selenite to selenium(0) in the form of nanoparticles. The effects of selenite (SeO32−) on the intensity of bacterial bioluminescence were studied, and its dependencies on exposure time and concentration of Na2SeO3 were analyzed. Bioluminescence activation and inhibition were revealed; dose–effect dependencies corresponded to the hormesis model. The toxicity of SeO32− was characterized by an effective concentration of 10−3 M. Effects of SeO32− on reactive oxygen species (ROS) in bacterial suspensions were studied. High positive correlations were found between the bioluminescence intensity and ROS content, which indicates the decisive role of ROS and associated redox processes in the bioeffects of selenite ions. Scanning and transmission electron microscopy revealed the presence of nano-structures in the bacteria exposed to selenite. The energy dispersion spectrum detected a high content of selenium in the nanoparticles. The particle size distribution depended on Na2SeO3 concentration; maxima of the distribution varied within 45–55 nm.

Winter indoor air quality in traditional Mongolian yurts, in a Ger district of Ulaanbaatar

In Ulaanbaatar roughly 60 % of the population live in traditional Mongolian yurts in the so-called Ger districts of the city. Winter indoor air quality is a serious concern in these districts as about 98 % of households consume solid fossil fuel (mainly coal). In our study, indoor air quality was assessed based on PAHs analysis and ecotoxicity testing of 24-hour samples collected in 4 yurts. Three of the selected yurts were equipped with conventional while the fourth one with improved stoves. Analysis of PAHs profiles showed the prevalence of higher molecular weight PAHs in all yurts. Concentrations of the 5-ring benzo(b)fluoranthene and 6-ring benzo(g.h.i)perylene were extremely high in one yurt using conventional stove, 8430 µg g−1 and 6320 µg g−1, respectively. Ecotoxicity of the samples was assessed using the kinetic version of the Vibrio fischeri bioluminescence inhibition bioassay. In concordance with PAHs concentrations, ecotoxicity was also the highest in that yurt.

From bacteria to fish: ecotoxicological insights into sulfamethoxazole and trimethoprim

Sulfamethoxazole (SMX) and trimethoprim (TRIM) are two of the most used antibiotics in the last 50 years, to prevent and treat bacterial infections; however, the available literature about toxicity to non-target organisms is quite discrepant and incomplete. This study aims to assess the SMX and TRIM ecotoxicological effects in standard species: Aliivibrio fischeri (bioluminescence inhibition), Escherichia coli ATCC 25922 (growth inhibition), Lemna minor (growth inhibition and biochemical biomarkers), Daphnia magna (immobilization/mortality, life history traits, and biochemical biomarkers), and Danio rerio (survival, hatching, abnormalities, and biochemical biomarkers). The species tested showed different acute sensitivities to SMX (A. fischeri < D. magna < E. coli < L. minor) and TRIM (L. minor < A. fischeri < D. magna < E. coli). Overall, TRIM reveals less toxicity than SMX, except for E. coli (Ecotoxicological approach based on Antimicrobial Susceptibility Testing – EcoAST procedure). Both antibiotics affect individually (e.g., growth and survival) and sub-individually (e.g., antioxidant defenses) L. minor, D. magna, and D. rerio. This study allowed us to generate relevant data and fill gaps in the literature regarding the effects of SMX and TRIM in aquatic organisms. The here-obtained results can be used to (i) complete and re-evaluate the Safety Data Sheet to improve the assessment of environmental safety and management of national and international entities; (ii) clarify the environmental risks of these antibiotics in aquatic ecosystems reinforcing the inclusion in the 4th Watch List of priority substances to be monitored in whole inland waters by the Water Framework Directive; and (iii) combat the development of antimicrobial resistance, as well as supporting the definition of environmental measurements in the context of European One Health Action Plan. However, it is essential to continue studying these antibiotics to better understand their toxicity at ecologically relevant concentrations and their long-term effects under different climatic change scenarios.

Bioluminescence Inhibition Bioassay for Estimation of Snow Cover in Urbanised Areas within Boreal Forests of Krasnoyarsk City

It has been proposed that the level of air pollution in a city should be estimated based on the accumulation of pollutants in the snow cover of urban forests. This study presents a bioluminescence method for estimating the extent of snow cover pollution in the urbanised areas of boreal forests in Krasnoyarsk city. A bioluminescent assay involving NAD(P)H:FMN oxidoreductase (Red) and luciferase with luminous bacteria (BLuc) was employed to measure the concentrations of six heavy metals (As, Cd, Zn, Co, Hg, and Pb) in the snow cover. The tested snow samples demonstrated a correlation between the reduced activity of the enzyme system and variations in Cd concentration. Furthermore, the research indicated that the period of unfavourable meteorological conditions in Krasnoyarsk city resulted in a notable decline in the activity of the BLuc–Red enzyme system, which may be associated with elevated air pollution levels. This study underscores the potential of the bioluminescence method for monitoring environmental pollution in urban forested areas.

