Anaerobic oxidation of ethane by archaea from a marine hydrocarbon seep.

Ethane is the second most abundant component of natural gas in addition to methane, and-similar to methane-is chemically unreactive. The biological consumption of ethane under anoxic conditions was suggested by geochemical profiles at marine hydrocarbon seeps1-3, and through ethane-dependent sulfate reduction in slurries4-7. Nevertheless, the microorganisms and reactions that catalyse this process have to date remained unknown8. Here we describe ethane-oxidizing archaea that were obtained by specific enrichment over ten years, and analyse these archaea using phylogeny-based fluorescence analyses, proteogenomics and metabolite studies. The co-culture, which oxidized ethane completely while reducing sulfate to sulfide, was dominated by an archaeon that we name 'Candidatus Argoarchaeum ethanivorans'; other members were sulfate-reducing Deltaproteobacteria. The genome of Ca. Argoarchaeum contains all of the genes that are necessary for a functional methyl-coenzyme M reductase, and all subunits were detected in protein extracts. Accordingly, ethyl-coenzyme M (ethyl-CoM) was identified as an intermediate by liquid chromatography-tandem mass spectrometry. This indicated that Ca. Argoarchaeum initiates ethane oxidation by ethyl-CoM formation, analogous to the recently described butane activation by 'Candidatus Syntrophoarchaeum'9. Proteogenomics further suggests that oxidation of intermediary acetyl-CoA to CO2 occurs through the oxidative Wood-Ljungdahl pathway. The identification of an archaeon that uses ethane (C2H6) fills a gap in our knowledge of microorganisms that specifically oxidize members of the homologous alkane series (CnH2n+2) without oxygen. Detection of phylogenetic and functional gene markers related to those of Ca. Argoarchaeum at deep-sea gas seeps10-12 suggests that archaea that are able to oxidize ethane through ethyl-CoM are widespread members of the local communities fostered by venting gaseous alkanes around these seeps.

Active antibiotic resistome in soils unraveled by single-cell isotope probing and targeted metagenomics.

Antimicrobial resistance (AMR) in soils represents a serious risk to human health through the food chain and human-nature contact. However, the active antibiotic-resistant bacteria (ARB) residing in soils that primarily drive AMR dissemination are poorly explored. Here, single-cell Raman-D2O coupled with targeted metagenomics is developed as a culture-independent approach to phenotypically and genotypically profiling active ARB against clinical antibiotics in a wide range of soils. This method quantifies the prevalence (contamination degree) and activity (spread potential) of soil ARB and reveals a clear elevation with increasing anthropogenic activities such as farming and the creation of pollution, thereby constituting a factor that is critical for the assessment of AMR risks. Further targeted sorting and metagenomic sequencing of the most active soil ARB uncover several uncultured genera and a pathogenic strain. Furthermore, the underlying resistance genes, virulence factor genes, and associated mobile genetic elements (including plasmids, insertion sequences, and prophages) are fully deciphered at the single-cell level. This study advances our understanding of the soil active AMR repertoire by linking the resistant phenome to the genome. It will aid in the risk assessment of environmental AMR and guide the combat under the One Health framework.

Canopy nitrogen deposition enhances soil ecosystem multifunctionality in a temperate forest.

Nitrogen (N) deposition affects ecosystem functions crucial to human health and well-being. However, the consequences of this scenario for soil ecosystem multifunctionality (SMF) in forests are poorly understood. Here, we conducted a long-term field experiment in a temperate forest in China, where N deposition was simulated by adding N above and under the canopies. We discover that canopy N addition promotes SMF expression, whereas understory N addition suppresses it. SMF was regulated by fungal diversity in canopy N addition treatments, which is largely due to the strong resistance to soil acidification and efficient resource utilization characteristics of fungi. While in understory N addition treatments, SMF is regulated by bacterial diversity, which is mainly because of the strong resilience to disturbances and fast turnover of bacteria. Furthermore, rare microbial taxa may play a more important role in the maintenance of the SMF. This study provides the first evidence that N deposition enhanced SMF in temperate forests and enriches the knowledge on enhanced N deposition affecting forest ecosystems. Given the divergent results from two N addition approaches, an innovative perspective of canopy N addition on soil microbial diversity-multifunctionality relationships is crucial to policy-making for the conservation of soil microbial diversity and sustainable ecosystem management under enhanced N deposition. In future research, the consideration of canopy N processes is essential for more realistic assessments of the effects of atmospheric N deposition in forests.

