Deciphering Microbe-Mediated Dissolved Organic Matter Reactome in Wastewater Treatment Plants Using Directed Paired Mass Distance.

Understanding the reaction mechanism of dissolved organic matter (DOM) during wastewater biotreatment is crucial for optimal DOM control. Here, we develop a directed paired mass distance (dPMD) method that constructs a molecular network displaying the reaction pathways of DOM. It couples direction inference and PMD analysis to extract the substrate-product relationships and delta masses of potentially paired reactants directly from sequential mass spectrometry data without formula assignment. Using this method, we analyze the influent and effluent samples from the bioprocesses of 12 wastewater treatment plants (WWTPs) and build a dPMD network to characterize the core reactome of DOM. The network shows that the first step of the transformation triggers reaction cascades that diversify the DOM, but the highly overlapped subsequent reaction pathways result in similar effluent DOM compositions across WWTPs despite varied influents. Mass changes exhibit consistent gain/loss preferences (e.g., +3.995 and -16.031) but different occurrences across WWTPs. Combined with genome-centric metatranscriptomics, we reveal the associations among dPMDs, enzymes, and microbes. Most enzymes are involved in oxygenation, (de)hydrogenation, demethylation, and hydration-related reactions but with different target substrates and expressed by various taxa, as exemplified by Proteobacteria, Actinobacteria, and Nitrospirae. Therefore, a functionally diverse community is pivotal for advanced DOM degradation.

A Graph Neural Network Model with a Transparent Decision-Making Process Defines the Applicability Domain for Environmental Estrogen Screening.

The application of deep learning (DL) models for screening environmental estrogens (EEs) for the sound management of chemicals has garnered significant attention. However, the currently available DL model for screening EEs lacks both a transparent decision-making process and effective applicability domain (AD) characterization, making the reliability of its prediction results uncertain and limiting its practical applications. To address this issue, a graph neural network (GNN) model was developed to screen EEs, achieving accuracy rates of 88.9% and 92.5% on the internal and external test sets, respectively. The decision-making process of the GNN model was explored through the network-like similarity graphs (NSGs) based on the model features (FT). We discovered that the accuracy of the predictions is dependent on the feature distribution of compounds in NSGs. An AD characterization method called ADFT was proposed, which excludes predictions falling outside of the model's prediction range, leading to a 15% improvement in the F1 score of the GNN model. The GNN model with the AD method may serve as an efficient tool for screening EEs, identifying 800 potential EEs in the Inventory of Existing Chemical Substances of China. Additionally, this study offers new insights into comprehending the decision-making process of DL models.

PBT assessment of chemicals detected in effluent of wastewater treatment plants by suspected screening analysis.

Wastewater treatment plants (WWTPs) are the major sources of contaminants discharged into downstream water bodies. Profiling the contaminants in effluent of WWTPs is crucial to assess the potential eco-risks toward downstream organisms. To this end, this study investigated the contaminants in effluent of 10 WWTPs locating in 10 cities of Yangtze River delta region of China by suspected screening analysis. Further, the persistence, bioaccumulation, toxicity (PBT) and the characteristics sub-structures of PBT-like chemicals were analyzed. Totally, 704 chemicals including 155 chemical products, 31 food additives, 52 natural substances, 112 personal care products, 123 pesticides, 192 pharmaceuticals, 17 hormones and 22 others were found. The results of PBT analysis suggested that 42 chemicals (5.97% among the detected chemicals in WWTPs) were with PBT property. Among them, 31 contaminants were not reported previously. 9 characteristics sub-structures (N-methyleneisobutylamine, 1-naphthaldehyde, 2,3,3-trimethylcyclohexene, cyclohexanol, N-sec-butyl-n-propylamine, (5E)-2,6-dimethylocta-1,5-diene, 2-ethylphenol, pentadecane and 6-methoxyhexane) were found for PBT-like chemicals. The sub-structures of highly linear alkyl partially explained the significantly higher PBT score for personal care products. Present study provides fundamental information on PBT properties of contaminants in effluent of WWTPs, which will benefit to prioritize contaminants with high concerns in effluent of WWTPs.