A homogeneous bioluminescent inhibition immunoassay to detect anti-interferon gamma antibodies.

Adult-onset immunodeficiency with antibodies to interferon-γ (AOID with AIGA), is a rare, acquired immunodeficiency causing susceptibility to disseminated non-tuberculous mycobacteria and other intracellular opportunistic infections. The diagnosis depends on demonstrating the presence of endogenous anti-interferon-γ antibodies (AIGA) that suppress Th1 cell mediated immunity. Bioluminescent immunoassays are a newly emerging immunoassay format which utilise the action of bioluminescent enzymes on a substrate for specific analyte detection. In-short, detecting antibodies are conjugated with a bioluminescent enzyme. The detecting antibodies bind the analyte of interest and produce light (luminescence) after addition of a substrate. The purpose of this study was to evaluate two newly developed bioluminescent immunoassays using Lumit® (Promega) technology as a diagnostic test for AOID with AIGA. Specific aims included the clinical validation of a new inhibition bioluminescent immunoassay technique to detect AIGA which block detection of interferon-γ (IFN-γ) in-vitro and correlation of inhibition bioluminescent immunoassay results with AOID with AIGA disease status. Two bioluminescent inhibition immunoassays were developed. One which adapted an existing kit from Promega (Lumit® Human IFN-γ Immunoassay) and one which was developed in-house. 87 healthy controls and 48 patients with previously diagnosed AOID with AIGA were recruited and tested using these two methods. Results showed both bioluminescent inhibition immunoassays were able to clearly discriminate between AOID with AIGA patients and healthy controls. The mean inhibition percentage between patient groups correlated with disease activity. Both assays appeared to be more sensitive when compared to the existing inhibition ELISA.

Equations for estimating binary mixture toxicity: 3-methyl-2-butanone with a series of electrophiles.

Mixture toxicity was determined for 32 binary combinations. One chemical was the non-reactive, non-polar narcotic 3-methyl-2-butanone (always chemical A) and the other was a potentially reactive electrophile (chemical B). Bioluminescence inhibition in Allovibrio fischeri was measured at 15-, 30-, and 45-minutes of exposure for A, B, and the mixture (MX). Concentration-response curves (CRCs) were developed for each chemical and used to develop predicted CRCs for the concentration addition (CA) and independent action (IA) mixture toxicity models. Also, MX CRCs were generated and compared with model predictions using the 45-minute data. Classification of observed mixture toxicity used three specific criteria: 1) predicted IA EC50 vs. CA EC50 values at 45-minutes, 2) consistency of 45-minute MX CRC fit to IA, CA, or otherwise at three effect levels (EC25, EC50 and EC75), and 3) the known/suspected mechanism of toxicity for chemical B. Mixture toxicity was then classified into one of seven groupings. As a result of the predicted IA EC50 being more toxic than the predicted CA EC50, IA represented the greater toxic hazard. For this reason, non-sham MXs having toxicity consistent with CA were classified as being "coincident" with CA rather than mechanistically-consistent with CA. Multiple linear regression analyses were performed to develop equations that can be used to estimate the toxicity of other 3M2B-containing binary mixtures. These equations were developed from the data for both IA and CA, at each exposure duration and effect level. Each equation had a coefficient of determination (r2) above 0.950 and a variance inflation factor <1.2. This approach can potentially reduce the need for mixture testing and is amenable to other model systems and to assays that evaluate toxicity at low effect levels.

Bacterial toxicity of Acetaminophen and Edaravone, and their binary mixtures: experimental and predicted values using traditional and novel Van Laar-based models

In recent years, the presence of Pharmaceutical Active Compounds (PhACs) in ecosystems has become a serious environmental problem due to their capacity to induce harmful effects at extremely low concentrations in both humans and wildlife. Water treatment plants have not been designed to remove these types of compounds efficiently. Thus, the detection of these pollutants is essential to evaluate their negative impacts and is one of the emerging issues in environmental chemistry. The main objective of this study is to determine the bacterial toxicity of two PhACs (both individually and as a mixture) through the quantification of bioluminescence inhibition in the marine bacteria Aliivibrio fischeri, a commonly used method in short-term toxicity tests. In this work, Acetaminophen and Edaravone, two drugs approved by the Food and Drug Administration, have been studied. The acute toxicity of these PhACs has been tested at two exposure times (5 and 15 min) and different concentrations, by estimation of the median effective concentration (EC50) for each individual compound or in combination at different concentrations. Moreover, the EC50 of the binary mixtures Acetaminophen/Edaravone have been forecast using two traditional predictive models, Concentration Addition and Independent Action. The results show that toxicity decreases with exposure time and depends on the concentration tested. Furthermore, a novel semi-empirical Van Laar-based model has been proposed and validated with the experimental data from this study and literature data, obtaining satisfactory estimations of the EC50 for binary mixtures.