Direct, Embedded, and Embodied Trade of Arsenic: 1990-2019.

International arsenic trade, physical and virtual, has resulted in considerable transfer of arsenic pollution across regions. However, no study has systematically captured, estimated, and compared physical and virtual arsenic trade and its relevant impacts. This study combines material flow analysis and embodied emission factors to estimate embedded (including direct and indirect trade) and embodied arsenic trade during 1990-2019, encompassing 18 arsenic-containing products among 244 countries. Global embedded arsenic trade increased considerably from 47 ± 7.3 to 450 ± 68 kilotonnes (kt) during this time and was dominated by indirect arsenic trade, contributing 94 and 90% to global arsenic trade in 1990 and 2019, respectively. Since the 1990s, global arsenic trade centers and the main flows have shifted from European and American markets to developing countries. The mass of arsenic involved in embodied trade increased from 87.5 ± 26 kt in 1990 to 800 ± 236 kt in 2019. Direct trade and indirect trade aggravate arsenic environmental emissions in major importing countries, like China, while embodied trade aggravates arsenic environmental emissions in major exporting countries, like Peru and Chile. The trade-related arsenic pollution transfer calls for a rational arsenic emission responsibility-sharing mechanism and corresponding policy recommendations for different trading countries.

Forest-to-Cropland Conversion Reshapes Microbial Hierarchical Interactions and Degrades Ecosystem Multifunctionality at a National Scale.

Conversion from natural lands to cropland, primarily driven by agricultural expansion, could significantly alter soil microbiome worldwide; however, influences of forest-to-cropland conversion on microbial hierarchical interactions and ecosystem multifunctionality have not been fully understood. Here, we examined the effects of forest-to-cropland conversion on intratrophic and cross-trophic microbial interactions and soil ecosystem multifunctionality and further disclosed their underlying drivers at a national scale, using Illumina sequencing combined with high-throughput quantitative PCR techniques. The forest-to-cropland conversion significantly changed the structure of soil microbiome (including prokaryotic, fungal, and protistan communities) while it did not affect its alpha diversity. Both intrakingdom and interkingdom microbial networks revealed that the intratrophic and cross-trophic microbial interaction patterns generally tended to be more modular to resist environmental disturbance introduced from forest-to-cropland conversion, but this was insufficient for the cross-trophic interactions to maintain stability; hence, the protistan predation behaviors were still disturbed under such conversion. Moreover, key soil microbial clusters were declined during the forest-to-cropland conversion mainly because of the increased soil total phosphorus level, and this drove a great degradation of the ecosystem multifunctionality (by 207%) in cropland soils. Overall, these findings comprehensively implied the negative effects of forest-to-cropland conversion on the agroecosystem, from microbial hierarchical interactions to ecosystem multifunctionality.

Sewage Sludge Promotes the Accumulation of Antibiotic Resistance Genes in Tomato Xylem.

Xylem serves as a conduit linking soil to the aboveground plant parts and facilitating the upward movement of microbes into leaves and fruits. Despite this potential, the composition of the xylem microbiome and its associated risks, including antibiotic resistance, are understudied. Here, we cultivated tomatoes and analyzed their xylem sap to assess the microbiome and antibiotic resistance profiles following treatment with sewage sludge. Our findings show that xylem microbes primarily originate from soil, albeit with reduced diversity in comparison to those of their soil microbiomes. Using single-cell Raman spectroscopy coupled with D2O labeling, we detected significantly higher metabolic activity in xylem microbes than in rhizosphere soil, with 87% of xylem microbes active compared to just 36% in the soil. Additionally, xylem was pinpointed as a reservoir for antibiotic resistance genes (ARGs), with their abundance being 2.4-6.9 times higher than in rhizosphere soil. Sludge addition dramatically increased the abundance of ARGs in xylem and also increased their mobility and host pathogenicity. Xylem represents a distinct ecological niche for microbes and is a significant reservoir for ARGs. These results could be used to manage the resistome in crops and improve food safety.

Spatiotemporal Changes of Antibiotic Resistance, Potential Pathogens, and Health Risk in Kindergarten Dust.