Effects of DOM characteristics from real wastewater on the degradation of pharmaceutically active compounds by the UV/H2O2 process.

The characteristics of dissolved organic matter (DOM) can significantly affect the degradation of target compounds by the advanced oxidation processes. In this study, the effects of the different hydrophobicity/hydrophilicity fractions, molecular weight (MW) fractions, fluorescence components and molecular components of DOM extracted from municipal wastewater on the degradation of 4 pharmaceutically active compounds (PhACs), including carbamazepine, clofibric acid, atenolol and erythromycin by the UV/H2O2 process were investigated. The results showed that the degradation rate constants of 4 PhACs decreased dramatically in the presence of DOM. The linear regressions of 4 PhACs degradation as a function of specific fluorescence intensity (SFI) are exhibited during the degradation of 4 PhACs and the SFI may be used to evaluate effect of DOM on target compounds in wastewater. The hydrophobic acid (HPO-A) exhibited the strongest inhibitory effect on degradation of 4 PhACs during oxidation process. The small MW fractions of DOM significantly inhibited the degradation of 4 PhACs during oxidation process. Among three fluorescence components, hydrophobic humic-like substances may significantly inhibit the degradation of 4 PhACs during oxidation process. At the molecular level, the formulas may be derived from terrestrial sources. CHO compound may significantly inhibit the degradation of 4 PhACs during oxidation process on formula classes. The unsaturated hydrocarbons, carbohydrates and tannins compounds may significantly inhibit the effectiveness of the UV/H2O2 process on compound classes.

Microbial Roles in Dissolved Organic Matter Transformation in Full-Scale Wastewater Treatment Processes Revealed by Reactomics and Comparative Genomics.

Understanding the degradation of dissolved organic matter (DOM) is vital for optimizing DOM control. However, the microbe-mediated DOM transformation during wastewater treatment remains poorly characterized. Here, microbes and DOM along full-scale biotreatment processes were simultaneously characterized using comparative genomics and high-resolution mass spectrometry-based reactomics. Biotreatments significantly increased DOM's aromaticity and unsaturation due to the overproduced lignin and polyphenol analogs. DOM was diversified by over five times in richness, with thousands of nitrogenous and sulfur-containing compounds generated through microbe-mediated oxidoreduction, functional group transfer, and C-N and C-S bond formation. Network analysis demonstrated microbial division of labor in DOM transformation. However, their roles were determined by their functional traits rather than taxa. Specifically, network and module hubs exhibited rapid growth potentials and broad substrate affinities but were deficient in xenobiotics-metabolism-associated genes. They were mainly correlated to liable DOM consumption and its transformation to recalcitrant compounds. In contrast, connectors and peripherals were potential degraders of recalcitrant DOM but slow in growth. They showed specialized associations with fewer DOM molecules and probably fed on metabolites of hub microbes. Thus, developing technologies (e.g., carriers) to selectively enrich peripheral degraders and consequently decouple the liable and recalcitrant DOM transformation processes may advance DOM removal.

Removal of pharmaceuticals by ammonia oxidizers during nitrification.

