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Antibiotic resistance (AR) is a major public health concern. Antibiotic intermediates (AIs) used in the production of semisynthetic antibiotics have the same bioactive structure as parent antibiotics and synthetic antibiotic production wastewater usually contains high concentrations of residual AIs; however, the effects of AIs and their interactive effects with antibiotics on the emergence of AR are unknown. In this study, antibiotic-sensitive E. coli K12 was exposed to five types of ß-lactam AIs and their parent antibiotic ampicillin to analyze their impact on the evolution of multiple AR. The results indicated that AI 6-APA inhibits bacterial growth and stimulates the production of reactive oxygen species, as well as induces AR and antibiotic persistence like the parent antibiotic AMP. Combined exposure to 6-APA and AMP synergistically stimulated the induction of multiple AR and antibiotic persistence. The resistance mutation frequency increased up to 6.1 × 106-fold under combined exposure and the combination index reached 1326.5, indicating a strong synergy of 6-APA and AMP. Phenotypic and genotypic analyses revealed that these effects were associated with the overproduction of reactive oxygen species, enhanced stress response signatures, and activation of efflux pumps. These findings provide evidence and mechanistic insights into AR induction by AIs in antibiotic production wastewater.

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Bioretention cells have emerged as a prominent strategy for mitigating pollutant loads within urban stormwater runoff. This study delves into the role of plant uptake in the simultaneous removal of nitrogen and phosphorus compounds within these systems. Three bioretention cells-CP, P1, and P2-were constructed using local soil, C33 sand, and gravel. CP served as the unvegetated control, while P1 and P2 were vegetated with vetiver and cattail, respectively. The removal efficiencies of NO3⁻-N, NH3⁻-N, NO2⁻-N, TN, TP, and COD from rainwater were evaluated under saturated and unsaturated conditions. The unvegetated control reactor (CP) achieved TN and TP removal rates of 40.44% and 82.52%, respectively. Reactor P1 (vetiver) demonstrated TN and TP removal rates of 62.92% and 97.19%, respectively. Reactor P2 (cattail) showed TN and TP removal rates of 49.71% and 87.78%, respectively. With the introduction of a saturation zone, TN removal efficiencies increased to 51.69%, 89.22%, and 79.91% for CP, P1, and P2, respectively. However, TP removal efficiencies decreased to 74.81%, 95.04%, and 84.58% for CP, P1, and P2, respectively. Plant tissue uptake tests indicated that vetiver could retain 5 times more TN and twice as much TP compared to cattail. This enhanced performance is attributed to vetiver's high photosynthetic potential as a C4 plant, resilience to varying environmental and nutrient conditions, extensive root network, secretion of oil sesquiterpenes from its root cortex, and the presence of arbuscular mycorrhizal fungi, which secrete glomalin, a substance that promotes water retention and nutrient uptake. Findings from this study indicate that the efficacy of traditional bioretention cells can be augmented through the strategic selection and integration of locally adapted plant species, coupled with the incorporation of saturation zones, to enhance pollutant removal capabilities and resilience to drought conditions.

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Tire wear particles (TWPs) in stormwater runoff have been widely detected and were generally classified into microplastics (MPs). TWPs and conventional MPs can be intercepted and accumulated in stormwater filtration systems, but their impacts on filtration, adsorption and microbial degradation processes of conventional pollutants (organic matters, nitrate and ammonium) have not been clarified. TWPs are different from MPs in surface feature, chemical components, adsorption ability and leaching of additives, which might lead to their different impacts on conventional pollutants removal. In this study, five different levels of aged polyethylene MPs (PEMPs) and aged TWPs contamination in stormwater filtration systems were simulated using thirty-three filtration columns. Results showed that ultraviolet aging treatment was less influential for the aging of TWPs than that of PEMPs, the specific surface area of aged PEMPs (1.603 m2/g) was over two times of unaged TWPs (0.728 m2/g) in the same size. Aged PEMPs and aged TWPs had different impacts on conventional pollutants removal performance and microbial communities, and the difference might be enlarged with exposure duration. The intensified aged PEMPs contamination generally promoted conventional pollutants removal, whereas aged TWPs showed an opposite trend. Mild contamination (0.01% and 0.1%, wt%) of aged PEMP/TWPs was beneficial to the richness and diversity of microbial communities, whereas higher contamination of aged PEMPs/TWPs was harmful. Aged PEMPs and TWPs had different impact on microbial community structure. Overall, the study found that TWPs were more detrimental than PEMPs in filtration systems. The research underscores the need for more comprehensive investigation into the occurrence, effects and management strategies of TWPs, as well as the importance of distinguishing between TWPs and MPs in future studies.