The microorganisms present in kindergartens are extremely important for children's health during their three-year preschool education. To assess the risk of outdoor dust in kindergartens, the antibiotic resistome and potential pathogens were investigated in dust samples collected from 59 kindergartens in Xiamen, southeast China in both the winter and summer. Both high-throughput quantitative PCR and metagenome analysis revealed a higher richness and abundance of antibiotic resistance genes (ARGs) in winter (P < 0.05). Besides, the bloom of ARGs and potential pathogens was evident in the urban kindergartens. The co-occurrence patterns among ARGs, mobile genetic elements (MGEs), and potential pathogens suggested some bacterial pathogens were potential hosts of ARGs and MGEs. We found a large number of high-risk ARGs in the dust; the richness and abundance of high-risk ARGs were higher in winter and urban kindergartens compared to in summer and peri-urban kindergartens, respectively. The results of the co-occurrence patterns and high-risk ARGs jointly reveal that urbanization will significantly increase the threat of urban dust to human beings and their risks will be higher in winter. This study unveils the close association between ARGs/mobile ARGs and potential pathogens and emphasizes that we should pay more attention to the health risks induced by their combination.

Understanding Stimulation of Conjugal Gene Transfer by Nonantibiotic Compounds: How Far Are We?

A myriad of nonantibiotic compounds is released into the environment, some of which may contribute to the dissemination of antimicrobial resistance by stimulating conjugation. Here, we analyzed a collection of studies to (i) identify patterns of transfer stimulation across groups and concentrations of chemicals, (ii) evaluate the strength of evidence for the proposed mechanisms behind conjugal stimulation, and (iii) examine the plausibility of alternative mechanisms. We show that stimulatory nonantibiotic compounds act at concentrations from 1/1000 to 1/10 of the minimal inhibitory concentration for the donor strain but that stimulation is always modest (less than 8-fold). The main proposed mechanisms for stimulation via the reactive oxygen species/SOS cascade and/or an increase in cell membrane permeability are not unequivocally supported by the literature. However, we identify the reactive oxygen species/SOS cascade as the most likely mechanism. This remains to be confirmed by firm molecular evidence. Such evidence and more standardized and high-throughput conjugation assays are needed to create technologies and solutions to limit the stimulation of conjugal gene transfer and contribute to mitigating global antibiotic resistance.

Divergent Fate and Roles of Dissolved Organic Matter from Spatially Varied Grassland Soils in China During Long-Term Biogeochemical Processes.

Terrestrial dissolved organic matter (DOM) is critical to global carbon and nutrient cycling, climate change, and human health. However, how the spatial and compositional differences of soil DOM affect its dynamics and fate in water during the carbon cycle is largely unclear. Herein, the biodegradation of DOM from 14 spatially distributed grassland soils in China with diverse organic composition was investigated by 165 days of incubation experiments. The results showed that although the high humified fraction (high-HS) regions were featured by high humic-like fractions of 4-25 kDa molecular weight, especially the abundant condensed aromatics and tannins, they unexpectedly displayed greater DOM degradation during 45-165 days. In contrast, the unique proteinaceous and 25-100 kDa fractions enriched in the low humified fraction (low-HS) regions were drastically depleted and improved the decay of bulk DOM but only during 0-45 days. Together, DOM from the high-HS regions would cause lower CO2 outgassing to the atmosphere but higher organic loads for drinking water production in the short term than that from the low-HS regions. However, this would be reversed for the two regions during the long-term transformation processes. These findings highlight the importance of spatial and temporal variability of DOM biogeochemistry to mitigate the negative impacts of grassland soil DOM on climate, waters, and humans.

Sustainable Blue Foods from Rice-Animal Coculture Systems.

Global interest grows in blue foods as part of sustainable diets, but little is known about the potential and environmental performance of blue foods from rice-animal coculture systems. Here, we compiled a large experimental database and conducted a comprehensive life cycle assessment to estimate the impacts of scaling up rice-fish and rice-crayfish systems in China. We find that a large amount of protein can be produced from the coculture systems, equivalent to ∼20% of freshwater aquaculture and ∼70% of marine wild capture projected in 2030. Because of the ecological benefits created by the symbiotic relationships, cocultured fish and crayfish are estimated to be carbon-negative (-9.8 and -4.7 kg of CO2e per 100 g of protein, respectively). When promoted at scale to displace red meat, they can save up to ∼98 million tons of greenhouse gases and up to ∼13 million hectares of farmland, equivalent to ∼44% of China's total rice acreage. These results suggest that rice-animal coculture systems can be an important source of blue foods and contribute to a sustainable dietary shift, while reducing the environmental footprints of rice production. To harvest these benefits, robust policy supports are required to guide the sustainable development of coculture systems and promote healthy and sustainable dietary change.

