Isolation, identification and whole-genome analysis of an Achromobacter strain with a novel sulfamethazine resistance gene and sulfamethazine degradation gene cluster.

A sulfamethazine (SM2) degrading strain, Achromobacter mucicolens JD417, was isolated from sulfonamide-contaminated sludge using gradient acclimation. Optimal SM2 degradation conditions were pH 7, 36 °C, and 5 % inoculum, achieving a theoretical maximum degradation rate of 48 % at 50 ppm SM2. Cell growth followed the Haldane equation across different SM2 concentrations. Whole-genome sequencing of the strain revealed novel functional annotations, including a sulfonamide resistance gene (sul4) encoding dihydropteroate synthase, two flavin-dependent monooxygenase genes (sadA and sadB) crucial for SM2 degradation, and unique genomic islands related to metabolism, pathogenicity, and resistance. Comparative genomics analysis showed good collinearity and homology with other Achromobacter species exhibiting organics resistance or degradation capabilities. This study reveals the novel molecular resistance and degradation mechanisms and genetic evolution of an SM2-degrading strain, providing insights into the bioremediation of sulfonamide-contaminated environments.

Degradation of sulfamethazine by microbial electrolysis cell with nickel-cobalt co-modified biocathode.

In this study, nickel-cobalt co-modified stainless steel mesh (Ni-Co@SSM) was prepared and used as the biocathode in microbial electrolysis cell (MEC) for sulfamethazine (SMT) degradation. The optimal electrochemical performance of the Ni-Co@SSM was obtained at the electrodeposition time of 600 s, electrodeposition current density of 20 mA cm-2, and nickel-cobalt molar ratio of 1:2. The removal of SMT in MEC with the Ni-Co@SSM biocathode (MEC-Ni-Co@SSM) was 82%, which increased by 30% compared with the conventional anaerobic reactor. Thirteen intermediates were identified and the potential degradation pathways of SMT were proposed. Proteobacteria, Firmicutes, Patescibacteria, Chloroflexi, Bacteroidetes, and Euryarchaeota are the dominant bacteria at the phylum level in the MEC-Ni-Co@SSM, which are responsible for SMT metabolism. Due to the electrical stimulation, there was an increase in the abundance of the metabolic function and the genetic information processing. This work provides valuable insight into utilizing MECs for effective treatment of antibiotic-containing wastewater.

Effect of subtherapeutic and therapeutic sulfamethazine concentrations on transcribed genes and translated proteins involved in Microbacterium sp. C448 resistance and degradation.

Microbacterium sp. C448, isolated from a soil regularly exposed to sulfamethazine (SMZ), can use various sulphonamide antibiotics as the sole carbon source for growth. The basis for the regulation of genes encoding the sulphonamide metabolism pathway, the dihydropteroate synthase sulphonamide target (folP), and the sulphonamide resistance (sul1) genes is unknown in this organism. In the present study, the response of the transcriptome and proteome of Microbacterium sp. C448 following exposure to subtherapeutic (33 µM) or therapeutic (832 µM) SMZ concentrations was evaluated. Therapeutic concentration induced the highest sad expression and Sad production, consistent with the activity of SMZ degradation observed in cellulo. Following complete SMZ degradation, Sad production tended to return to the basal level observed prior to SMZ exposure. Transcriptomic and proteomic kinetics were concomitant for the resistance genes and proteins. The abundance of Sul1 protein, 100-fold more abundant than FolP protein, did not change in response to SMZ exposure. Moreover, non-targeted analyses highlighted the increase of a deaminase RidA and a putative sulphate exporter expression and production. These two novel factors involved in the 4-aminophenol metabolite degradation and the export of sulphate residues formed during SMZ degradation, respectively, provided new insights into the Microbacterium sp. C448 SMZ detoxification process.

Immobilization of Bacillus Thuringiensis and applicability in removal of sulfamethazine from soil.