The adverse effect of pharmaceuticals on ecosystem and human health raises great interest for the removal of pharmaceuticals in wastewater treatment plants (WWTPs). Enhanced removal of pharmaceuticals by ammonia oxidizers (AOs) has been observed during nitrification. This review provides a comprehensive summary on the removal of pharmaceuticals by AOs-ammonia oxidizing bacteria (AOB), ammonia oxidizing archaea (AOA), and complete ammonia oxidizer (comammox) during nitrification in pure ammonia oxidizing culture and mixed microbes systems. The superior removal of pharmaceuticals by AOs in conditions with nitrifying activity compared with the conditions without nitrifying activity was proposed. The contribution of AOs on pharmaceuticals removal in pure and mixed microbe systems was discussed and activated sludge modeling was suggested as the proper measure on assessing the contribution of AOs on the removal of pharmaceuticals in mixed microbe culture. Three transformation processes and the involved reaction types of pharmaceuticals transformation during nitrification were reviewed. The present paper provides a systematical summary on pharmaceuticals removal by different AOs across pure and mixed microbes culture during nitrification, which opens up the opportunity to optimize the wastewater biological treatment systems for enhanced removal of pharmaceuticals. KEY POINTS: • The superior removal of pharmaceuticals by ammonia oxidizers (AOs) was summarized. • The removal contribution of pharmaceuticals attributed by AOs was elucidated. • The transformation processes and reaction types of pharmaceuticals were discussed.

Metagenomic insights into the "window" effect of static magnetic field on nitrous oxide emission from biological nitrogen removal process at low temperature.

This study aimed to explore whether the "window" effect of static magnetic field (SMF) on nitrous oxide (N2O) emission from the biological nitrogen removal process at low temperature existed and reveal its biological mechanism at the gene level. Four sequencing batch reactors (SBRs) with SMFs of 0, 10, 45, and 75 mT were operated continuously for 110 days at 10 °C and the lowest N2O-Gas cumulative emission (5.50 mg N/day) and N2O conversion rate (4.28 %) in 45 mT SMF-SBR verified the existence of the "window" effect. In 45 mT SMF-SBR, nearly all enzymatic activities related to N2O reduction and corresponding functional gene abundances improved significantly. Metagenomic high-throughput sequencing analysis revealed that Alicycliphilus denitricans, Paracoccus denitrificans, Rhodopseudomonas palustris, Pseudomonas stutzeri, and Dechloromonas aromatica, as species related to N2O reduction, could be separately enriched by applying suitable SMF intensity. Gene functions annotation based on KEGG and CAZy databases indicated that SMF not only accelerated the rate of free ammonia into ammonia-oxidizing bacteria and electrons delivered to the corresponding denitrification reductases, but also enhanced the degradation of complex organic matter into smaller molecules, and thus reducing the production of N2O via nitrifier denitrification and incomplete denitrification pathways at 10 °C. These findings provided a guideline and presented a blueprint of ecophysiology for the future application of magnetic field to the reduction of N2O emission in wastewater treatment plants in the cold region.

Inhibition effect of magnetic field on nitrous oxide emission from sequencing batch reactor treating domestic wastewater at low temperature.

This study aims to investigate the effect of a magnetic field on nitrous oxide (N2O) emission from a sequencing batch reactor treating low-strength domestic wastewater at low temperature (10°C). After running for 124 days in parallel, results indicated that the conversion rate of N2O for a magnetic field-sequencing batch reactor (MF-SBR) decreased by 34.3% compared to that of a conventional SBR (C-SBR). Meanwhile, the removal efficiencies for total nitrogen (TN) and ammonia nitrogen (NH4-N) of the MF-SBR were 22.4% and 39.5% higher than those of the C-SBR. High-throughput sequencing revealed that the abundances of AOB (Nitrosomonas), NOB (Nitrospira) and denitrifiers (Zoogloea), which could reduce N2O to N2, were promoted significantly in the MF-SBR. Enzyme activities (Nir) and gene abundances (nosZ nirS and nirK) for denitrification in the MF-SBR were also notably higher compared to C-SBR. Our study shows that application of a magnetic field is a useful approach for inhibiting the generation of N2O and promoting the nitrogen removal efficiency by affecting the microbial characteristics of sludge in an SBR treating domestic wastewater at low temperature.

Removal Characteristics of Dissolved Organic Nitrogen and Its Bioavailable Portion in a Postdenitrifying Biofilter: Effect of the C/N Ratio.