Biological Interactions Mediate Soil Functions by Altering Rare Microbial Communities.

Soil microbes, the main driving force of terrestrial biogeochemical cycles, facilitate soil organic matter turnover. However, the influence of the soil fauna on microbial communities remains poorly understood. We investigated soil microbiota dynamics by introducing competition and predation among fauna into two soil ecosystems with different fertilization histories. The interactions significantly affected rare microbial communities including bacteria and fungi. Predation enhanced the abundance of C/N cycle-related genes. Rare microbial communities are important drivers of soil functional gene enrichment. Key rare microbial taxa, including SM1A02, Gammaproteobacteria, and HSB_OF53-F07, were identified. Metabolomics analysis suggested that increased functional gene abundance may be due to specific microbial metabolic activity mediated by soil fauna interactions. Predation had a stronger effect on rare microbes, functional genes, and microbial metabolism compared to competition. Long-term organic fertilizer application increased the soil resistance to animal interactions. These findings provide a comprehensive understanding of microbial community dynamics under soil biological interactions, emphasizing the roles of competition and predation among soil fauna in terrestrial ecosystems.

Lipid Metabolites as Potential Regulators of the Antibiotic Resistome in Tetramorium caespitum.

Antibiotic resistance genes (ARGs) are ancient but have become a modern critical threat to health. Gut microbiota, a dynamic reservoir for ARGs, transfer resistance between individuals. Surveillance of the antibiotic resistome in the gut during different host growth phases is critical to understanding the dynamics of the resistome in this ecosystem. Herein, we disentangled the ARG profiles and the dynamic mechanism of ARGs in the egg and adult phases of Tetramorium caespitum. Experimental results showed a remarkable difference in both gut microbiota and gut resistome with the development of T. caespitum. Meta-based metagenomic results of gut microbiota indicated the generalizability of gut antibiotic resistome dynamics during host development. By using Raman spectroscopy and metabolomics, the metabolic phenotype and metabolites indicated that the biotic phase significantly changed lipid metabolism as T. caespitum aged. Lipid metabolites were demonstrated as the main factor driving the enrichment of ARGs in T. caespitum. Cuminaldehyde, the antibacterial lipid metabolite that displayed a remarkable increase in the adult phase, was demonstrated to strongly induce ARG abundance. Our findings show that the gut resistome is host developmental stage-dependent and likely modulated by metabolites, offering novel insights into possible steps to reduce ARG dissemination in the soil food chain.

Effects of wet-dry alternation on organic phosphorus dynamics and sediment characteristics in the intertidal zone of Nansi Lake.

The study of the fractions and distribution characteristics of organic phosphorus in the sediment of the water level fluctuating zone of Nansi Lake is conducive to revealing the transformation of phosphorus in the lake, and has important scientific significance for controlling the eutrophication of Nansi Lake. Based on the sediment of the water level fluctuation zone of Nansi Lake. The improved Hedley continuous grading extraction, ultraviolet-visible spectroscopy and three-dimensional fluorescence spectroscope were used to characterize the structural characteristics and stability of organic molecules in the sediment, and to reflect the differences in the structure and stability of organophosphate in the water level fluctuating zone. Principal component analysis (PCA), Redundancy analysis (RDA) and correlation heat map analysis were used to analyze the correlation between phosphorus and physicochemical index. The results showed that the alternation between wet-dry conditions was more favorable for the release of phosphorus from sediment, compared to continuous inundation conditions. Moreover, the higher the frequency of wet-dry alternations, the greater the release of phosphorus in different forms from the sediment. Wet-dry alternation resulted in a reduction of substituent on the aromatic rings of sediment DOM (dissolved organic matter), and the continuous drying would increase the molecular weight and humidification degree of DOM in the sediment. Correlation analysis showed that NaOH-Po content in sediment was significantly negatively correlated with TP, IP, OP and various organophosphorus forms, indicating a close transformation relationship between phosphorus forms in sediment. The results can provide a scientific basis for controlling the release of endogenous phosphorus and the risk of eutrophication in Nansi Lake.

