Mechanistic Aspects of Photodegradation of Deoxynucleosides Induced by Triplet State of Effluent Organic Matter.

Excited triplet states of wastewater effluent organic matter (3EfOM*) are known as important photo-oxidants in the degradation of extracellular antibiotic resistance genes (eArGs) in sunlit waters. In this work, we further found that 3EfOM* showed highly selective reactivity toward 2'-deoxyguanosine (dG) sites within eArGs in irradiated EfOM solutions at pH 7.0, while it showed no photosensitizing capacity toward 2'-deoxyadenosine, 2'-deoxythymidine, and 2'-deoxycytidine (the basic structures of eArGs). The 3EfOM* contributed to the photooxidation of dG primarily via one-electron transfer mechanism, with second-order reaction rate constants of (1.58-1.74) × 108 M-1 s-1, forming the oxidation intermediates of dG (dG(-H)•). The formed dG(-H)• could play a significant role in hole hopping and damage throughout eArGs. Using the four deoxynucleosides as probes, the upper limit for the reduction potential of 3EfOM* is estimated to be between 1.47 and 1.94 VNHE. Compared to EfOM, the role of the triplet state of terrestrially natural organic matter (3NOM*) in dG photooxidation was minor (∼15%) mainly due to the rapid reverse reactions of dG(-H)• by the antioxidant moieties of NOM. This study advances our understanding of the difference in the photosensitizing capacity and electron donating capacity between NOM and EfOM and the photodegradation mechanism of eArGs induced by 3EfOM*.

Effects of pH on the triplet state dissolved organic matter induced free available chlorine decay and radical formation.

Triplet state dissolved organic matter (3DOM*) plays a significant role in inducing oxidant decay and radical generation in light-based advanced oxidation processes. However, the effects of pH still need investigation. This work quantitatively analyzed the pH-dependent free available chlorine (FAC) decay and radical formation (i.e., HO• and Cl•) induced by 3DOM* or triplet state photosensitizer (3PS*). Upon UV irradiation at 254 nm, the decay rate of FAC by 3DOM* or 3PS* was the highest at neutral pH, while those by dark reaction of DOM and the direct photolysis of FAC were the highest at acidic conditions. This is attributed to the variation of FAC species, 3DOM* or 3PS* formation, and the reaction rate constants of FAC with 3DOM* or 3PS* at pH 5.0-10.0. 3DOM* and 3PS* formed increasingly with pH varying from 5.0 to 10.0, while their reactivity with FAC decreased due to the speciation from HOCl to OCl-. Radical formation (i.e., HO• and Cl•) from FAC reaction with 3DOM* or 3PS* occurred at all the testing pH range (5.0-10.0). This work highlighted the pH-dependent role of 3DOM* in oxidant decay and radical formation in treating DOM containing waters through oxidant photolysis. ENVIRONMENTAL IMPLICATIONS: Triplet state dissolved organic matter (3DOM*) plays a significant role in inducing oxidant decay and radical generation in light-based AOPs. This study revealed the effects of pH in 3DOM* induced free available chlorine (FAC) decay and radical formation (i.e., HO• and Cl•). With DOM at 3 mgC L-1, FAC decayed fastest under neutral conditions and radical formation (i.e., HO• and Cl•) was enhanced at 5.0-10.0 due to 3DOM* reaction with FAC. These results highlighted the pH-dependent role of 3DOM* in oxidant transformation and radical formation in treating DOM containing waters by AOPs based on oxidant photolysis.

Copper Safeguards Dissolved Organic Matter from Sunlight-Driven Photooxidation.

