Effects of Advanced Treatment Systems on the Removal of Antibiotic

Jun 26, 2013 - Effects of Advanced Treatment Systems on the Removal of Antibiotic Resistance Genes in Wastewater Treatment Plants from Hangzhou, China...
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Effects of Advanced Treatment Systems on the Removal of Antibiotic Resistance Genes in Wastewater Treatment Plants from Hangzhou, China Hong Chen* and Mingmei Zhang Department of Environmental Engineering, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, People’s Republic of China S Supporting Information *

ABSTRACT: This study aimed at quantifying the concentration and removal of antibiotic resistance genes (ARGs) in three municipal wastewater treatment plants (WWTPs) employing different advanced treatment systems [biological aerated filter, constructed wetland, and ultraviolet (UV) disinfection]. The concentrations of tetM, tetO, tetQ, tetW, sulI, sulII, intI1, and 16S rDNA genes were examined in wastewater and biosolid samples. In municipal WWTPs, ARG reductions of 1−3 orders of magnitude were observed, and no difference was found among the three municipal WWTPs with different treatment processes (p > 0.05). In advanced treatment systems, 1−3 orders of magnitude of reductions in ARGs were observed in constructed wetlands, 0.6−1.2 orders of magnitude of reductions in ARGs were observed in the biological aerated filter, but no apparent decrease by UV disinfection was observed. A significant difference was found between constructed wetlands and biological filter (p < 0.05) and between constructed wetlands and UV disinfection (p < 0.05). In the constructed wetlands, significant correlations were observed in the removal of ARGs and 16S rDNA genes (R2 = 0.391−0.866; p < 0.05). Constructed wetlands not only have the comparable ARG removal values with WWTP (p > 0.05) but also have the advantage in ARG relative abundance removal, and it should be given priority to be an advanced treatment system for further ARG attenuation from WWTP.

1. INTRODUCTION Various antibiotics have been widely used for humans and animals, but the overuse and misuse of such drugs potentially contribute to the emergence and spread of antibiotic resistance genes (ARGs) in the environment.1 A widespread distribution of AGRs is likely due to selective pressure exerted by prolonged exposure to corresponding antibiotics.2,3 ARGs may persist in the environment even after the selective pressure has been removed.4 Given that ARGs are recognized as emerging contaminants,5 an increasing number of reports have been published on the dissemination of ARGs and the environmental pollution that they cause. Tetracycline resistance genes, including tetA, tetB, tetC, tetD, tetE, tetG, tetK, tetL, tetM, tetO, tetS, tetQ, tetW, and tetX have been detected in wastewater treatment plants (WWTPs),6−8 river water and sediments,3,5 soil,9,10 and aquaculture farms.4 Sulfonamide resistance genes, including sul1, sul2, sul3, and sulA, have likewise been detected in WWTPs,11 river water and sediments,3,12 and aquaculture farms.13 WWTPs are considered to be among the significant reservoirs for various ARG encoding resistances, and high microbial density and diversity of activated sludge may contribute to genetic exchange in WWTPs.14,15 Although other studies indicated that municipal WWTPs play an active © 2013 American Chemical Society

role in ARG removal, the elimination of ARGs in WWTPs remains incomplete, and the ARGs may finally enter the aquatic and terrestrial environment via wastewater discharge and the land application of biosolids.2,7 Previous studies in China showed high detection frequency of four tet genes (tetM, tetO, tetQ, and tetW) and two sul genes (sulI and sulII) found in different environmental compartments.3,8,10 Given their capability to capture exogenous gene cassettes and convert these cassettes by site-specific recombination,16 the genetic element of the integrase gene (intI1) of class 1 integrons was believed to contribute significantly to the evolution and proliferation of multiple antibiotic-resistant bacteria.17 Class 1 integrons generally contain an intI1 gene encoding a site-specific integrase responsible for integration and a conserved segment (sulI gene) encoding resistance to sulfonamide.18 The occurrence of intI1 has been reported in influent and effluent samples of WWTPs.19,20 Over the past decade, a steep increase in the number of WWTPs was observed in China.21 WWTPs have been built in Received: Revised: Accepted: Published: 8157