Microbial degradation is considered an essential and promising treatment for sulfadimidine contamination of soil. To address the low colonization rates and inefficiencies of typical antibiotic-degrading bacteria, sulfamethazine (SM2)-degrading strain H38 is converted into immobilized bacteria in this study. Results show that the removal rate of SM2 by immobilized strain H38 reaches 98% at 36 h, whereas the removal rate of SM2 by free bacteria reaches 75.2% at 60 h. In addition, the immobilized bacteria H38 exhibits tolerance to a wide range of pH (5-9) and temperature (20 °C-40 °C). As the amount of inoculation increases and the initial concentration of SM2 decreases, the removal rate of SM2 by the immobilized strain H38 increases gradually. Laboratory soil remediation tests show that the immobilized strain H38 can remove 90.0% of SM2 from the soil on the 12th day, which exceeds the removal by free bacteria by 23.9% in the same period. Additionally, the results show that the immobilized strain H38 enhances the overall activity of microorganisms in SM2-contaminated soil. Compared with the SM2 only (control group containing no bacteria) and free bacterial treatment groups, the gene expression levels of ammonia-oxidizing archaea, ammonia-oxidizing bacteria, cbbLG, and cbbM increased significantly in the treatment group with immobilized strain H38. This study shows that immobilized strain H38 can reduce the effect of SM2 on soil ecology to a greater extent than free bacteria, while providing safe and effective remediation.

Functional fungal pellets self-immobilized by mycelium fragments of Irpex lacteus WRF-IL for efficient degradation of sulfamethazine as the sole carbon source.

In order to achieve an efficient microbial material with dual functions of self-immobilization and sulfamethazine (SMZ) degradation, this study explored the pelletization technique utilizing mycelium fragments of Irpex lacteus WRF-IL and systematically examined the pellets formation conditions and degradation capability. The Box-Behnken design results demonstrated that pure mycelium fragments, broken by frosted glass beads, could be rapidly self-immobilized to form white rot mycelial pellets (WRMPs) within 24 h, serving as the pelleting core. These WRMPs could completely remove SMZ as the sole carbon source within 20 h. The addition of sucrose expedited this process, achieving complete removal within only 14 h. Kinetic analysis showed that WRMPs could potentially remove SMZ at higher concentrations (>25 mg/L). Biodegradation was the primary pathway of SMZ removal. Seven intermediates were identified by QTOF LC/MS, and three transformation pathways initiated by SO2 overflow, molecular rearrangement, and aniline moiety oxidation were deduced.

Biochemical Toxicity and Potential Detoxification Mechanisms in Earthworms Eisenia fetida Exposed to Sulfamethazine and Copper.

The present study investigated the biochemical toxicity and potential detoxification mechanisms in earthworms Eisenia fetida exposed to sulfamethazine (SMZ) (7.5, 15 and 30 mg kg-1) either alone or in combination with Copper (Cu) (100 mg kg-1) in soil. The results showed that increasing concentrations of SMZ in soil activated superoxide dismutase, catalase and glutathione peroxidase isozymes, suggesting reactive oxygen species (ROS) burst in earthworms. Treatment with SMZ and Cu separately or in combination caused protein oxidation and damage, elevating the synthesis of ubiquitin, the 20S proteasome, cytochrome P450 (CYP450), and heat shock protein 70 (HSP70). Such treatments also induced the activities of proteases, endoproteinase (EP) and glutathione S-transferases (GSTs). The results suggested that the ubiquitin-20S proteasome, proteases, EP and HSP70 were involved in degradation or remediation of oxidatively damaged proteins. Elevated levels of CYP450 and GSTs also participated in the detoxification of the earthworms.

Biodiversity, isolation and genome analysis of sulfamethazine-degrading bacteria using high-throughput analysis.

Sulfamethazine (SM2) is one of the sulfonamide antibiotics that is frequently detected in aquatic environment. Given the complex structure of SM2 and its potential threat to the environment, it is necessary to determine the degradation behavior of high-concentration SM2. The mechanisms of community structure and diversity of activated sludge were analyzed. A novel SM2-degrading strain YL1 was isolated which can degrade SM2 with high concentration of 100 mg L-1. Strain YL1 was identified as Paenarthrobacter ureafaciens and there was also a significant increase in the genus during acclimation. Additional SM2 metabolic mechanisms and genomic information of YL1 were analyzed for further research. The succession of the community structure also investigated the effect of SM2 on the activated sludge. This result not only advances the current understanding of microbial ecology in activated sludge, but also has practical implications for the design and operation of the environmental bioprocesses for treatment of antimicrobial-bearing waste streams.

N-acetyltransferase 2 acetylator genotype-dependent N-acetylation of 4-aminobiphenyl in cryopreserved human hepatocytes.