Sunlight plays a crucial role in the transformation of dissolved organic matter (DOM) and the associated carbon cycle in aquatic environments. This study demonstrates that the presence of nanomolar concentrations of copper (Cu) significantly decreases the rate of photobleaching and the rate of loss of electron-donating moieties of three selected types of DOM (including both terrestrial and microbially derived DOM) under simulated sunlight irradiation. Employing Fourier transform ion cyclotron resonance mass spectrometry, we further confirm that Cu selectively inhibits the photooxidation of lignin- and tannin-like phenolic moieties present within the DOM, in agreement with the reported inhibitory impact of Cu on the photooxidation of phenolic compounds. On the basis of the inhibitory impact of Cu on the DOM photobleaching rate, we calculate the contribution of phenolic moieties to DOM photobleaching to be at least 29-55% in the wavelength range of 220-460 nm. The inhibition of loss of electrons from DOM during irradiation in the presence of Cu is also explained quantitatively by developing a mathematical model describing hydrogen peroxide (a proxy measure of loss of electrons from DOM) formation on DOM irradiation in the absence and presence of Cu. Overall, this study advances our understanding of DOM transformation in natural sunlit waters.

Triplet Photochemistry of Effluent Organic Matter in Degradation of Extracellular Antibiotic Resistance Genes.

Wastewater effluent is a major source of extracellular antibiotic resistance genes (eArGs) in the aquatic environment, a threat to human health and biosecurity. However, little is known about the extent to which organic matter in the wastewater effluent (EfOM) might contribute to photosensitized oxidation of eArGs. Triplet states of EfOM were found to dominate the degradation of eArGs (accounting for up to 85%). Photo-oxidation proceeded mainly via proton-coupled electron transfer reactions. They broke plasmid strands and damaged bases. O2•- was also involved, and it coupled with the reactions' intermediate radicals of eArGs. The second-order reaction rates of blaTEM-1 and tet-A segments (209-216 bps) with the triplet state of 4-carboxybenzophenone were calculated to be (2.61-2.75) × 108 M-1 s-1. Besides as photosensitizers, the antioxidant moieties in EfOM also acted as quenchers to revert intermediate radicals back to their original forms, reducing the rate of photodegradation. However, the terrestrial origin natural organic matter was unable to photosensitize because it formed less triplets, especially high-energy triplets, so its inhibitory effects predominated. This study advances our understanding of the role of EfOM in the photo-oxidation of eArGs and the difference between EfOM and terrestrial-origin natural organic matter.

Reactions of neonicotinoids with peroxydisulfate: The generation of neonicotinoid anion radicals and activation pathway to form sulfate radicals.

To activate persulfate to generate reactive species such as sulfate radical (SO4•-) for micropollutants abatement, external energy or chemicals are often needed. In this study, a novel SO4•- formation pathway was reported during the oxidation of neonicotinoids by peroxydisulfate (S2O82-, PDS) without any other chemical additions. Thiamethoxam (TMX) was used as a representative neonicotinoid and SO4•- was the dominant specie contributing to its degradation during PDS oxidation at neutral pH. TMX anion radical (TMX•-) was found to activate PDS to generate SO4•- with the second-order reaction rate constant determined to be (1.44 ± 0.47)× 106 M-1s-1 at pH 7.0 by using laser flash photolysis. TMX•- was generated from the TMX reactions with superoxide radical (O2•-), which was formed from the hydrolysis of PDS. This indirect PDS activation pathway via anion radicals was also applicable to other neonicotinoids. The formation rates of SO4•- were found to negatively linearly correlated with Egap (LUMO-HOMO). The DFT calculations indicated the energy barrier of anion radicals to activate PDS was greatly reduced compared to the parent neonicotinoids. The pathway of anion radicals' activation of PDS to form SO4•- improved the understanding of PDS oxidation chemistry and provided some guidance to enhance oxidation efficiency in field applications.

Reducing properties of triplet state organic matter (3DOM*) probed via the transformation from chlorine dioxide to chlorite.