Application of luminescent Photobacterium Phosphoreum T3 for the detection of zearalenone and estimating the efficiency of their enzymatic degradation

Zearalenone (ZEN), a nonsteroidal estrogenic mycotoxin, causes enormous economic losses in the food and feed industries. Simple, rapid, low-cost, and quantitative analysis of ZEN is particularly urgent in the fields of food safety and animal husbandry. Using the bioluminescent bacterium Photobacterium phosphoreum T3, we propose a bioluminescence inhibition assay to evaluate ZEN levels quickly. The limit of detection (LOD), limit of quantification (LOQ), and quantitative working range of this bioluminescence inhibition assay were 0.1 µg/mL, 5 µg/mL, and 5-100 µg/mL, respectively. The concentration-response curve of the bioluminescence inhibition rate and ZEN concentration was plotted within the range 5 to 100 μg/mL, as follows: y = 0.0069x2 – 0.0190x + 7.9907 (R2 = 0.9943, y is luminescence inhibition rate, x is ZEN concentration). First, we used the bioluminescence inhibition assay to detect the remaining ZEN in samples treated with purified lactonohydrolase ZHD101. The bioluminescence inhibition assay results showed a strong correlation with the HPLC analysis. Furthermore, we successfully evaluated the overall toxicity of samples treated with purified peroxidase Prx and H2O2 using the P. phosphoreum T3 bioluminescence inhibition assay. The results indicate that the degradation products of ZEN created by purified peroxidase Prx and H2O2 showed little toxicity to P. phosphoreum T3. In this study, a simple, rapid, and low-cost assay method of zearalenone by bioluminescent P. phosphoreum T3 was developed. The bioluminescence inhibition assay could be used to estimate the efficiency of enzymatic degradation of ZEN.

Aqueous photolysis of naproxen exposed to UV and natural sunlight: Formation of excited triplet and photosensitizing product

Photochemical transformation is an important attenuation process for the non-steroidal anti-inflammatory drug naproxen (NPX) in both engineered and natural waters. Herein, we investigated the photolysis of NPX in aqueous solution exposed to both ultraviolet (UV, 254 nm) and natural sunlight irradiation. Results show that N2 purging significantly promoted NPX photolysis under UV irradiation, suggesting the formation of excited triplet state (3NPX*) as a critical transient. This inference was supported by benzophenone photosensitization and transient absorption spectra. Sunlight quantum yield of NPX was only one fourteenth of that under UV irradiation, suggesting the wavelength-dependence of NPX photochemistry. 3NPX* formed upon irradiation of NPX underwent photodecarboxylation leading to the formation of 2-(1-hydroxyethyl)-6-methoxynaphthalene (2HE6MN), 2-(1-hydroperoxyethyl)-6-methoxynaphthalene (2HPE6MN), and 2-acetyl-6-methoxynaphthalene (2A6MN). Notably, the conjugation and spin-orbit coupling effects of carbonyl make 2A6MN a potent triplet sensitizer, therefore promoting the photodegradation of the parent NPX. In hospital wastewater, the photolysis of NPX was influenced because the photoproduct 2A6MN and wastewater components could competitively absorb photons. Bioluminescence inhibition assay demonstrated that photoproducts of NPX exhibited higher toxicity than the parent compound. Results of this study provide new insights into the photochemical behaviors of NPX during UV treatment and in sunlit surface waters.

Bio-screening and quantification of methyl paraben in vinegar and coconut juice separated by HPTLC

As a widely used food preservative, methyl paraben was experimentally evidenced with serious hormone-like adverse effects. Herein, a high performance thin-layer chromatography platformed bioluminescent bioautography and image analysis for the selective quantification and confirmation of methyl paraben was proposed and validated in vinegar and coconut juice. First, the detectability of the bioautography to the analyte on different layer materials was estimated, revealing that normal silica gel was the best choice. After that, the liquid of sample extract and working solution were separated to overcome the background noises due to co-extracted matrices. The separation result was then coupled to the optimized bioautography, enabling instant and straightforward screening of the targeted compound. For accurate quantification, bioluminescent inhibition pattern caused by the analyte was processed by image analysis, giving useful sensitivity (LOD > 16 mg/kg), precision (RSD < 10.1 %) and accuracy (spike-recovery rate 76.9 %–112.2 %). Finally, the suspected result was confirmed by determining its MS fingerprint, further strengthening the reliability of screening.

The Cocktail Effects on the Acute Cytotoxicity of Pesticides and Pharmaceuticals Frequently Detected in the Environment.