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Table 1. Characteristics of Wastewater Treatment Plants and Advanced Treatment Processes plant name

capacity (tons)

serving population

wastewater treatment process

HRT (h)

advanced treatment process

plant A plant B plant C

60000 20000 600000

285000 94500 2750000

oxidation ditch oxidation ditch anaerobic−anoxic−oxic

14 14 12

constructed wetland biological aerated filter UV

Figure 1. Sampling sites and processes of three WWTPs and advanced treatment processes. Plant A, (1) the influent of the grid screen as the influent of the main process, (2) the influent of the OX, (3) the mixture in the OX, (4) the effluent of the OX, (5−8) the effluent samples of each subsystem in the constructed wetland, and (9) the effluent of constructed wetland. Plant B, (1−4) same as plant A and (5) the effluent of the biological aerated filter. Plant C, (1) the effluent of the grid screen as the influent of the main process, (2) the effluent of the primary clarifier, (3) the mixture in the anoxic tank, (4) the mixture in the anaerobic tank, (5) the mixture in the oxic tank, (6) the second clarifier effluent, and (7) the disinfection effluent by UV.

Linan county, Yuhang district, and Hangzhou city. The characteristics and locations of the WWTPs are shown in Table 1 and Figure S1 of the Supporting Information, respectively. Plants A and B represent small- and mediumsized WWTPs using the triple OX process. Plant C represents large WWTPs using the A/A/O process. The prescribing habit of antibiotics and the percentage of health infrastructure sewage of each WWTP are similar because Linan county, Yuhang district, and Hangzhou city belong to the same region (Hangzhou region). Plants A and C were sampled in April and August 2012 to conduct a time series comparison. Plant B was sampled only in April 2012 for a comparison to plant A. 2.2. Advanced Treatment System. Three different advanced treatment systems were employed by three WWTPs, i.e., constructed wetlands for plant A, biological aerated filter for plant B, and UV disinfection for plant C. Approximately 3000 m3/day of effluent from plant A was further treated using the constructed wetland system. The constructed wetlands contain five subsystems, including four stabilization ponds and one horizontal subsurface flow wetland. It has a total area of 3675 m2 and a hydraulic retention time (HRT) of 1.6 days. The effluent of plant B was further treated by biological aerated filter, which covers an area of 1500 m2 with a HRT of 3 h. The effluent of plant C was further treated by UV disinfector, with UV transmittance of 45%, total power of 900 kW, and light intensity great than 1 mW/mm2. 2.3. Sample Collection. Samples of raw influent wastewater, effluent before advanced treatment, and final effluent were collected, along with activated sludge and excess sludge. Except for excess sludge, other samples from WWTPs were

more than 90% of Chinese cities, and these WWTPs have a treatment capacity of 125 000 000 m3/day.21 WWTPs use a combination of physical, chemical, and biological procedures to eliminate or reduce suspended solids, organic matter loads, nitrogen, and phosphate.14 With more stringent environmental requirements, most current municipal WWTPs were required to upgrade and strengthen their treatment processes to reduce pollutant emissions, such that the advanced treatment system became the most preferred process. The removal of organic substrates and nutrients is the priority for advanced treatment systems,22 but limited attention has been directed toward the fate of emerging contaminants, such as ARGs. In this study, the presence of four tet genes (tetM, tetO, tetQ, and tetW) and two sul genes (sulI and sulII) in municipal WWTPs employing different advanced treatment processes was investigated. The objective of this study was (1) to quantify the presence of six ARGs and the intI1 gene in each treatment stage of three WWTPs using the oxidation ditch (OX) and anaerobic−anoxic−oxic (A/A/O) technologies, (2) to investigate the fate of ARGs in three different advanced treatment processes [i.e., biological aerated filter, constructed wetland, and ultraviolet (UV) disinfection], and (3) to assess whether the advanced treatment system can effectively attenuate ARGs from WWTP effluent.