The triplet states of dissolved organic matter (3DOM*) have been well known to oxidize various organic contaminants, but evidence of their reducing properties are largely scarce. In this work, chlorine dioxide (ClO2) as a single-electron oxidant was used as a probe to evaluate the reduction property of 3DOM*. The reduction of ClO2 to chlorite was observed in the solutions of model photosensitizers (i.e., 4-carboxybenzophenone, benzophenone, acetophenone, 3-methoxyacetophenone, naphthalene, and xanthone) during UV irradiation with the presence of ClO2, though they are resistant to ClO2 oxidation in the dark. The reducing property of the triplet states of photosensitizers was verified and their second-order reaction rate constants with ClO2 were determined to be in the range of 1.45(± 0.03)× 109 - 2.18(± 0.06) × 109 M-1 s-1 at pH 7.0. The quenching tests excluded the role of other reactive species (e.g., HO•, O(3P), Cl•, ClO• and HOCl/OCl-, O2•- and eaq-) in ClO2 reduction to chlorite when using model photosensitizers and DOM isolates. Chlorite formation was 48.1-90.4% and 4812.8-7721.8% higher during UV irradiation with the presence of ClO2 and DOM than those without UV irradiation or without DOM present, respectively. The enhancement was attributed to the enhanced electron donating capacity (chlorite precursors) of DOM upon UV irradiation and also to 3DOM* acting as an electron donor reducing ClO2 to chlorite. This study highlighted the important role of 3DOM* as a reductant.

Integrated comparison of growth and oxidative stress induced by tylosin in two freshwater algae Chlorella vulgaris and Raphidocelis subcapitata.

Two model algae, Chlorella vulgaris (C. vulgaris) and Raphidocelis subcapitata (R. subcapitata), are commonly used in registration procedures to evaluate compounds with antimicrobial capacity. However, it has been found that these two algae show considerable differences in sensitivity when exposed to antibiotics. The selection of a suitable test species plays a crucial role in assessing the environmental hazards and risks of a compound, as the balance between oxidative stress and antioxidants is a key factor for alga growth. This study was conducted to investigate the status of oxidative stress and mechanism of antioxidant defense system of algae under antibiotic stress. Different tylosin (TYN) exposure-concentrations were used for the tests in this study. Oxidative stress biomarkers (malondialdehyde (MDA)), non-enzymatic antioxidants (reduced glutathione (GSH)), antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GP), glutathione S-transferase (GST)) and photosynthetic pigments were measured to determine the status of the antioxidant defense system. With increasing TYN concentration, the growth of R. subcapitata was significantly inhibited, while there was no effect on C. vulgaris. When the growth of R. subcapitata was inhibited, the content of MDA was significantly increased and the antioxidant system was activated, which indicated a significant increase in the activity of SOD and CAT.

Prediction of adverse effects of effluents containing phenolic compounds in the Ba River on the ovary of fish (Hemiculter leucisculus) using transcriptomic and metabolomic analyses.

The aim of this work was to evaluate the endocrine disrupting effects on the ovarian development of sharpbelly (Hemiculter leucisculus) caused by effluents containing phenolic compounds. This was achieved using integrated transcriptomic and metabolomic analyses, along with histopathological examinations. Sharpbelly, an indigenous freshwater fish widely distributed in East Asia, were collected by pole fishing from three sampling sites in the Ba River. These sampling sites include a mid-stream site near a wastewater outfall and a reference site located upstream and a far field comparison site located downstream. In sharpbelly collected near the wastewater discharge, the oocyte development was activated, compared to the other two sites. Histopathological alterations in the fish ovaries were likely due to the upregulated steroid hormone biosynthesis process, as suggested by the differentially expressed genes (e.g., hsd3b, hsd17b1) and differentially accumulated metabolites (e.g., pregnenolone). Additionally, under the stress of effluents containing phenolic compounds, genes related to the signaling pathways for oxidative phosphorylation and leukocyte transendothelial migration were dysregulated, suggesting the potential induction of inflammation and several ovarian diseases. Overall, these findings suggest that effluents containing phenolic compounds influence ovary development and reproductive function of female sharpbelly. Whether there is any resulting dysfunction of folliculogenesis, abnormality of ovulation, production of premature eggs and/or potential induction of ovarian cancers remains to be determined by further studies, for a better evaluation on effluents containing phenolic compounds to the fish fertility and the health of their offspring, and even the stability of the wild fish population. Notably, the integration of transcriptomics and metabolomics can complement the routine chemical analysis to comprehensively monitor the effects of wastewater treatment plant effluents on the health of wild fish.