Arylamine N-acetyltransferases are xenobiotic-metabolizing enzymes responsible for detoxification of many drugs and carcinogens. Two N-acetyltransferase proteins (NAT1 and NAT2) are expressed in humans and they both N-acetylate aromatic amine carcinogens such as 4-aminobiphenyl. Arylamines such as 4-aminobiphenyl represent a large class of chemical carcinogens. Exposure to 4-aminobiphenyl occurs in the chemical, dye and rubber industries as well as in hair dyes, paints, and cigarette smoke. NAT2 is subject to a genetic polymorphism resulting in rapid, intermediate and slow acetylator phenotypes. We investigated the role of the NAT2 genetic polymorphisms on the N-acetylation of 4-aminobiphenyl in cryopreserved human hepatocytes in which NAT2 genotype and deduced phenotype were determined. Differences in sulfamethazine (selectively N-acetylated via NAT2) and 4-aminobiphenyl (N-acetylated by both NAT1 and NAT2) N-acetylation rates among rapid, intermediate, and slow NAT2 acetylator genotypes were tested for significance by one-way analysis of variance. In vitro 4-aminobiphenyl N-acetyltransferase activities differed significantly between rapid, intermediate and slow acetylators at 10 µM (P = 0.0102) or 100 µM (P = 0.0028). N-acetylation of 4-aminobiphenyl in situ also differed significantly between human hepatocytes from rapid, intermediate, and slow acetylators at 10 µM (P = 0.0015) and 100 µM (P = 0.0216). A gene dose-response relationship was exhibited as intermediate acetylators catalyzed 4-aminobiphenyl N-acetylation both in vitro and in situ at rates arithmetically between rapid and slow acetylators. In conclusion, N-acetylation of 4-aminobiphenyl is NAT2 genotype-dependent in human hepatocytes. These results suggest refinement of the exposure limit and safety for arylamine carcinogens according to NAT2 genotype.

Plant and microalgae consortium for an enhanced biodegradation of sulfamethazine.

Pharmaceutical contamination in diverse water resources has been recognized as an emerging concern in environment because of its wide distribution and adverse effects on aquatic microorganisms and human health. Plant remediation with augmentation of microorganisms is a cost-effective and environmentally friendly approach toward an efficient treatment of pollutants, which can be easily applied in situ. (Bio)degradation of sulfamethazine (SMZ) by Iris pseudacorus, microalgal consortium, and plant-microalgal consortium was investigated. I. pseudacorus and microalgae could remove 63.5, and 25.8% of 1 mg SMZ L-1, respectively, whereas, the plant-microalgal consortium achieved 74% removal. The identified intermediates extracted after plant remediation indicated (bio)degradation of SMZ was through ring cleavage, hydroxylation, and dehydroxylation. Pigment content (total chlorophyll and carotenoid) of I. pseudacorus was significantly influenced by SMZ stress. A phytoreactor (20 L) constructed with I. pseudacorus achieved 30.0% and 71.3% removal of 1 mg SMZ L-1 from tap water and nutrient medium. This study has provided a better understanding of the metabolic mechanisms of SMZ in plants and showed the potential development of a plant-microalgal consortium as an advanced technology for treatment of these emerging contaminants. Graphical abstract.

Multiresidue Determination of 27 Sulfonamides in Poultry Feathers and Its Application to a Sulfamethazine Pharmacokinetics Study on Laying Hen Feathers and Sulfonamide Residue Monitoring on Poultry Feathers.

A method for the simultaneous determination of 27 sulfonamides in poultry feathers using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was established in this study. The samples were extracted using 0.1 mol/L HCl solutions in a 60 °C water bath for 2 h, purified using hydrophilic-lipophilic balance solid-phase extraction, nitrogen-dried, and then reconstituted for UPLC-MS/MS analysis, which was performed with a CSH-C18 column. Linearity, limit of detection, limit of quantification, recovery, and precision were calculated in accordance with Commission Decision 2002/657/EC. For linearity, all standard curves showed a standard coefficient greater than 0.99, and the recoveries and coefficient of variation were 89-115% and <20%, respectively. The limit of detection and limit of quantification were 0.2-5 and 0.5-20 ng/g, respectively. The method was successfully applied to sulfamethazine (SMZ) residue accumulation monitoring in laying hen feathers and sulfonamide residue monitoring on poultry feathers. SMZ residue accumulation in the laying hen feathers was studied after administration with 100 mg/kg of SMZ for 21 consecutive days. SMZ residues were still detected in feathers 14 days after drug administration and persisted for up to 85 days. Results from 42 poultry feather samples showed that the feather is a suitable medium to monitor the illegal use of sulfonamides in poultry production.

Genomic characterization, kinetics, and pathways of sulfamethazine biodegradation by Paenarthrobacter sp. A01.

