Autotrophic growth activity of complete ammonia oxidizers in an upflow biological contact filter for drinking water treatment.

Biological filters effectively remove ammonium from drinking water via nitrification. In a pilot-scale upflow biological contact filter (U-BCF), complete ammonia oxidizers (comammox), which are capable of oxidizing ammonia to nitrate in one cell, were more abundant than ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). However, little information is available on the contribution of comammox to nitrification. In this study, we evaluated the autotrophic growth activity of comammox associated with biological activated carbon (BAC) in a U-BCF by DNA-stable isotope probing (DNA-SIP). BAC samples collected from the U-BCF were continuously fed mineral medium containing 0.14 mg N L-1 ammonium and 12C- or 13C-labeled bicarbonate for 20 days. DNA-SIP analysis revealed that comammox (clades A and B) as well as AOA assimilated bicarbonate after 10 days of incubation, proving that dominant comammox could contribute to nitrification. Contrarily, AOB remained inactive throughout the observation period. Amplicon sequencing of the 13C-labeled DNA fractions of comammox revealed that specific genotypes other than the most dominant genotype in the original sample were more enriched under the incubation condition for the DNA-SIP experiment. Thus, dominant genotypes of comammox in a U-BCF might utilize organic nitrogen to fuel nitrification in ammonia-limited environments.

Identification of new eligible indicator organisms for combined sewer overflow via 16S rRNA gene amplicon sequencing in Kanda River, Tokyo.

Fecal indicator bacteria (FIB) are commonly used to evaluate the pollution impact of combined sewer overflows (CSOs) in urban rivers. Although water quality assessment with FIB has a long tradition, recent studies demonstrated that FIB have a low correlation with pathogens and therefore are not accurate enough for the assessment of potential human hazards in water. Consequently, new eligible and more specific indicators have to be identified, which was done in this study via sequencing of genetic markers from total community DNA. To identify potential microbiome-based indicators, microbial communities in samples from an urban river in Tokyo under different climatic conditions (dry and rainy) were compared with the influent and effluent of three domestic wastewater treatment plants (WWTPs) by analyzing 16 S rRNA gene amplicon libraries. In the first part of this study, physicochemical parameters and FIB quantification with selective culture techniques facilitated the identification of samples contaminated with CSO, sewage, or both. This allowed the grouping of samples into CSO-contaminated and non-contaminated samples, an essential step prior to the microbiome comparison between samples. Increased turbidity, ammonia concentrations, and E. coli [up to (9.37 ± 0.95) × 102 CFU/mL after 11.5 mm of rainfall] were observed in CSO-contaminated river samples. Comparison of dry weather (including WWTP samples) and rainy weather samples showed a reduction in microbial diversity in CSO-contaminated samples. Furthermore, the results of this study suggest Bacteroides spp. as a novel indicator of sewage pollution in surface waters.

Temperature-Dependent Ammonium Removal Capacity of Biological Activated Carbon Used in a Full-Scale Drinking Water Treatment Plant.

Nitrification is a key function of biological activated carbon (BAC) filters for drinking water treatment. It is empirically known that the nitrification activity of BAC filters depends on water temperature, potentially resulting in the leakage of ammonium from BAC filters when the water temperature decreases. However, the ammonium removal capacity of BAC filters and factors governing the capacity remain unknown. This study employed a bench-scale column assay to determine the volumetric ammonium removal rate (VARR) of BAC collected from a full-scale drinking water treatment plant. VARR was determined at a fixed loading rate under different conditions. Seasonal variations of the VARR as well as impacts of the water matrix and water temperature on ammonium removal were quantitatively analyzed. While the VARR in an inorganic medium at 25 °C was maintained even during low water temperature periods and during breakpoint chlorination periods, the water matrix factor reduced the VARR in ozonated water at 25 °C by 33% on average. The VARR in ozonated water was dependent on water temperature, indicating that the microbial activity of BAC did not adapt to low water temperature. The Arrhenius equation was applied to reveal the relationship between VARR and water temperature. The actual ammonium removal performance of a full-scale BAC filter was predicted. VARR is useful for water engineers to reexamine the loading and filter depth of BAC filters.