Transcriptomic analysis of Raphidocelis subcapitata exposed to erythromycin: The role of DNA replication in hormesis and growth inhibition.

The occurrence of hormesis in the algal growth inhibition test is a major challenge in the dose-response characterization, whereas the molecular mechanism remains unraveled. The aim of this study is therefore to investigate the changes in the molecular pathways in a model green alga Raphidocelis subcapitata treated with erythromycin (ERY; 4, 80, 120 µg L-1) by transcriptomic analysis. After 7 day exposure, ERY at 4 µg L-1 caused hormetic effects (21.9 %) on cell density, whereas 52.0 % and 65.4 % were inhibited in two higher exposures. By using adj p < 0.05 and absolute log2 fold change> 1 as a cutoff, we identified 218, 950, and 2896 differentially expressed genes in 4, 80, 120 µg L-1 treatment groups, respectively. In two higher ERY treated groups, genes involved in phases I, II & III metabolism processes and porphyrin and chlorophyll metabolism pathway were consistently suppressed. Interestingly, genes (e.g., pri2, mcm2, and mcm6) enriched in DNA replication process were up-regulated in 4 µg L-1 group, whereas these genes were all repressed in 120 µg L-1 group. Alteration trend in gene expression was consistent with algal growth. Taken together, our results unveiled the molecular mechanism of action in ERY- stimulated/ inhibited growth in green alga.

Integrative analyses of transcriptomics and metabolomics in Raphidocelis subcapitata treated with clarithromycin.

As a macrolide antibiotic, clarithromycin (CLA) has a high detection rate in surface water and sewage treatment plant effluents worldwide, posing a considerably high ecological risk to aquatic ecosystem. However, algal transcriptome and metabolome in response to CLA remains largely unknown. In this study, a model alga Raphidocelis subcapitata (R. subcapitata), was exposed to CLA at the concentrations of 0, 3, 10, and 15 µg L-1. Transcriptomic analysis was performed for all the treatment groups, whereas metabolomics was merely applied to 0, 3, and 10 µg L-1 groups because of the limited amount of algal biomass. After 7 d cultivation, the growth of R. subcapitata was significantly hindered at the concentrations above 10 µg L-1. A total of 115, 1833, 2911 genes were differentially expressed in 3, 10, and 15 µg L-1 groups, respectively; meanwhile, 134 and 84 differentially accumulated metabolites (DAMs) were found in the 3 and 10 µg L-1 groups. Specifically, expression levels of DEGs and DAMs related to xenobiotic metabolism, electron transport and energy synthesis were dysregulated, leading to the produced reactive oxygen species (ROS). To confront the CLA-induced injury, the biosynthesis of unsaturated fatty acids and carotenoids of R. subcapitata in 3 µg L-1 were up-regulated; although the photosynthesis was up-regulated in both 10 µg L-1 and 15 µg L-1 groups, the energy synthesis and the ability to resist ROS in these two groups were down-regulated. Overall, this study shed light on the mechanism underlying the inhibitory effects of macrolide antibiotics in algae.

Involvement of oxidative stress in the sensitivity of two algal species exposed to roxithromycin.