Fundamentals and application in phytoremediation of an efficient arsenate reducing bacterium Pseudomonas putida ARS1.

Arsenic is a ubiquitous environmental pollutant. Microbe-mediated arsenic bio-transformations significantly influence arsenic mobility and toxicity. Arsenic transformations by soil and aquatic organisms have been well documented, while little is known regarding effects due to endophytic bacteria. An endophyte Pseudomonas putida ARS1 was isolated from rice grown in arsenic contaminated soil. P. putida ARS1 shows high tolerance to arsenite (As(III)) and arsenate (As(V)), and exhibits efficient As(V) reduction and As(III) efflux activities. When exposed to 0.6 mg/L As(V), As(V) in the medium was completely converted to As(III) by P. putida ARS1 within 4 hr. Genome sequencing showed that P. putida ARS1 has two chromosomal arsenic resistance gene clusters (arsRCBH) that contribute to efficient As(V) reduction and As(III) efflux, and result in high resistance to arsenicals. Wolffia globosa is a strong arsenic accumulator with high potential for arsenic phytoremediation, which takes up As(III) more efficiently than As(V). Co-culture of P. putida ARS1 and W. globosa enhanced arsenic accumulation in W. globosa by 69%, and resulted in 91% removal of arsenic (at initial concentration of 0.6 mg/L As(V)) from water within 3 days. This study provides a promising strategy for in situ arsenic phytoremediation through the cooperation of plant and endophytic bacterium.

Temporal dynamics of soil bacterial network regulate soil resistomes.

Soil bacteria are diverse and form complicated ecological networks through various microbial interactions, which play important roles in soil multi-functionality. However, the seasonal effects on the bacterial network, especially the relationship between bacterial network topological features and soil resistomes remains underexplored, which impedes our ability to unveil the mechanisms of the temporal-dynamics of antibiotic resistance genes (ARGs). Here, a field investigation was conducted across four seasons at the watershed scale. We observed significant seasonal variation in bacterial networks, with lower complexity and stability in autumn, and a wider bacterial community niche in summer. Similar to bacterial communities, the co-occurrence networks among ARGs also shift with seasonal change, particularly with respect to the topological features of the node degree, which on average was higher in summer than in the other seasons. Furthermore, the nodes with higher betweenness, stress, degree, and closeness centrality in the bacterial network showed strong relationships with the 10 major classes of ARGs. These findings highlighted the changes in the topological properties of bacterial networks that could further alter antibiotic resistance in soil. Together, our results reveal the temporal dynamics of bacterial ecological networks at the watershed scale, and provide new insights into antibiotic resistance management under environmental changes.

Movement of protistan trophic groups in soil-plant continuums.

Protists, functionally divided into consumers, phototrophs, and parasites act as integral components and vital regulators of microbiomes in soil-plant continuums. However, the drivers of community structure, assembly mechanisms, co-occurrence patterns, and the associations with human pathogens and different protistan trophic groups remain unknown. Here, we characterized the phyllosphere and soil protistan communities associated with three vegetables under different fertilization treatments (none and organic fertilization) at five growth stages. In this study, consumers were the most diverse soil protist group, had the role of inter-kingdom connector, and were the primary biomarker for rhizosphere soils which were subjected to decreasing deterministic processes during plant growth. In contrast, phototrophs had the greatest niche breadth and formed soil protistan hubs, and were the primary biomarkers for both bulk soils and the phyllosphere. Parasites had minimal input to microbial co-occurrence networks. Organic fertilization increased the relative abundance (RA) of pathogenic protists and the number of pathogen-consumer connections in rhizosphere soils but decreased protistan richness and the number of internal protistan links. This study advances our understanding of the ecological roles and potential links between human pathogens and protistan trophic groups associated with soil-plant continuums, which is fundamental to the regulation of soil-plant microbiomes and maintenance of environmental and human health.

Soil organic matter dynamics mediated by arbuscular mycorrhizal fungi - an updated conceptual framework.