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To protect agro-systems and food security, study on the effect of microplastics and heavy metals on edible plants is of great significance. Existing studies mostly used virgin microplastics to evaluate their effects on plants, effects of naturally aged microplastics and their combined effects with heavy metals are rarely explored. In this study, single and combined effect of polyethylene microplastics (PE, both virgin and naturally aged) and cadmium (Cd) on pakchoi under seedling and mature stages were analyzed from perspectives of growth inhibition, oxidative damage, nutrition content and soil enzyme activities. Results showed that inhibiting effects of naturally aged PE (PEa) on the growth of pakchoi were stronger than virgin PE (PEv), whereas co-contamination of PEa and Cd was less toxic than that of PEv and Cd. The co-contamination of PE and Cd could inhibit pakchoi dry biomass by over 85 %. Both single and combined contamination of PE and Cd promoted soil fluorescein diacetate hydrolase (FDA) activities, which were 1.11 to 2.04 times of that in control group. Soluble sugar contents under co-contamination of PEa and Cd were 14 % to 22 % higher than those in control group. PEa and PEv showed different effects on oxidative damage of pakchoi. Compared with PEv, catalase (CAT) activities were more sensitive with PEa, whereas PEa had lower effect on superoxide dismutase (SOD) activities. The response of pakchoi to PE and Cd changed with growth stage. Chlorophyll contents in pakchoi under seedling stage were generally higher than those under mature stage. For Cd contaminated soils, PE benefited pakchoi growth under seedling stage, i.e. antagonistic effect between Cd and PE but hindered their growth under mature stage, i.e. synergistic effect. The results unraveled here emphasized PE, especially PEa, could trigger negative effects on agro-systems, whereas PE could be beneficial for heavy metal contaminated agro-systems under specific situations.

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Methane （CH4） and nitrous oxide （N2O） are concerning greenhouse gases. Urban rivers have been important emission sources of CH4 and N2O in recent years. It is meaningful for city greenhouse gas reduction to provide a systematic analysis of spatiotemporal characteristics, mechanisms, and influencing factors of the production and emission of CH4 and N2O from urban rivers. This study combed measured data of urban river CH4 and N2O dissolution concentrations and emission fluxes from related literature published in the past 20 years and also concluded the spatiotemporal characteristics of urban river CH4 and N2O emissions. This study estimated that CH4 and N2O emissions （expressed by CO2-eq） from urban rivers in Beijing were 234.63 and 59.53 Gg CO2-eq in 2018, whereas CH4 and N2O emissions （expressed by CO2-eq） from urban rivers in Shanghai were 159.86 and 260.24 Gg CO2-eq in 2018, respectively. These results demonstrated that urban rivers have become important CH4 and N2O emission sources. This study summarized the production/consumption processes and import/export pathways of CH4 and N2O in urban rivers. What is more, this study discussed the main influencing factors of urban river CH4 and N2O production and emissions from the perspectives of river environmental conditions and urbanization effects. At last, the present work prospected the future research trends of urban river CH4 and N2O emissions and provides urban rivers with scientific support for greenhouse gas reduction.

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Plastic weathering in the natural environment is a dynamic and complex process, where the release of microplastics, nanoplastics and additives poses potential threats to ecosystems. Understanding the release of different weathering products from plastics is crucial for predicting and assessing the environmental hazards of plastics. This study systematically explored these phenomena by exposing polystyrene (PS) to UV irradiation and mechanical agitation for different durations (1 day, 5 days, 10 days, 20 days). The degree of aging, yellowing, brittleness, and the abundance of carbonyl (CO) functional groups in PS were all gradually increasing over time. The weathering pattern of PS surfaces manifested as initial particle oxidation followed by later cracks or flakes formation. The release of products was positively correlated with the aging degree of plastics, as well as among the various released products. Laser infrared and Raman tests indicated that, for microplastics, the size range of 10-20 µm consistently dominated over time, while the primary size range of nanoplastics shifted towards smaller sizes. Additives and other soluble products were prone to release from weathering plastics, with 20 different chemicals detected after 20 d. The release of plastic additives was closely related to aging time, additive type, and quantity. This study contributes to our understanding of the weathering process of plastics, clarifies the release patterns of products over time, and the relationships among different products. It helps predict and assess the environmental pollution caused by plastics.