Addition of external carbon sources to postdenitrification biofilters (DNFs) is frequently used in municipal wastewater treatment plants to enhance dissolved inorganic nitrogen removal. However, little is known about its influence on the removal of dissolved organic nitrogen (DON). This study investigated the effect of the carbon-to-nitrogen (C/N) ratio (3, 4, 5, and 6) on the removal characteristics of DON and bioavailable DON (ABDON) in the pilot-scale DNFs treating real secondary effluent. Results showed that DNFs effluent DON accounted for 31.2-39.8% of the effluent total nitrogen. The maximum effluent DON and ABDON concentrations both occurred in DNF operated at a C/N ratio of 3. There was no significant difference in effluent DON concentrations in DNFs at C/N ratios of 4, 5, and 6; however, effluent ABDON and DON bioavailability significantly decreased with C/N ratios (p < 0.05, t-test). According to the chemical composition analysis, effluent DON at high C/N ratios tends to contain less % molecular weight < 1 kDa nitrogenous organic compounds and proteins/amino sugars-like nitrogenous organic formulas, which is likely responsible for its low bioavailability. Overall, this study indicates the benefit of a high C/N ratio during the DNF process in terms of controlling the DON forms that readily stimulate algal growth.

Engineering application of anaerobic ammonium oxidation process in wastewater treatment.

Anaerobic ammonium oxidation (Anammox), a promising biological nitrogen removal process, has been verified as an efficient, sustainable and cost-effective alternative to conventional nitrification and denitrification processes. To date, more than 110 full-scale anammox plants have been installed and are in operation, treating industrial NH4+-rich wastewater worldwide, and anammox-based technologies are flourishing. This review the current state of the art for engineering applications of the anammox process, including various anammox-based technologies, reactor selection and attempts to apply it at different wastewater plants. Process control and implementation for stable performance are discussed as well as some remaining issues concerning engineering application are exposed, including the start-up period, process disturbances, greenhouse gas emissions and especially mainstream anammox applications. Finally, further development of the anammox engineering application is proposed in this review.

Uptake and Accumulation of Polystyrene Microplastics in Zebrafish (Danio rerio) and Toxic Effects in Liver.

Microplastics have become emerging contaminants, causing widespread concern about their potential toxic effects. In this study, the uptake and tissue accumulation of polystyrene microplastics (PS-MPs) in zebrafish were detected, and the toxic effects in liver were investigated. The results showed that after 7 days of exposure, 5 µm diameter MPs accumulated in fish gills, liver, and gut, while 20 µm diameter MPs accumulated only in fish gills and gut. Histopathological analysis showed that both 5 µm and 70 nm PS-MPs caused inflammation and lipid accumulation in fish liver. PS-MPs also induced significantly increased activities of superoxide dismutase and catalase, indicating that oxidative stress was induced after treatment with MPs. In addition, metabolomic analysis suggested that exposure to MPs induced alterations of metabolic profiles in fish liver and disturbed the lipid and energy metabolism. These findings provide new insights into the toxic effects of MPs on fish.

Degradation of conjugated estrogen in visible light-driven intimately coupled photocatalysis and biodegradation system.

Visible light-driven intimately coupled photocatalysis and biodegradation (VDICPB) is an efficient technology for removing recalcitrant contaminants, but the degradation pathway on 17ß-estradiol 3-Sulfate (E2-3S) is still not clear. In this study, VDICPB based on N-doped TiO2 as a photocatalyst was established to investigate the removal and transformation of E2-3S in synthetic wastewater. VDICPB showed a satisfactory removal efficiency of 97.8 ± 0.4 %, which was much higher than that of independent photocatalysis (84.0 ± 2.2 %) or biodegradation system (71.4 ± 1.8 %). Steroid C/D-rings of E2-3S was broken in VDICPB since the transformation process reached terminal central pathway. Primary metabolites did not accumulate in VDICPB, resulting in a low expression of functional genes. E2-3S was mainly removed by cooperative interaction of photocatalysis and co-metabolism of biofilm. Photocatalysis led to deconjugation and microbes acted to mineralization. This study provides technical reference and theoretical support for the removal of new pollutants.