2. MATERIALS AND METHODS 2.1. Wastewater Treatment Plants. Three WWTPs (plants A, B, and C) with different advanced treatment systems in Hangzhou, China were studied. Plants A, B, and C receive domestic sewage, including health infrastructure sewage from 8158

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(TaKaRa), 0.3 μL of ROX reference dye, 0.2 μM of each primer, 2 μL of template DNA, and 4.6 μL of ddH2O. All of the qPCR amplification and quantification were conducted using a StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA). The PCR protocol was as follows: 30 s at 95 °C, followed by 40 cycles of 5 s at 95 °C, 30 s at the annealing temperatures, 72 °C extension for 30 s, a fluorescence acquisition step at 72 °C, and then a final melt curve stage with temperature ramping from 60 to 95 °C. Each reaction was run in triplicate. R2 values were more than 0.990 for all standard curves (see Table S2 of the Supporting Information). 2.6. Statistical Analysis. Copy numbers were log-transformed as needed to normalize the distributions prior to statistical analysis. Data were analyzed by OriginPro 8.0 (OriginLab Corporation, Northampton, MA). UNIANOVA (specifies the removal value as the dependent variable and wastewater treatment processes as a factor) was conducted to analyze the results between different sampling sites with SPSS, version 17.5. p < 0.05 was considered statistically significant.

collected in roughly equal volumes per hour with composite samplers, i.e., about 30 mL from primary and secondary treatments and 100 mL from the effluent of WWTP and advanced treatment system, and the mixed samples of 24 h were used for ARG determination. The sampling locations of municipal treatment plants and the advanced treatment processes are shown in Figure 1. In plants A and B, the sampling points were positioned at (1) the influent of the grid screen as the influent of the main process, (2) the influent of the OX, (3) the mixture in the OX, and (4) the effluent of the OX. In plant A, numbers 5−9 represent the effluent samples of each subsystem in the constructed wetland, whereas in plant B, number 5 represents the effluent of the biological aerated filter. In plant C, the sampling points were positioned at (1) the effluent of the grid screen as the influent of the main process, (2) the effluent of the primary clarifier, (3) the mixture in the anoxic tank, (4) the mixture in the anaerobic tank, (5) the mixture in the oxic tank, (6) the second clarifier effluent, and (7) the disinfection effluent by UV. The liquid samples were stored at 4 °C and analyzed within 24 h. The solid samples were stored at −80 °C. 2.4. Sample Pretreatment and DNA Extraction. Samples were concentrated by a vacuum filtration apparatus onto 0.22 μm filters until the filter clogged, then the filters was stored at −80 °C until DNA extraction was performed for molecular analyses. Total DNA was extracted using an UltraClean Water DNA Kit (MoBio Laboratories). For excess sludge, DNA from 0.1 g of lyophilized samples was extracted using the FastDNA SPIN Kit for Soil (MP, Biomedicals). DNA extraction was performed following the protocol of the manufacturer. The quality and concentration of the purified DNA were determined by 1.5% agarose gel electrophoresis and spectrophotometer analysis (NanoDrop ND-2000c, Thermo). 2.5. Quantitative Polymerase Chain Reaction (PCR). Four tet genes (tetM, tetO, tetQ, and tetW) and two sul genes (sulI and sulII) were selected for quantitative detection using SYBR Green I qPCR, as well as the integrase gene of class 1 integrons (intI1) and 16S rDNA genes. Primers, annealing temperature, and amplification size were described in Table S1 of the Supporting Information. Plasmids carrying target genes were used to generate standard curves. Specifically, after PCR amplification, fresh PCR products with the confirmed presence of the target genes were excised and purified using an Agarose GelDNA Purification Kit (TaKaRa). As per the instructions of the manufacturer, the purified PCR products were ligated into pMD19-T vector (TaKaRa) and then cloned into Escherichia coli DH5α (TaKaRa). Positive clones were screened by PCR to verify cloning of the target genes and sequenced. With the use of the BLAST alignment tool (http://www.ncbi.nlm.nih.gov/ blast/), positive clones with the target gene inserts were chosen as the standards for the real-time qPCR. Plasmids carrying target genes were extracted according to a QIAprep Spin Miniprep Kit (QIAGEN). The concentration and quality of the plasmid was determined by agarose gel electrophoresis and spectrophotometer analysis (NanoDrop ND-2000c, Thermo). The copy numbers of ARGs genes per microliter of plasmid solution were calculated according to Zhang et al.23 Five- or sixpoint standard curves for qPCR were generated using 10-fold serial dilutions of the plasmid carrying target genes. According to the standard curves, the Ct value of unknown samples was used to calculate the number of corresponding gene copies. qPCRs were conducted in 96-well plates with a final volume of 15 μL, consisted of 7.5 μL of SYBR Premix Ex Taq