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Effluent quality deterioration caused by seasonal low temperature is a great challenge to the application of anammox technology. Here, the effects of different graphene materials on anammox process were investigated under both optimal temperature and low-temperature. The batch tests showed that at 30 °C, 300 mg/L of reduced graphene oxide­sodium alginate gel (RGOSA) had the most significant promoting effect, reaching nitrogen removal efficiency (NRE) and nitrogen removal rate (NRR) of 95 % and 8.88 mgN/L/d, respectively. The changes of EPS secretion patterns and increasing of key enzymes activity might contribute to the enhanced anammox activity. During the long-term operation of anammox reactor, the NRE and NRR of the reactor decreased when the temperature dropped to 15 °C, showing an NRE of 50 %-57 % with the addition of 200 mg/L of reduced graphene oxide (RGO) and 40 %-45 % with the addition of 20 mg/L of RGO. Furthermore, specific anammox activity (SAA) of the RGO200 reactor at 15 °C increased by 57.1 % compared to the UASB reactor without graphene addition. Additionally, 16S rRNA and metagenomic analysis results revealed anammox bacteria Ca. Kuenenia was the dominant bacteria. Moreover, the RGO can significantly increase the relative abundance of N-converting functional genes. This study demonstrates the graphene materials can help anammox process adapting to low temperatures, providing a possible solution for the application of anammox technology.

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Antibiotic wastewater contains a variety of pollutant stressors that can induce and promote antibiotic resistance (AR) when released into the environment. Although these substances are mostly in concentrations lower than those known to induce AR individually, it is possible that antibiotic wastewater discharge might still promote the AR transmission risk via additive or synergistic effects. However, the comprehensive effect of antibiotic wastewater on AR development has rarely been evaluated, and its treatment efficiency remains unknown. Here, samples were collected from different stages of a cephalosporin production wastewater treatment plant, and the potential AR induction effect of their chemical mixtures was explored through the exposure of the antibiotic-sensitive Escherichia coli K12 strain. Incubation with raw cephalosporin production wastewater significantly promoted mutation rates (3.6 × 103-9.3 × 103-fold) and minimum inhibition concentrations (6.0-6.7-fold) of E. coli against ampicillin and chloramphenicol. This may be attributed to the inhibition effect and oxidative stress of cephalosporin wastewater on E. coli. The AR induction effect of cephalosporin wastewater decreased after the coagulation sedimentation treatment and was completely removed after the full treatment process. A Pearson correlation analysis revealed that the reduction in the AR induction effect had a strong positive correlation with the removal of organics and biological toxicity. This indicates that the antibiotic wastewater treatment had a collaborative processing effect of conventional pollutants, toxicity, and the AR induction effect. This study illustrates the potential AR transmission risk of antibiotic wastewater and highlights the need for its adequate treatment.

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Sulfide produced from sewers is considered one of the dominant threats to public health and sewer lifespan due to its toxicity and corrosiveness. In this study, we developed an environmentally friendly strategy for gaseous sulfide control by enriching indigenous sulfur-oxidizing bacteria (SOB) from sewer sediment. Ceramics acted as bio-carriers for immobilizing SOB for practical use in a lab-scale sewer reactor. 16 S rRNA gene sequences revealed that the SOB consortium was successfully enriched, with Thiobacillus, Pseudomonas, and Alcaligenes occupying a dominant abundance of 64.7% in the microbial community. Metabolic pathway analysis in different acclimatization stages indicates that microorganisms could convert thiosulfate and sulfide into elemental sulfur after enrichment and immobilization. A continuous experiment in lab-scale sewer reactors confirmed an efficient result for sulfide removal with hydrogen sulfide reduction of 43.9% and 85.1% under high-sulfur load and low-sulfur load conditions, respectively. This study shed light on the promising application for sewer sulfide control by biological sulfur oxidation strategy.