Changes in Dissolved Organic Matter Composition and Disinfection Byproduct Precursors in Advanced Drinking Water Treatment Processes.

Molecular changes in dissolved organic matter (DOM) from treatment processes at two drinking water treatment plants in Japan were investigated using unknown screening analysis by Orbitrap mass spectrometry. DOM formulas with carbon, hydrogen and oxygen (CHO-DOM) were the most abundant class in water samples, and over half of them were commonly found at both plants. Among the treatment processes, ozonation induced the most drastic changes to DOM. Mass peak intensities of less saturated CHO-DOM (positive (oxygen subtracted double bond equivalent per carbon (DBE-O)/C)) decreased by ozonation, while more saturated oxidation byproducts (negative (DBE-O)/C) increased and new oxidation byproducts (OBPs) were detected. By Kendrick mass analysis, ozone reactions preferred less saturated CHO-DOM in the same alkylation families and produced more saturated alkylation families of OBPs. Following ozonation, biological activated carbon filtration effectively removed <300 Da CHO-DOM, including OBPs. Following chlorination, over 50 chlorinated formulas of disinfection byproducts (DBPs) were found in chlorinated water samples where at least half were unknown. Putative precursors of these DBPs were determined based on electrophilic substitutions and addition reactions. Ozonation demonstrated better decomposition of addition reaction-type precursors than electrophilic substitution-type precursors; over half of both precursor types decreased during biological activated carbon filtration.

Incorporation of 13C-HCO3- by ammonia-oxidizing archaea and bacteria during ammonia oxidation of sludge from a municipal wastewater treatment plant.

Ammonia-oxidizing archaea (AOA) have recently been proposed as potential players for ammonia removal in wastewater treatment plants (WWTPs). However, there is little evidence directly showing the contribution of AOA to ammonia oxidation in these engineered systems. In this study, DNA-stable isotope probing (DNA-SIP) with labeled 13C-HCO3- was introduced to sludge from a municipal WWTP. Quantitative PCR demonstrated that AOA amoA genes outnumbered AOB amoA genes in this WWTP sludge. AOA amoA gene sequence analysis revealed that AOA present in this WWTP were specific to one subcluster within the group 1.1b Thaumarchaeota. When ammonia was supplied to DNA-SIP incubation, the DNA-SIP profiles demonstrated the incorporation of the 13C into AOA and AOB. However, the 13C was not found to be assimilated into both microorganisms in the incubation without ammonia. Specific primers were designed to target amoA genes of AOA belonging to the subcluster found in this WWTP. Applying the primers to DNA-SIP experiment revealed that AOA of this subcluter most likely utilized inorganic carbon during ammonia oxidation under the studied conditions.

Quantification of Chloroflexi Eikelboom morphotype 1851 for prediction and control of bulking events in municipal activated sludge plants in Japan.

The dominant filamentous bacteria associated with bulking incidents in Japanese activated sludge plants with nutrient removal were identified and their quantitative correlations with sludge settleability were assessed, with the aim of controlling bulking incidents by specifically suppressing bacterial growth. Fluorescence in situ hybridization (FISH) analyses using existing oligonucleotide FISH probes indicated that the presence of Eikelboom type 1851 filamentous bacteria belonging to the phylum Chloroflexi is correlated with biomass settleability in the municipal wastewater treatment plants examined. Real-time quantitative PCR (qPCR) assays developed in this study also showed a linear correlation between type 1851 filament members and sludge settleability, with the exception of some winter samples. The real-time qPCR assays and 16S ribosomal RNA gene amplicon sequencing to reveal the microbial community of activated sludge showed that the abundance of type 1851 at 200 mL g-1 of sludge volume index was estimated to be about 1.9% of the total microbial cells. The abundance of type 1851 served as a bulking indicator in plants where type 1851 was dominant.