3. RESULTS AND DISCUSSION 3.1. Occurrence and Effects of Wastewater Treatment on ARGs. Figure 2 summarizes the gene copy numbers (defined as the gene copy numbers per milliliter of wastewater) of the six ARGs, intI1 genes, and 16S rDNA genes in all stages of the wastewater treatment processes, and the four selected tet genes and two sul genes were detected in all wastewater samples. High detection frequencies of different ARGs in the influent of WWTPs were expected because these genes are commonly found in Gram-negative bacteria24,25 and carried by mobile elements,12,24−26 such as plasmids, transposons, and integrons, which are common in WWTPs,27 rivers,3,28 and soils.29 In the influent samples, the concentrations of different genes varied between plants A and C but the differences were not significant (p > 0.05). The concentrations of tet, sul, and intI1 genes in the influent of plants A and C generally ranged from 2.80 × 106 to 6.91 × 107 copies/mL, from 1.17 × 107 to 3.06 × 107 copies/mL, and from 5.76 × 107 to 8.44 × 107 copies/mL, respectively. A similar result was reported by other studies on the WWTPs in Michigan,7 where the gene copies of tetW, tetO, and sulI ranged from 106 to 108 copies/mL. In the study by Auerbach et al.,6 the gene copies of tetQ are approximately 1 order of magnitude higher than that in our research, ranging from 107 to 108 copies/mL. On the basis of previous studies3−5,9 and this research, the high detection frequencies and gene copy numbers of ARGs in the raw influent of WWTPs from different areas indicate that the ARGs widely exist. In the primary treatment stage, suspended solids were removed by gravity. A2, B2, and C2 were the water samples after the primary treatment processes, and ARG removal was negligible. In the secondary treatment processes, an attenuation of tet genes in WWTPs was observed, whereas an increase in the gene copy numbers of sul genes or even an increase along the treatment processes was observed. On the basis of the mass balance approach used by Kim et al.,30 the activated sludge process of WWTPs neither amplified nor attenuated the ARG fractions. In this study, no obvious change in ARG concentrations was observed in the biological process, with either the Tri-OX or A/A/O technologies. However, after the dewatering of the secondary clarifier, the ARG copy numbers 8159

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Figure 3. Log concentration (copies/g) of ARGs in biosolid samples from three WWTPs. The x-axis labels indicated different genes. Rectangular boxes indicated the interquartile range of the data. The median value was indicated by the horizontal line inside the box. A small “□” represented the mean values. Gene quantities were presented as the copy number normalized to grams of dry biosolid (n = 5).