Biodegradation is an important route for the removal of sulfamethazine (SMZ), one of the most commonly used sulfonamide antibiotics, in the environment. However, little information is known about the kinetics, products, and pathways of SMZ biodegradation owing to the complexity of its enzyme-based biotransformation processes. In this study, the SMZ-degrading strain A01 belonging to the genus Paenarthrobacter was isolated from SMZ-enriched activated sludge reactors. The bacterial cells were rod-shaped with transient branches 2.50-4.00 µm in length with most forming in a V-shaped arrangement. The genome size of Paenarthrobacter sp. A01 had a total length of 4,885,005 bp with a GC content of 63.5%, and it contained 104 contigs and 55 RNAs. The effects of pH, temperature, initial substrate concentration and additional carbon source on the biodegradation of SMZ were investigated. The results indicated that pH 6.0-7.8, 25 °C and the addition of 0.2 g/L sodium acetate favored the biodegradation, whereas a high concentration of SMZ, 500 mg/L, had an inhibitory effect. The biodegradation kinetics with SMZ as the sole carbon source or 0.2 g/L sodium acetate as the co-substrate fit the modified Gompertz model well with a correlation coefficient (R2) of 0.99. Three biodegradation pathways were proposed involving nine biodegradation products, among which C6H9N3O2S and C12H12N2 were two novel biodegradation products that have not been reported previously. Approximately 90.7% of SMZ was transformed to 2-amino-4, 6-dimethylpyrimidine. Furthermore, sad genes responsible for catabolizing sulfonamides were characterized in A01 with high similarities of 96.0%-100.0%. This study will fill the knowledge gap in the biodegradation of this ubiquitous micropollutant in the aquatic environment.

Evaluating the net effect of sulfadimidine on nitrogen removal in an aquatic microcosm environment.

Antibiotics enter into aquatic pond sediments by wastewater and could make detrimental effects on microbial communities. In this study, we examined the effects of sulfadimidine on nitrogen removal when added to experimental pond sediments. We found that sulfadimidine increased the number of sulfadimidine resistant bacteria and significantly increased the abundance of sul2 at the end of the incubation time (ANOVA test at Tukey HSD, P < 0.05). In addition, sulfadimidine decreased the N2O reduction rate as well as the amount of nitrate reduction. Pearson correlation analysis revealed that the N2O reduction rate was significantly and negatively correlated with narG (r = -0.679, P < 0.05). In contrast, we found a significant positive correlation between the amount of nitrate reduction and the abundance of narG (r = 0.609, P < 0.05) and nirK (r = 0.611, P < 0.05). High-throughput sequencing demonstrated that Actinobacteria, Euryarchaeota, Gemmatimonadetes, Nitrospirae, Burkholderiaceae (a family of Proteobacteria), and Thermoanaerobaculaceae (a family of Firmicutes) decreased with sulfadimidine exposure. In sediments, Actinobacteria, Bacteroidetes, Cyanobacteria, Epsilonbacteraeota, Euryarchaeota, Firmicutes, Gemmatimonadetes, and Spirochaetesat may play key roles in nitrogen transformation. Overall, the study exhibited a net effect of antibiotic exposure regarding nitrogen removal in an aquatic microcosm environment through a combination of biochemical pathways and molecular pathways, and draws attention to controlling antibiotic pollution in aquatic ecosystems.

Electrodes boost microbial metabolism to mineralize antibiotics in manure.

Livestock manures are potential sources of antibiotics in the environment. Sulfamethazine (SMZ), frequently used in veterinary medicine, can enter the environment by using manure as soil fertilizer due to its incomplete absorption in the animal gut and its unmetabolized excretion. The objective of this study was to evaluate the mineralization of 14C-labelled SMZ in manure under a new redox scenario provided by microbial electrochemical reactors, termed microbial electroremediating cells (MERC). These devices aim to overcome the electron acceptor limitation in bacterial oxidative metabolism by means of using electrodes to enhance the biodegradation of pollutants in the environment. Our results revealed that the total degradation of 14C-SMZ reached 43.5% in short term batch laboratory scale experiments under reducing conditions (-400 mV vs. Ag/AgCl). Actually, SMZ mineralization was enhanced up to 10-fold in the early stages (after 2 weeks) in comparison with an electrode-free natural attenuation assay. Moreover, mineralization showed a dependence on electrode potential, with negligible results for conditions set to +400 mV vs Ag/AgCl. The impact of merging electrodes and microorganisms for manure bioremediation suggests a promising future for this emerging technology to treat polluted livestock wastes and prevent soil and groundwater pollution.