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Decentralized wastewater treatment warrants considerable development in numerous countries and regions. Owing to the unique characteristics of high ammonia nitrogen concentrations and low carbon/nitrogen ratio, nitrogen removal is a key challenge in treating expressway service area sewage. In this study, an anoxic/oxic-moving bed biofilm reactor (A/O-MBBR) and a traditional A/O bioreactor were continuously operated for 115 days and their outcomes were compared to investigate the enhancement effect of carriers on the total nitrogen removal (TN) for expressway service area sewage. Results revealed that A/O-MBBR required lower dissolved oxygen, exhibited higher tolerance toward harsh conditions, and demonstrated better shock load resistance than traditional A/O bioreactor. The TN removal load of A/O-MBBR reached 181.5 g‧N/(m3‧d), which was 15.24% higher than that of the A/O bioreactor. Furthermore, under load shock resistance, the TN removal load of A/O-MBBR still reached 327.0 g‧N/(m3‧d), with a TN removal efficiency of above 80%. Moreover, kinetics demonstrated that the denitrification rate of the A/O-MBBR was 121.9% higher than that of the A/O bioreactor, with the anoxic tank biofilm contributing 60.9% of the total denitrification rate. Community analysis results revealed that the genera OLB8, uncultured_f_Saprospiraceae and OLB12 were the dominant in biofilm loaded on carriers, and OLB8 was the key for enhanced denitrification. FAPROTAX and PICRUSt2 analyses confirmed that more bacteria associated with nitrogen metabolism were enriched by the A/O-MBBR carriers through full denitrification metabolic pathway and dissimilatory nitrate reduction pathway. This study offers a perspective into the development of cost-effective and high-efficiency treatment solutions for expressway service area sewage.

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Biodegradable microplastics (BMPs) and cadmium (Cd) are posing threats to agro-systems especially to plants and current studies mostly used virgin BMPs to explore their ecological effects. However, effects of naturally aged BMPs and their combined effects with Cd on pakchoi are yet to be unraveled. Therefore, this study incubated naturally aged polylactic acid (PLA) MPs through soil aging process and investigated the single and combined effects of Cd and PLA MPs (virgin and aged) on pakchoi (Brassica rapa subsp. chinensis) morphology, antioxidant systems and soil microbial activities. Our results found that after being deposited in soil for six months, aged PLA (PLAa) MPs formed with a fractured surface, demonstrating more detrimental effects on pakchoi than virgin ones. PLA/PLAa MPs and Cd stunted pakchoi growth, caused oxidative stress and altered the biophysical environment in soil, separately. Moreover, co-existence of PLA/PLAa MPs and Cd caused greater damages to pakchoi than applied alone. The co-presence of PLAa MPs and Cd inhibited pakchoi biomass accumulation rate by 92.2 % compared with the no-addition group. The results unraveled here emphasized BMPs, especially aged BMPs, could trigger negative effects on agro-systems with heavy metals. These findings will give reference to future holistic assessments of BMPs' ecological effects.

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Anaerobic digestion (AD) is promising for treating high-strength wastewater. However, the effect of operational parameters on microbial communities of AD with sulfate is not yet fully understood. To explore this, four reactors were operated under rapid- and slow-filling modes with different organic carbons. Reactors in the rapid-filling mode generally exhibited a fast kinetic property. For example, the degradation of ethanol was 4.6 times faster in ASBRER than in ASBRES, and the degradation of acetate was 11.2 times faster in ASBRAR than in ASBRAS. Nevertheless, reactors in the slow-filling mode could mitigate propionate accumulation when using ethanol as organic carbon. Taxonomic and functional analysis further supported that rapid- and slow-filling modes were suitable for the growth of r-strategists (e.g., Desulfomicrobium) and K-strategists (e.g., Geobacter), respectively. Overall, this study provides valuable insights into microbial interactions of AD processes with sulfate through the application of the r/K selection theory.

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This review aims to provide a comprehensive understanding of the potential of CMs-dominated DIET in the degradation of recalcitrant organic pollutants in AD. The review covers the mechanisms and efficiencies of recalcitrant organic pollutant degradation by CMs-dominated DIET, the comparison of degradation pathways between DIET and chemical treatment, recent insights on DIET-enhanced degradation, and the evaluation of the potential and future development of CMs-dominated DIET. The review emphasizes the importance of coupled syntrophic microorganisms, electron flux, and physicochemical properties of CMs in enhancing the degradation performance of AD. Additionally, it highlights the advantages of DIET-led syntrophic metabolism over traditional oxidation technologies in terms of environmental friendliness and efficiency. Finally, the review acknowledges the potential risks associated with introducing CMs into AD systems and provides guidance for waste treatment and energy recovery.