1010 copies/g, from 5.39 × 1010 to 5.60 × 1010 copies/g, and 6.16 × 1011 copies/g, respectively. Earlier studies discovered the occurrence of ARGs in environments influenced by the effluent of WWTPs27,28,31 and the application of biosolids.9 Higher levels of ARGs than background were observed in the soil amended with biosolids.9 In this study, the concentrations of these ARGs in the effluent and biosolids remain higher than those in natural water and soil.3,9,11 Thus, the continuous input of resistance genes from the WWTP effluent and the application of biosolids should be conducted with caution, because these activities may be a highly significant external cause of the introduction of ARGs into the natural environment in eastern China. 3.2. ARG Removal in WWTPs and Advanced Treatment Processes. The removal of ARGs, intI1 genes, and 16S rDNA genes in WWTPs as well as their advanced treatment processes were determined, as shown in Figure 4. In the

Figure 2. Occurrence and changes of ARGs in plants A, B, and C, with plants A and B using the triple OX process and plant C using the A/A/ O process. A1−A4 represent the samples collected in plant A (n = 2). B1−B4 represent the samples collected in plant B (n = 10). C1−C6 represent the samples collected in plant C (n = 2). To compare absolute reduction of ARGs in WWTPs, gene quantities are presented normalized to milliliters of wastewater. Error bars indicate the standard deviation of three replicates.

were significantly lower than those in the effluent of the anaerobic or oxic processes. In the effluent, the concentrations of tet, sul, and intI1 genes in plants A and C ranged from 2.46 × 104 to 1.21 × 106 copies/mL, from 6.61 × 105 to 3.13 × 106 copies/mL, and from 2.37 × 106 to 2.82 × 106 copies/mL, respectively. A previous study determined a significant correlation between the absolute tet gene copies and bacterial 16S rDNA gene copies in sediment samples,10 such that, under the effect of sludge sedimentation, a larger amount of ARGcontaining bacteria was removed with the removal of the biomass. Significant reductions of ARGs in the secondary clarifier were likely related to sludge sedimentation. This implied that the removal of ARGs in WWTPs partly relied upon sludge sedimentation and also indirectly proved that the biosolids will contain a large amount of ARGs. The occurrence and concentration of ARGs, intI1 genes, and 16S rDNA genes in biosolids were also detected, as shown in Figure 3. The concentrations of all target genes were relatively higher, and the mean values of the biosolids ranged from 2.36 × 108 to 1.50 ×

Figure 4. Log removals of tetM, tetO, tetQ, tetW, sulI, sulII, intI1, and 16S rDNA genes by WWTPs and advanced treatment systems. Plant A, oxidation ditch (OX) as the wastewater treatment process and constructed wetland (CW) as the advanced treatment system; plant B, oxidation ditch (OX) as the wastewater treatment process and biological aerated filter (BF) as the advanced treatment system; and plant C, anaerobic−anoxic−oxic (A/A/O) as the wastewater treatment process and ultraviolet radiation disinfection (UV) as the advanced treatment system. n = 2 for plant A and C, and n = 1 for plant B. 8160

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Figure 5. Concentrations of ARGs, intI1 genes, and 16S rDNA genes in different sampling sites of constructed wetlands. A4 (n = 2) represented the effluent samples from plant A. A5 (n = 2) represented the effluent samples from subsystem I of constructed wetlands. A6 (n = 2) represented the effluent samples from subsystem II of constructed wetlands. A7 (n = 2) represented the effluent samples from subsystem III of constructed wetlands. A8 (n = 2) represented the effluent samples from subsystem IV of constructed wetlands. A9 (n = 2) represented the effluent samples from subsystem V of constructed wetlands. Error bars indicate the standard deviation of three replicates.