Arbuscular mycorrhizal (AM) fungi play an important role in soil organic matter (SOM) formation and stabilization. Previous studies have emphasized organic compounds produced by AM fungi as persistent binding agents for aggregate formation and SOM storage. This concept overlooks the multiple biogeochemical processes mediated by AM fungal activities, which drive SOM generation, reprocessing, reorganization, and stabilization. Here, we propose an updated conceptual framework to facilitate a mechanistic understanding of the role of AM fungi in SOM dynamics. In this framework, four pathways for AM fungi-mediated SOM dynamics are included: 'Generating', AM fungal exudates and biomass serve as key sources of SOM chemodiversity; 'Reprocessing', hyphosphere microorganisms drive SOM decomposition and resynthesis; 'Reorganizing', AM fungi mediate soil physical changes and influence SOM transport, redistribution, transformation, and storage; and 'Stabilizing', AM fungi drive mineral weathering and organo-mineral interactions for SOM stabilization. Moreover, we discuss the AM fungal role in SOM dynamics at different scales, especially when translating results from small scales to complex larger scales. We believe that working with this conceptual framework can allow a better understanding of AM fungal role in SOM dynamics, therefore facilitating the development of mycorrhiza-based technologies toward soil health and global change mitigation.

Distinct patterns of soil bacterial and fungal community assemblages in subtropical forest ecosystems under warming.

Climate change globally affects soil microbial community assembly across ecosystems. However, little is known about the impact of warming on the structure of soil microbial communities or underlying mechanisms that shape microbial community composition in subtropical forest ecosystems. To address this gap, we utilized natural variation in temperature via an altitudinal gradient to simulate ecosystem warming. After 6 years, microbial co-occurrence network complexity increased with warming, and changes in their taxonomic composition were asynchronous, likely due to contrasting community assembly processes. We found that while stochastic processes were drivers of bacterial community composition, warming led to a shift from stochastic to deterministic drivers in dry season. Structural equation modelling highlighted that soil temperature and water content positively influenced soil microbial communities during dry season and negatively during wet season. These results facilitate our understanding of the response of soil microbial communities to climate warming and may improve predictions of ecosystem function of soil microbes in subtropical forests.

Artificial Sweetener Enhances the Spread of Antibiotic Resistance Genes During Anaerobic Digestion.

Artificial sweeteners have been frequently detected in the feedstocks of anaerobic digestion. As these sweeteners can lead to the shift of anaerobic microbiota in the gut similar to that caused by antibiotics, we hypothesize that they may have an antibiotic-like impact on antibiotic resistance genes (ARGs) in anaerobic digestion. However, current understanding on this topic is scarce. This investigation aimed to examine the potential impact of acesulfame, a typical artificial sweetener, on ARGs in anaerobic digestion by using metagenomics sequencing and qPCR. It was found that acesulfame increased the number of detected ARG classes and the abundance of ARGs during anaerobic digestion. The abundance of typical mobile genetic elements (MGEs) and the number of potential hosts of ARGs also increased under acesulfame exposure, suggesting the enhanced potential of horizontal gene transfer of ARGs, which was further confirmed by the correlation analysis between absolute abundances of the targeted ARGs and MGEs. The increased horizontal dissemination of ARGs may be associated with the SOS response induced by the increased ROS production, and the increased cellular membrane permeability. These findings indicate that artificial sweeteners may accelerate ARG spread through digestate disposal, thus corresponding strategies should be considered to prevent potential risks in practice.

Enhanced Formation of 6PPD-Q during the Aging of Tire Wear Particles in Anaerobic Flooded Soils: The Role of Iron Reduction and Environmentally Persistent Free Radicals.

Rapid urbanization drives increased emission of tire wear particles (TWPs) and the contamination of a transformation product derived from tire antioxidant, termed as N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-Q), with adverse implications for terrestrial ecosystems and human health. However, whether and how 6PPD-Q could be formed during the aging of TWPs in soils remains poorly understood. Here, we examine the accumulation and formation mechanisms of 6PPD-Q during the aging of TWPs in soils. Our results showed that biodegradation predominated the fate of 6PPD-Q in soils, whereas anaerobic flooded conditions were conducive to the 6PPD-Q formation and thus resulted in a ∼3.8-fold higher accumulation of 6PPD-Q in flooded soils than wet soils after aging of 60 days. The 6PPD-Q formation in flooded soils was enhanced by Fe reduction-coupled 6PPD oxidation in the first 30 days, while the transformation of TWP-harbored environmentally persistent free radicals (EPFRs) to superoxide radicals (O2•-) under anaerobic flooded conditions further dominated the formation of 6PPD-Q in the next 30 days. This study provides significant insight into understanding the aging behavior of TWPs and highlights an urgent need to assess the ecological risk of 6PPD-Q in soils.