Xenobiotics never appear as single, isolated substances in the environment but instead as multi-component mixtures. However, our understanding of the ecotoxicology of mixtures is far from sufficient. In this study, three active pharmaceutical ingredients (carbamazepine, diclofenac, and ibuprofen) and three pesticides (S-metolachlor, terbuthylazine, and tebuconazole) from the most frequently detected emerging micropollutants were examined for their acute cytotoxicity, both individually and in combination, by bioluminescence inhibition in Aliivibrio fischeri (NRRL B-11177). Synergy, additive effects, and antagonism on cytotoxicity were determined using the combination index (CI) method. Additionally, PERMANOVA was performed to reveal the roles of these chemicals in binary, ternary, quaternary, quinary, and senary mixtures influencing the joint effects. Statistical analysis revealed a synergistic effect of diclofenac and carbamazepine, both individually and in combination within the mixtures. Diclofenac also exhibited synergy with S-metolachlor and when mixed with ibuprofen and S-metolachlor. S-metolachlor, whether alone or paired with ibuprofen or diclofenac, increased the toxicity at lower effective concentrations in the mixtures. Non-toxic terbuthylazine showed great toxicity-enhancing ability, especially at low concentrations. Several combinations displayed synergistic effects at environmentally relevant concentrations. The application of PERMANOVA was proven to be unique and successful in determining the roles of compounds in synergistic, additive, and antagonistic effects in mixtures at different effective concentrations.

Toxicity of substituted p-phenylenediamine antioxidants and their derived novel quinones on aquatic bacterium: Acute effects and mechanistic insights

Substituted para-phenylenediamines (PPDs) are synthetic chemicals used globally for rubber antioxidation, with their quinone derivatives (PPD-Qs) raising particular environmental concerns due to their severe toxicity to aquatic organisms. Emerging research has identified a variety of novel PPD-Qs ubiquitously detected in the environment, yet experimental proof for the toxicity of PPD-Qs has not been forthcoming due to the unavailability of bulk standards, leaving substantial gaps in the prioritization and mechanistic investigation of such novel pollutants. Here, we use synthesized chemical standards to study the acute toxicity and underlying mechanism of 18 PPD-Qs and PPDs to the aquatic bacterium V. fischeri. Bioluminescence inhibition EC50 of PPD-Qs ranged from 1.76–15.6 mg/L, with several emerging PPD-Qs demonstrating significantly higher toxicity than the well-studied 6PPD-Q. This finding suggests a broad toxicological threat PPD-Qs pose to the aquatic bacterium, other than 6PPD-Q. Biological response assays revealed that PPD-Qs can reduce the esterase activity, cause cell membrane damage and intracellular oxidative stress. Molecular docking unveiled multiple interactions of PPD-Qs with the luciferase in V. fischeri, suggesting their potential functional impacts on proteins through competitive binding. Our results provided crucial toxicity benchmarks for PPD-Qs, prioritized these novel pollutants and shed light on the potential toxicological mechanisms.

Nitrogen-reliant degradation behaviors of characteristic NHCs by UV-based advanced oxidation processes: Kinetics, pathways and toxicity assessment

In recent decades, nitrogen-containing heterocyclic compounds (NHCs) have been widely synthesized and applied due to their excellent coordination capability of heterocyclic N atoms. However, their high toxicity and poor biodegradability pose significant hazards to aquatic ecosystems. Three characteristic NHCs (cNHCs) with similar molecular structures but minor differences in the number and position of N atoms, namely benzotriazole (BTA), benzimidazole (BMZ), and indazole (IDZ), were selected to evaluate impacts of these differences on their degradation behaviors during UV/H2O2 treatments. It was found that the key mechanism for the removal of BTA, BMZ, and IDZ in the UV/H2O2 processes was the generation of ∙OH through UV-excited H2O2 decomposition. Density functional theory (DFT) calculations revealed that the variation in the number and position of heterocyclic ring N atoms significantly affected the electron cloud distribution of the cNHCs, thereby resulting in different reactivities. Mineralization results and N evolution experiments indicated that N elements were initially released in the form of ammonia nitrogen during UV/H2O2 treatments, which then underwent oxidation to form nitrite and nitrate. The two ortho-N atoms were most easily mineralized and release inorganic nitrogen, followed by the two meta-N atoms. However, the BTA compound containing three N atoms, exhibited inertness towards radicals after hydroxylation, thereby hindering mineralization. A dual-directional transformation products (TPs) identification protocol was employed to identify 30 TPs of the cNHCs and propose recommended degradation pathways. Furthermore, Vibrio fischeri bioluminescence inhibition bioassay and QSAR prediction models were used to preliminarily screen and identify 13 toxic TPs with potential ecological risks.