Unveiling intricate transformation pathways of emerging contaminants during wastewater treatment processes through simplified network analysis.

As the key stage for purifying wastewater, elimination of emerging contaminants (ECs) is found to be fairly low in wastewater treatment plants (WWTPs). However, less knowledge is obtained regarding the transformation pathways between various chemical structures of ECs under different treatment processes. This study unveiled the transformation pathways of ECs with different structures in 15 WWTPs distributed across China by simplified network analysis (SNA) we proposed. After treatment, the molecular weight of the whole component of wastewater decreased and the hydrophilicity increased. There are significant differences in the structure of eliminated, consistent and formed pollutants. Amino acids, peptides, and analogues (AAPAs) were detected most frequently and most removable. Benzenoids were refractory. Triazoles were often produced. The high-frequency reactions in different WWTPs were similar, (de)methylation and dehydration occurred most frequently. Different biological treatment processes performed similarly, while some advanced treatment processes differed, such as a significant increase of -13.976 (2HO reaction) paired mass distances (PMDs) in the chlorine alone process. Further, the common structural transformation was uncovered. 4 anti-hypertensive drugs, including irbesartan, valsartan, olmesartan, and losartan, were identified, along with 22 transformation products (TPs) of them. OH2 and H2O PMDs occurred most frequently and in 80.81 % of the parent-transformation product pairs, the intensity of the product was higher than parent in effluents, whose risk should be considered in future assessment activity. Together our results provide a macrography perspective on the transformation processes of ECs in WWTPs. In the future, selectively adopting wastewater treatment technology according to structures is conductive for eliminating recalcitrant ECs in WWTPs.

Phosphite in sedimentary interstitial water of Lake Taihu, a large eutrophic shallow lake in China.

The seasonal occurrence and distribution of phosphite (HPO3(2-), P) in sedimentary interstitial water from Lake Taihu was monitored from 2011 to 2012 to better understand its possible link to P cycle in the eutrophic shallow lake. Phosphite concentrations ranged from < MDL to 14.32 ± 0.19 µg P/kg with a mean concentration of 1.58 ± 0.33 µg P/kg, which accounts for 5.51% total soluble P (TSP(s)) in surficial sediments (0-20 cm). Spatially, the concentrations of sedimentary phosphite in the lake's northern areas were relatively higher than those in the southern areas. Higher phosphite concentrations were always observed in seriously polluted sites. Generally, phosphite in the deeper layers (20-40 cm and 40-60 cm) showed minor fluctuations compared to that in the surficial sediments, which may be associated with the frequent exchange at the sediment-water interface. Phosphite concentrations in surficial or core sediments decreased as spring > autumn > summer > winter. Higher phosphite levels occurred in the areas with lower redox (Eh), higher P contents, and particularly when metal bonded with P to form Al-P(s) and Ca-P(s). Phosphite may be an important media in the P biogeochemical cycle in Lake Taihu and contribute to its internal P transportation.

Inhibition effect of phosphorus-based chemicals on corrosion of carbon steel in secondary-treated municipal wastewater.

Secondary-treated municipal wastewater (MWW) could supply a viable alternative water resource for cooling water systems. Inorganic salts in the concentrated cooling water pose a great challenge to corrosion control chemicals. In this study, the inhibition effect of 1-hydroxy ethylidene-1,1-diphosphonic acid (HEDP), trimethylene phosphonic acid (ATMP) and 2-phosphonobutane-1,2,4-tricarboxylic acid (PBTCA) on corrosion of carbon steel in secondary-treated MWW was investigated by the means of potentiodynamic polarization and electrochemical impedance spectroscopy. The inhibition effect increased with increasing concentration of inhibitors. The corrosion rates of carbon steel were 1.5, 0.8, 0.2 and 0.5 mm a(-1) for blank, HEDP, ATMP and PBTCA samples at 50 mg L(-1), respectively. The phosphorus-based chemicals could adsorb onto the surface of the carbon steel electrode, form a coat of protective film and then protect the carbon steel from corrosion in the test solution.