The contribution of selected organic substrates to the anaerobic cometabolism of sulfamethazine.

Biodegradation of organic micropollutants is likely to occur due to cometabolism by particular microbial groups. In an effort to identify the stages of anaerobic digestion potentially involved in the biodegradation of the veterinary antimicrobial sulfamethazine (SMZ), the influence of selected carbon sources (sucrose, glucose, fructose, ethanol, meat extract, cellulose, soluble starch, soy oil, acetic acid, propionic acid and butyric acid) on SMZ removal by anaerobic sludge was evaluated in short-term batch experiments. Adsorption to the granular sludge constituted a significant removal mechanism, accounting for 39% of SMZ removal in control experiments. The presence of glucose, fructose, sucrose and meat extract exerted an inducing effect on SMZ degradation, resulting in removal efficiencies of 54, 53, 58 and 61%, respectively, indicating the occurrence of cometabolism. Time courses of sucrose and meat extract degradation revealed markedly distinct organic acid profiles but resulted in similar SMZ removals. Temporal profiles of acetic and propionic acid degradation were not associated with SMZ removal, as changes in SMZ concentration were observed even after the organic acids had been completely removed. The experimental results suggest that SMZ cometabolism is not associated to sucrose hydrolysis, acetoclastic methanogenesis and acetogenesis from propionic acid.

Effects of individual and combined zinc oxide nanoparticle, norfloxacin, and sulfamethazine contamination on sludge anaerobic digestion.

This work investigated the individual and combined effects of zinc oxide, norfloxacin, and sulfamethazine on sludge anaerobic digestion-associated methane production, protein and carbohydrate metabolism, and microbial diversity. Norfloxacin and sulfamethazine (500 mg/kg) did not inhibit methane production, but inhibited its production rate. Zinc oxide nanoparticles with antibiotics inhibited hydrolysis, fermentation, and methanogenesis over varying digestion periods. Complex pollution had a greater impact on methane production than zinc oxide alone, with acute, synergistic toxicity to methanogenesis over short periods. Complex pollution also had varying effects on bacterial and archaeal communities during digestion. These results aid understanding of the toxicity of emerging contaminants in sludge digestion, with the potential to improve pollution removal and reduce associated risks.

Biofiltration of the antibacterial drug sulfamethazine by the species Chenopodium quinoa and its further biodegradation through anaerobic digestion.

The biofiltering capacity, distribution patterns and degradation of the antimicrobial sulfamethazine (SMT) by halophyte Chenopodium quinoa under hydroponic conditions and its further biodegradation through anaerobic digestion were evaluated. C. quinoa was cultivated for a complete life cycle under different concentrations of SMT (0, 2 and 5mg/L) and sodium chloride (0 and 15g/L). C. quinoa is able to uptake and partially degrade SMT. The higher the SMT concentration in the culture medium, the higher the SMT content in the plant tissue. SMT has different distribution patterns within the plant organs, and no SMT is found in the seeds. Dry crop residues containing SMT have a great potential to produce methane through anaerobic digestion and, in addition, SMT is further biodegraded. The highest specific methane yields are obtained using crop residues of the plants cultivated in the presence of salt and SMT with concentrations between 0 and 2mg/L.

Removal kinetics of sulfamethazine and its transformation products formed during treatment using a horizontal flow-anaerobic immobilized biomass bioreactor.

Sulfamethazine (SMZ) is an antibiotic from sulfonamides class widely used in veterinary medicine and reported in wastewater and sewage. Thus, it is essential to study technologies to reduce SMZ present in the aquatic environment. Anaerobic bioreactors are a low-cost technology applied for wastewater treatment. The objective of this paper is to study kinetics parameters related to SMZ removal using a horizontal flow-anaerobic immobilized biomass reactor (HAIB) and to evaluate its transformation products formed during this treatment. The bioreactor was operated at mesophilic condition with a hydraulic retention time of 12 h. The removal of SMZ was evaluated at three different concentrations: 200 ng L-1 (phase I), 400 ng l-1 (phase II) and 600 ng L-1 (phase III). The apparent first-order removal constant obtained for chemical oxygen demand was 0.885 ± 0.094 h-1 while SMZ showed a removal constant of 0.356 h-1. SMZ was removed with an efficiency of 56.0 ± 13.0 % (phase I); 62.0 ± 12.0 % (phase II) and 62.0 ± 6.00 % (phase III). Seven transformation products were detected and one of these with m/z 233 is reported for the first-time. The HAIB bioreactor has a potential to assist in wastewater treatment to remove contaminants at ng L-1 concentration level.