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Achieving simultaneous carbon and nitrogen removal with sludge-liquid separation in a single reactor offers a solution to land shortages and improves treatment efficiency in municipal wastewater treatment plants of megacities. This study proposes a novel air-lifting continuous-flow reactor configuration with an alternative-aeration strategy that creates multi-functional zones for anoxic, oxic, and settlement processes. The optimal operating conditions for the reactor include a long anoxic hydraulic retention time, low dissolved oxygen (DO) in the oxic zone, and no specific reflux for external nitrifying liquid, which exhibit a high nitrogen removal efficiency of over 90% in treating real sewage with C/N < 4 in the pilot-scale study. Results show that a high sludge concentration and a low DO concentration facilitate simultaneous nitrification and denitrification, and a well mixing of sludge and substrate in different reaction zones promotes mass transfer and microbial activity. The long-term operation enriches functional microbes for carbon storage and nutrient removal.

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The study assessed the occurrence and distribution of microbial community and antibiotic resistance genes (ARGs) in food waste, anaerobic digestate, and paddy soil samples, and revealed the potential hosts of ARGs and factors influencing their distribution. A total of 24 bacterial phyla were identified, of which 16 were shared by all samples, with Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria accounting for 65.9-92.3 % of the total bacterial community. Firmicutes was the most abundant bacteria in food waste and digestate samples, accounting for 33-83 % of the total microbial community. However, in paddy soil samples with digestate, Proteobacteria had the highest relative abundance of 38-60 %. Further, 22 ARGs were detected in food waste and digestate samples, with multidrug, macrolide-lincosamide-streptogramin (MLS), bacitracin, aminoglycoside, tetracycline, vancomycin, sulfonamide, and rifamycin resistance genes being the most abundant and shared by all samples. The highest total relative abundance of ARGs in food waste, digestate, and soil without and with digestate was detected in samples from January 2020, May 2020, October 2019, and May 2020, respectively. The MLS, vancomycin, tetracycline, aminoglycoside, and sulfonamide resistance genes had higher relative abundance in food waste and anaerobic digestate samples, whereas multidrug, bacteriocin, quinolone, and rifampin resistance genes were more abundant in paddy soil samples. Redundancy analysis demonstrated that aminoglycoside, tetracycline, sulfonamide, and rifamycin resistance genes were positively correlated with total ammonia nitrogen and pH of food waste and digestate samples. Vancomycin, multidrug, bacitracin, and fosmidomycin resistance genes had positive correlations with potassium, moisture, and organic matter in soil samples. The co-occurrence of ARG subtypes with bacterial genera was investigated using network analysis. Actinobacteria, Proteobacteria, Bacteroidetes, and Acidobacteria were identified as potential hosts of multidrug resistance genes.

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This study performed a long-term operation to achieve efficient medium-chain fatty acids (MCFAs) production by anaerobic fermentation of food waste without external electron donors. The results show that total MCFAs reached the highest concentration of 29,886.10 mg COD/L, and n-caproate was the primary product, reaching the current maximum concentration of 28,191.66 mg COD/L. Microbial composition analysis demonstrated Lactobacillus, Bifidobacterium, Sporanaerobacter, and Caproiciproducens constituted the core community throughout the process. Metagenomic analysis suggested that two pathways, reverse ß-oxidization (RBO) and fatty acid biosynthesis (FAB), were observed, and the FAB pathway was the main CE pathway. Unclassified_f_Ruminococcaceae and Limosilactobacillus were the main participants in the FAB pathway. This study is expected to provide new insights into MCFAs production from organic waste.