Ammonium removal from wastewater via struvite pyrolysate recycling with Mg(OH)2 addition.

Magnesium ammonium phosphate (MAP) pyrolysate recycling technology was investigated with Mg(OH)2-mediated pyrolysis. The results revealed that the removal ratio of ammonium was stable at about 75%, and could be increased to 79% after additional acidolysis. The phosphate concentration in the supernate was low at 2 mg/L. The optimum conditions for ammonia release were a 1:1 molar ratio of Mg(OH)2:NH4(+), a heating temperature of 110 °C and a heating time of 3 h. With continual additions of Mg(OH)2 to release ammonia, magnesium phosphate (Mg3(PO4)2) was suggested as a possible derivative. However, with Mg(OH)2-mediated pyrolysis, the growth and nucleation of MAP was inhibited during MAP pyrolysate recycling.

Enhanced degradation of pharmaceuticals in wastewater by coupled radical and non-radical pathways: Further unravelling kinetics and mechanism.

Advanced oxidation processes based on radicals and/or non-radical catalysis are emerging as promising technologies for eliminating pharmaceuticals (PhACs) from wastewater. However, the respective contributions of different removal pathways (radicals or non-radical) for PhAC degradation still lacks quantitative investigation. Zero-valent iron and carbon nanotubes are frequently used to generate both radicals and non-radical species via the activation of persulfate (Fe0/SWCNT/PDS). Herein, the removal kinetics of 1 µM PhACs are depicted, and the corresponding synergistic mechanism of the Fe0/SWCNT/PDS process is discussed. Coupled removal pathways showed the higher degradation of PhACs than the individual pathways. Radicals quenching studies combined with electron spin resonance characterisation suggested that the radical-based removal pathway tends to attack electron-deficient organics, whereas its counterpart is more likely to work on electron-rich organics. From the perspectives of the contribution rate, the redox cycles of conjugated Fe species play a more important role in the generation of radicals than free Fe species, and the faster electron transfer in the conductive bridge offered by SWCNT is responsible for the effective corrosion of Fe0 and the decomposition of PDS. Six real wastewater samples were used to prove the generality of the above removal contribution, regardless of the wastewater samples, and the results suggested that identical attack patterns were obtained in all real wastewater samples, although coexistence matrix slightly suppressed PhAC removal. This work provides a deeper insight into the high-performance working mechanism on synergistic interactions and contaminant removal in a combined catalysis system.

Fate of progesterone and norgestrel in anaerobic/anoxic/oxic (A/A/O) process: Insights from biotransformation and mass flow.

Progesterone (P4) and norgestrel (NGT) are two steroid progestogens that can pose adverse effects on aquatic organisms at ng/L levels. Despite increasing concern on their occurrence and removal in wastewater, their fate in the wastewater treatment process has not been well documented. This study identified the transformation products (TPs) of P4 and NGT in anaerobic/anoxic/oxic (A/A/O) process. Potential functional genes involved in biotransformation of P4 and NGT were explored. The elimination or formation behavior of P4, NGT and convinced TPs along various units of A/A/O process was revealed through the mass flow. Results showed that 12 and 13 TPs were identified in the P4 and NGT groups respectively, wherein 10 identical TPs and C-19 structures transformation pathways were observed in both groups. Six genes were found that may be involved in dehydrogenation and isomerization reactions in the pathways. Mass flow indicated that P4 and NGT were mainly eliminated in anaerobic and anoxic units, while convinced TPs mainly formed in anaerobic and anoxic units and were then eliminated in aerobic unit. Further, the ecological risks of the effluent should not be ignored as residual compounds including P4 or NGT and their TPs in the effluent still posed adverse effects on zebrafish transcript levels.