municipal WWTPs, the removal of tet genes reached 1.5−2.5 orders of magnitude, whereas the removal values of sul genes was relatively low at approximately 0.9−1.9 orders of magnitude. Previous research discovered that tetracyclineresistant bacteria were easier to remove than sulfamethoxazole-resistant bacteria,2 which may explain why tet genes were easier to remove than sul genes. UNIANOVA was conducted to analyze the removal values of the ARGs, intI1 genes, and 16S rDNA genes, and no significant difference (p > 0.05) could be found among the removal values of ARGs in plants A, B, and C. The relative abundance of tet genes (defined as the gene copy numbers of ARGs normalized to the gene copy numbers of 16S rDNA, as shown in Figure S2 of the Supporting Information) indicated a reduction during processing in WWTPs. However, for sul and intI1 genes, the relative abundance increased during processing in WWTPs. A big reduction in ARG copy numbers has been reported throughout the wastewater treatment process,2,6,7 but the relative abundance of sulI (gene abundances of sulI normalized to that of 16S rRNA genes) remained stable along the treatment process.2 Other studies suggest that wastewater treatment processes contribute to the selective increase in antibiotic-resistant bacteria19 while stimulating horizontal gene transfer among microbial species.32 A significant reduction in ARG copy numbers was observed in our study, but the relative abundance of sul and intI1 genes in the effluent of municipal WWTPs increased slightly. In the advanced treatment process, the constructed wetland was observed to contribute significantly to the removal of ARGs (1.3−2.1 orders of magnitude for tet genes and 1.5 orders of magnitude for the sulI gene) and intI1 genes (about 1.7 orders of magnitude), followed by the biological aerated filter (1.0− 1.21 orders of magnitude for tet genes and approximately 0.65 orders of magnitude for sul and intI1 genes). Only a slight change in ARG concentrations was observed between the preand post-UV disinfected effluent samples (0.5−0.7 orders of magnitude for tet genes and approximately 0.3 orders of magnitude for sul and intI1 genes). McKinney and Pruden33 found that the UV disinfection only had limited potential to damage ARGs in water and wastewater effluents in a batch-scale experiment, which was consistent with our result. UNIANOVA was conducted to analyze the removal values of ARGs, intI1

genes, and 16S rDNA genes in different advanced treatment processes, and a significant difference was observed (p < 0.05) between constructed wetland and UV disinfection as well as between constructed wetland and biological aerated filter (p < 0.05). In terms of relative abundance, reductions in tet, sulI, and intI1 genes were found in constructed wetland (see Figure S2 of the Supporting Information). In the biological aerated filter and UV disinfection processes, the relative abundance of sul and intI1 genes increased. In terms of removal values, no significant difference (p > 0.05) was found between WWTPs and constructed wetlands. Comparing the relative abundance of ARGs in WWTPs and in constructed wetland, we found that the ARG (except sulII) abundance in the final effluent of constructed wetland was lower than that in the effluent of WWTPs. This finding suggests that the constructed wetland as an advanced treatment process positively influences the further attenuation of ARGs from the WWTP effluent. 3.3. Removal of ARGs in Constructed Wetland. To investigate further the removal effects of constructed wetland on ARGs, intI1 genes, and 16S rDNA genes, samples from each subsystem of constructed wetland were collected. An analysis of these samples was motivated by the fact that the relative ARG abundance was lower in constructed wetland effluent samples than in other advanced treatment processes. The constructed wetlands with five subsystems have been running for 3 years, and the annual performance of this constructed wetland indicated that the average removal rates of total nitrogen, total phosphorus, ammonia nitrogen, and chemical oxygen demand were 75, 76, 95, and 54%, respectively.34 Subsystem I was planted with Hydrocotyle verticillata and Myriophyllum spicatum, with biological stuffing below the plants, which served as a microbial membrane purification system with an area of 190 m2. Subsystem I had an active function in ARG removal; the removal values of tet, sul, and intI1 genes were 0.35−0.54, 0.52−1.54, and 0.37 orders of magnitude, respectively. However, for subsystem II with an area of 253 m2 and planted with Cyperus haspan, a limited effect was observed in terms of ARG removal. Better ARG removal values were found in subsystem III, with an area of 1014 m2 and planted with Cyperus alternifolius, Thalia dealbata, Canna lily, 8161

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parameters of wetland, floristics, and geographical environment on the removal of ARGs.