Degradation of sulfadiazine, sulfachloropyridazine and sulfamethazine in aqueous media.

Antibiotics discharged to the environment constitute a main concern for which different treatment alternatives are being studied, some of them based on antibiotics removal or inactivation using by-products with adsorbent capacity, or which can act as catalyst for photo-degradation. But a preliminary step is to determine the general characteristics and magnitude of the degradation process effectively acting on antibiotics. A specific case is that of sulfonamides (SAs), one of the antibiotic groups most widely used in veterinary medicine, and which are considered the most mobile antibiotics, causing that they are frequently detected in both surface- and ground-waters, facilitating their entry in the food chain and causing public health hazards. In this work we investigated abiotic and biotic degradation of three sulfonamides (sulfadiazine -SDZ-, sulfachloropyridazine -SCP-, and sulfamethazine -SMT-) in aqueous media. The results indicated that, in filtered milliQ water and under simulated sunlight, the degradation sequence was: SCP > SDZ ≈ SMT. Furthermore, the rate of degradation clearly increased with the raise of pH: at pH 4.0, half-lives were 1.2, 70.5 and 84.4 h for SCP, SDZ and SMT, respectively, while at pH 7.2 they were 2.3, 9.4 and 13.2 h for SCP, SMT and SDZ. The addition of a culture medium hardly caused any change in degradation rates as compared to experiments performed in milliQ water at the same pH value (7.2), suggesting that in this case sulfonamides degradation rate was not affected by the presence of some chemical elements and compounds, such as sodium, chloride and phosphate. However, the addition of bacterial suspensions extracted from a soil and from poultry manure increased the rate of degradation of these antibiotics. This increase in degradation cannot be attributed to biodegradation, since there was no degradation in the dark during the time of the experiment (72 h). This indicates that photo-degradation constitutes the main removal mechanism for SAs in aqueous media, a mechanism that in this case was favored by humic acids supplied with the extracts from soil and manure. The overall results could contribute to the understanding of the environmental fate of the three sulfonamides studied, aiding to program actions that could favor their inactivation, which is especially relevant since its dissemination can involve serious environmental and public health risks.

Role of Neuropeptides in the Regulation of the Insect Immune System - Current Knowledge and Perspectives.

Despite much research on the insect immune system, hormonal regulation of its activity is not well-understood. Previous research on insect neuroendocrinology suggests that neuropeptides may play an important role in the regulation of the insect immune system. Especially recent studies dealing for example with adipokinetic hormones, bursicon or insulin-like peptides provided deeper insights on this issue showing that neuropeptides can modulate various aspects of insect immune responses, both at the molecular and cellular level. The presented review summarizes the current knowledge about the role of neuropeptides regulating the insect immune system activity. Based on structural and functional homology of some vertebrate and insect neuropeptide families, several propositions of insect neuropeptides that might also possess immunotropic activities, but have not been examined for this aspect, are discussed.

Metabolomic analysis of short-term sulfamethazine exposure on marine medaka (Oryzias melastigma) by comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry.

Toxicological effects of sulfamethazine (SM2) have garnered increasing concern due to its wide applications in aquaculture and persistence in the aquatic environment. Most studies have main focused on freshwater fish (i.e. zebrafish), while information regarding effects of SM2 on marine species is still scarce. Here, the hepatotoxicities in marine medaka (Oryzias melastigma) with an increasing SM2 concentration exposures (0.01 mg/L, 0.1 mg/L and 1 mg/L) were assessed by comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC × GC-TOF/MS) based metabolomics. Significant metabolites belonging to different metabolites classes were identified by multivariate statistical analysis. The increases levels of amino acids including alanine, asparagine, ornithine, proline, threonine, glutamic acid, lysine, tyrosine and phenylalanine were found in at least two exposure levels. Pathway analysis revealed that amino acids played important biological roles during SM2 exposure: up-regulation of high energy-related amino acids for energy alteration; immune function disorder, oxidative stress and corresponding toxicities defenses. The down regulations of sugar and fatty acid metabolism were observed with an increasing level of SM2 exposure, suggesting that extra energy for cellular defense and detoxification was demanded in terms of different stress request. This study provided an innovative perspective to explore possible SM2 induced hepatic damages at three exposure levels on a nontarget aquatic specie.