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Rainfall runoff and combined sewer overflow (CSO) converge with organic waste, nutrients, and microbes from the ground and wastewater. These pollutants promote the spread and transformation of antibiotic resistance genes (ARGs). In this study, four rainfall runoff and one CSO outfall were chosen, and samples were collected to explore the occurrence and distribution of ARGs. The ARGs were extracted from suspended solids and analyzed using metagenomic sequencing. A total of 888 ARG subtypes, belonging to 17 ARG types, were detected in all samples. Eleven ARG types were shared by all the samples. Multidrug resistance genes had the highest relative abundance. Their total relative abundance reached 1.07 ratio (ARG copy number/16S rRNA gene copy number) and comprised 46.6% of all the ARGs. In all samples, the CSO outfall had the highest total relative abundance (8.25 × 10-1 ratio) of ARGs, with a ratio ranging ND (not detected)-3.78 × 10-1 ratio. Furthermore, the relationship between ARG types and environmental factors was determined using redundancy analysis. The results showed that chemical organic demand (COD) and bacterial abundance were positively correlated with most ARG types, including multidrug, bacitracin, aminoglycoside, ß-lactam, tetracycline, and sulfonamide. NH3-N, TN, and TP were positively correlated with rifamycin, fosmidomycin, and vancomycin resistance genes. The relationship among the ARG subtypes was investigated using network analyses. The multidrug resistance gene subtypes had the highest frequency of co-occurrence. This study provides insights into the occurrence and distribution of ARGs under non-point source pollution and may contribute to the control of ARGs.

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With the continuous development of nanomaterials in recent years, the application of nanocatalysts in catalytic ozone oxidation has attracted more and more researchers' attention due to their excellent catalytic properties. In this review, we systematically summarized the current research status of nanocatalysts mainly involving material categories, mechanisms and catalytic efficiency. Based on summary and analysis, we found most of the reported nanocatalysts were in the stage of laboratory research, which was caused by the nanocatalysts defects such as easy aggregation, difficult separation, and easy leakage. These defects might result in severe resource waste, economic loss and potentially adverse effects imposed on the ecosystem and human health. Aiming at solving these defects, we further analyzed the reasons and the existing reports, and revealed that coupling nano-catalyst and membrane, supported nanocatalysts and magnetic nanocatalysts had promising potential in solving these problems and promoting the actual application of nanocatalysts in wastewater treatment. Furthermore, the advantages, shortages and our perspectives of these methods are summarized and discussed.

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Hydrogenotrophic denitrification (HD) is a promising autotrophic biological process for advanced nitrogen removal, while sludge granulation was seldom reported. This study aimed to cultivate granular sludge to improve capacity and stability of HD process. The resulting HD granular sludge performed high nitrogen removal rate (NRR) of 0.42 ± 0.0.4 kgN/(m3·d) with low accumulation of nitrite and nitrous oxide emission. HD granular sludge reactor performed over 3 times higher NRR compared to that in HD fixed-bed biofilm reactor (0.13 ± 0.01 kgN/(m3·d). Besides, granular sludge reactor could treat groundwater well even at the low temperature of 15 °C. The dominant genera were Hydrogenophaga and Comamonas in granular sludge, and Dechloromonas in biofilm. Noticeably, sulfate in the groundwater stimulated the growth of sulfur converting microbes with increasing abundances of sulfite reductase gene and sulfate-reducing bacteria Desulfovibrio. This study highlights the potential implementation of HD process in granular sludge reactor for advance nitrogen removal from impaired groundwater.

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Microplastics (MPs), which are widely present in the natural environment, may be harmful to the growth and health of aquatic organisms, though studies in this area are lacking. In this study, the crucian carp (Carassius carassius), a type of omnivorous freshwater fish, was chosen as the target, which was fed with fish food containing different concentrations of MPs for a 30-day food exposure experiment to study the effects of MPs on crucian growth, liver damage, and gut microbiome composition. Compared with that in the control group, the body length of the crucians in the environmental groups did not change significantly. The weight of the crucians in the low PE-MPs group increased significantly, but the weight of crucians in the medium and high PE-MPs groups decreased markedly. The liver tissues of the low PE-MPs group of crucians were basically normal, whereas crucians in the medium and high PE-MPs groups had varying degrees of liver damage, and crucians in the high PE-MPs group had the most serious liver damage. At the phylum level, Proteobacteria, Fusobacteria, Firmicutes, and Bacteroides were the dominant species in the gut of the crucians. Pathogens such as Staphylococcus and Ralstonia were present in the crucian gut of environmental groups. Alpha diversity results showed that the gut microbiome of crucians in the high PE-MPs group was the most abundant. PCoA results indicated that the gut microbiome of crucians in the control and environmental groups had obvious clustering characteristics.

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