Prediction of pharmaceuticals removal in activated sludge system under different operational parameters using an extended ASM-PhACs model.

Removal of pharmaceuticals is essential in wastewater treatment systems due to their release and accumulation in the environment, which are raising issues for the environment and human health. A mathematical model could be used to predict pharmaceuticals removal under various operational parameters and assess the contributions of different removal pathways to pharmaceuticals removal. Here an ASM-PhACs model was established to describe pharmaceuticals removal including diclofenac (DCF), erythromycin (ERY), gemfibrozil (GEM) and carbamazepine (CBZ) removal in activated sludge system. The pharmaceuticals removal processes linked to co-metabolic biodegradation through the growth of ammonia oxidizing bacteria (AOB), metabolic biodegradation through AOB, metabolic biodegradation through heterotrophic bacteria (HB) and sludge adsorption were incorporated into activated sludge model (ASM1) framework. The kinetic equations were established for each pharmaceuticals removal process. To provide the experimental data for model calibration and validation, two sets of batch tests were designed and conducted in the laboratory scale using SBR technology. According to the batch test data and results of sensitivity analysis, the newly added parameters and some original default parameters affecting pharmaceuticals removal processes were screened and calibrated. The model could accurately simulate all the dynamics of chemical oxygen demand, nitrogen and pharmaceuticals under various conditions. To explore the effect of operational parameters on pharmaceuticals removal efficiency, the wide range of operational parameters was analyzed during model simulation. According to the simulation results, both influent NH4+-N concentration and DO were found to be the significant parameters that impact the removal of DCF, ERY and GEM. AOB biodegradation played an important role in DCF, ERY and GEM removal. The developed model framework helps to investigate the removal mechanisms and key influencing factors of pharmaceuticals removal, thus providing guidelines for reactor design, operation and optimization aiming at pharmaceuticals removal.

Nontarget screening based on molecular networking strategy to identify transformation products of citalopram and sertraline in wastewater.

Citalopram (CIT) and sertraline (SER) are highly consumed antidepressants worldwide and have been extensively detected in wastewater. Due to the incomplete mineralization, transformation products (TPs) of them can be detected in wastewater. Comparing with parent compounds, knowledge on TPs are limited. To fill these research gaps, lab-scale batch experiments, WWTPs sampling and in silico toxicity prediction were implemented to investigate the structure, occurrence and toxicity of TPs. Based on molecular networking nontarget strategy, 13 TPs of CIT and 12 TPs of SER were tentatively identified. Among them, 4 TPs from CIT and 5 TPs from SER were newly found in present study. TPs identification results compared with results obtained from previous nontarget strategies demonstrated that the excellent performances for molecular networking strategy on candidate TPs prioritizing and new TPs finding, especially for low abundance TPs. Further, transformation pathways for CIT and SER in wastewater were proposed. Newly identified TPs provided insights on defluorination, formylation and methylation for CIT and dehydrogenation, N-malonylation and N-acetoxylation for SER transformed in wastewater. Nitrile hydrolysis and N-succinylation were found to be the dominant transformation pathways for CIT and SER in wastewater, respectively. WWTPs sampling results shown the concentrations of SER and CIT ranged from 0.46 to 28.66 ng/L and 17.16 to 58.36 ng/L. In addition, 7 TPs of CIT and 2 TPs of SER found in lab-scale wastewater samples were found in WWTPs. In silico results suggested 2 TPs of CIT may be more toxic than CIT toward all three trophic levels organisms. Present study provides new insights into the transformation processes of CIT and SER in wastewater. In addition, the necessity of paying more attention on TPs was further highlighted from the aspects of toxicity for TPs of CIT and SER in effluent of WWTPs.