Algal species Raphidocelis subcapitata and Chlorella vulgaris are commonly used to test the chemicals with an antibacterial mode of action during marketing authorization process. However, significant differences in the sensitivity toward antibiotic exposure have been reported. The selection of an inappropriate test species would thus underestimate the environmental hazard of target chemicals and pose a potential threat to the ecosystem. Since oxidative stress is a crucial factor determining the inhibition of algal growth, an investigation on oxidative stress and antioxidant defense mechanisms in these two species was performed to explore its roles in species sensitivity. Here, roxithromycin (ROX), a macrolide antibiotic extensively used to treat respiratory, urinary and soft tissue infections, was used for testing. After 7 days exposure to ROX at the low (0.01 mg L-1) and high (0.09 mg L-1) concentrations, R. subcapitata was inhibited while the growth of C. vulgaris was stimulated. We investigated the roles of oxidative stress in algae by measuring the oxidative stress biomarkers (MDA), non-enzymatic antioxidants (GSH), and antioxidant enzymes (SOD, CAT, GP, GST). The results suggested that when the growth of algae is inhibited, MDA content as well as activities of oxidative stress enzymes would increase, and thus, activating the antioxidant system. On the contrary, it was inferred that when the growth is stimulated, MDA content and oxidative stress enzymes activities would decrease.

Comparison of oxidative stress induced by clarithromycin in two freshwater microalgae Raphidocelis subcapitata and Chlorella vulgaris.

Clarithromycin (CLA), a macrolide antibiotic, has been frequently detected in the global surface waters. Concerns have been raised over the potential impacts of CLA on the non-target aquatic species, particularly algae acting as the primary producers in the ecosystem. This study therefore evaluated the toxicological effects of CLA at a range of concentration levels (0, 5, 20, 40, 80 µg L-1) on two green algae, Raphidocelis subcapitata (R. subcapitata) and Chlorella vulgaris (C. vulgaris). The algal growth, photosynthetic pigment contents, lipid peroxidation biomarker malondialdehyde (MDA), responses of antioxidants including superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), glutathione peroxidase (GP), and glutathione S-transferase (GST) were measured. After 7 d exposure, the growth of R. subcapitata was inhibited with the CLA exposure levels higher than 20 µg L-1, whereas the inhibition in C. vulgaris was detected at the concentration level of 80 µg L-1. The MDA contents in both species were elevated. To cope with the increased levels of ROS, the activities of enzymatic antioxidants (SOD, CAT, GP, and GST) and the content of non-enzymatic antioxidant (GSH) in R. subcapitata were all enhanced. However, in C. vulgaris, enhancement was detected only in the activities of antioxidant enzymes (SOD, CAT, and GP). In addition, chlorophyll a, b, and carotenoid contents were all significantly increased in R. subcapitata but decreased in C. vulgaris. The results suggested that R. subcapitata is more sensitive to CLA exposure than C. vulgaris. This study provides insights into the CLA - oxidative stress process in two algae.

Effects of chironomid larvae and Limnodrilus hoffmeisteri bioturbation on the distribution and flux of chromium at the sediment-water interface.

The impacts of chironomid larvae and the tubificid worm Limnodrilus hoffmeisteri on the distribution and flux of the heavy metal chromium (Cr) across the sediment-water interface were investigated with a 21-day laboratory microcosm experiment. The two studied species feature different bioturbation modes involving bioirrigation and upward bioconveyance. The Cr concentrations in the overlying water and pore water were measured and compared using treatments with bioturbation by a single species and by combinations of both species and a treatment with no organisms. The results indicated that both bioturbation modes significantly increased the Cr concentrations in the overlying water and pore water. The overlying water had lower Cr concentrations than the pore water. Little variation in the Cr concentrations was observed in the treatment without organisms. Both species enhanced the Cr flux from the pore water to the overlying water. The worm treatments had a great impact on the Cr concentration in the overlying water through intensive upward conveyance activity, while the chironomid larvae treatments exerted significant effects on the Cr variation in the pore water and Cr flux across the interface via bioirrigation activity. These findings reveal the importance of bioturbation in biogeochemical processes in freshwater ecosystems.