Iris pseudacorus, Herba saururi chinensis, and Oenanthe javanica. The removal value of tet genes ranged from 0.83 to 1.00 orders of magnitude, whereas that of sul genes ranged from 0.57 to 1.02 orders of magnitude in subsystem III. Subsystem IV was planted with Nymphaea, Herba saururi, and Pontederia cordata and has an area of 1267 m2. The removal of tet genes in subsystem IV reached 0.38−0.75 orders of magnitude, and that for sulI genes was 0.21 orders of magnitude. The limited effect was observed in the removal of the sulII and intI1 genes. Subsystem V was a horizontal subsurface flow wetland with an area of 950 m2 and planted with vetiver grass, Typha latifolia, and Arundo donax L. A limited effect was observed in terms of ARG removal. Subsystems I and III effectively removed ARGs, whereas for subsystems II, IV, and V, the removal values varied among different sampling seasons (p < 0.05, as shown in Figure S3 of the Supporting Information). The difference between seasons and subsystems may be attributed to the temperature and plant types. The temperature would affect the growth of plants and microorganisms, given that the biological stuffing and plant rhizosphere biofilms in constructed wetland would have an active function in the interception of bacteria. A previous study on a surface flow constructed wetland also showed that the system has good reliability of approximately 2 orders of magnitude for fecal coliform removal.35 The removal of Salmonella, E. coli, and Enterococcus was also observed in integrated constructed wetlands used to treat agricultural wastewater.36 Munir et al.8 found significantly higher ARG removal rates in a membrane biological reactor than in conventional treatment plants. We further studied the relationships among the removal of ARGs, intI1 genes, and 16S rDNA genes in the wastewater samples, as shown in Figure S4 of the Supporting Information, and statistically significant correlations were observed in the removal of ARGs and 16S rDNA genes (tetM, R2 = 0.391 and p < 0.05; tetO, R2 = 0.521 and p < 0.05; tetQ, R2 = 0.430 and p < 0.05; tetW, R2 = 0.583 and p < 0.01; sulI, R2 = 0.866 and p < 0.01; sulI, R2 = 0.778 and p < 0.01; and intI1, R2 = 0.845 and p < 0.01). ARG removal could be attributed to the efficient removal of microorganisms in constructed wetland. The ARG concentrations in the sediment samples in each subsystem were also measured and analyzed (Figure 5). The ARG concentrations in the sediment samples of each subsystem showed seasonal variations, which were not significant (p > 0.05). The relationships between ARG concentrations in the wastewater samples and corresponding sediment samples are shown in Figure S5 of the Supporting Information, and statistically significant correlations were observed (tetW, R2 = 0.881 and p < 0.01; tetM, R2 = 0.791 and p < 0.01; and sulI, R2 = 0.879 and p < 0.01). No significant correlations (p > 0.05) were found between the concentrations of other ARGs detected in the water samples and sediment samples, and the linearity was extremely weak with a high degree of scatter. The constructed wetlands, as an advanced treatment system of WWTP, demonstrated almost the same performance in the removal of ARG copy numbers with WWTPs (OX or A/A/O) and a better reduction of relative abundance. Therefore, the constructed wetlands seemed to be a considerable advanced treatment system to attenuate the discharge of ARGs from the important reservoirs (WWTP) into an aqueous ecosystem in tropical and subtropical zones. Given the complexity of constructed wetlands, we conclude by underscoring the need for further research on the factors such as type and design

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ASSOCIATED CONTENT

* Supporting Information S

Detailed descriptions of ARG analyses, PCR conditions, primers, annealing temperature, and standard curves (Tables S1 and S2), sampling site location (Figure S1), relative abundance ARGs in three WWTPs (Figure S2), concentrations of ARGs, intI1 genes, and 16S rDNA genes in wastewater sediment samples (Figure S3), correlation between the log removal of ARGs, intI1 genes, and 16S rDNA genes in wastewater samples of the constructed wetland (Figure S4), and correlation between the concentration of ARGs in wastewater and sediment samples in the constructed wetland (Figure S5). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Telephone/Fax: 86-571-8898-2028. E-mail: chen_hong@zju. edu.cn. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the managers of wastewater treatment plants for providing the samples and information needed for this study. This work was supported by the Natural Science Foundation of China (2127712 and 21210008) and Science and Technology Department of Zhejiang Province (2012C23043).

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REFERENCES

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dx.doi.org/10.1021/es401091y | Environ. Sci. Technol. 2013, 47, 8157−8163