Abundance and diversity of ammonia-oxidizing archaea and bacteria on granular activated carbon and their fates during drinking water purification process.

Ammonia is a precursor to trichloramine, which causes an undesirable chlorinous odor. Granular activated carbon (GAC) filtration is used to biologically oxidize ammonia during drinking water purification; however, little information is available regarding the abundance and diversity of ammonia-oxidizing archaea (AOA) and bacteria (AOB) associated with GAC. In addition, their sources and fates in water purification process remain unknown. In this study, six GAC samples were collected from five full-scale drinking water purification plants in Tokyo during summer and winter, and the abundance and community structure of AOA and AOB associated with GAC were studied in these two seasons. In summer, archaeal and bacterial amoA genes on GACs were present at 3.7 × 10(5)-3.9 × 10(8) gene copies/g-dry and 4.5 × 10(6)-4.2 × 10(8) gene copies/g-dry, respectively. In winter, archaeal amoA genes remained at the same level, while bacterial amoA genes decreased significantly for all GACs. No differences were observed in the community diversity of AOA and AOB from summer to winter. Phylogenetic analysis revealed high AOA diversity in group I.1a and group I.1b in raw water. Terminal-restriction fragment length polymorphism analysis of processed water samples revealed that AOA diversity decreased dramatically to only two OTUs in group I.1a after ozonation, which were identical to those detected on GAC. It suggests that ozonation plays an important role in determining AOA diversity on GAC. Further study on the cell-specific activity of AOA and AOB is necessary to understand their contributions to in situ nitrification performance.

Sensitivity and quantitativeness of large-volume injection combined with liquid chromatography/high-resolution mass spectrometry for target screening analysis of emerging contaminants.

Solid phase extraction (SPE) has been widely used for pretreatment in target screening (TS) analysis. However, some compounds are difficult to recover by SPE or their recovery is unstable for environmental samples. In this study, we tested large-volume injection (LVI) without SPE for TS analysis of 103 compounds listed by the Ministry of the Environment (Japan)-so-called 'items to be surveyed'-using liquid chromatography high-resolution mass spectrometry. We evaluated the limit of quantification (LOQ) by LVI and compared this LOQ with the LOQ by SPE pretreatment using a hydrophilic-lipophilic balance (HLB) combined with activated carbon, which was found previously to afford the best SPE cartridges for target compounds recovery. The LOQ generally decreased as the injection volume increased, and the LOQ was at least 250 times lower for a 500-µL injection than for a 2-µL injection for half of the compounds. LVI provided LOQs lower than the predicted no effect concentration for more compounds than the SPE method. The average matrix effect (ME) by LVI was in the range 70%-130% for 69 out of 97 compounds. The ME was higher or lower for some of the remaining compounds, but the ME was in the range 10%-1000% for all 18 water samples for 84 of the 97 compounds. Comparing the ME by LVI and the recovery ratio by the SPE method showed that LVI achieved more accurate quantitation than the SPE method for a larger number of compounds. Therefore, LVI provides better sensitivity and quantitativeness than the SPE method using HLB and activated carbon for TS analysis of as many 'items to be surveyed' as possible.

Non-target liquid chromatography high-resolution mass spectrometry screening to prioritize unregulated micropollutants that persist through domestic wastewater treatment.

Efforts to regulate and monitor emerging contaminants are insufficient because new chemicals are continually brought to market, and many are unregulated and potentially harmful. Domestic wastewater treatment plants are not designed to remove micropollutants and are important sources of emerging contaminants in the aquatic environment. In this study, non-target screening, an unbiased method for analyzing compounds without prior information, was used to identify compounds that may be emitted in wastewater treatment plant effluent and should be monitored. Nine wastewater treatment plants using different treatment methods were studied, and a non-target screening data-processing method was used. The frequencies at which the contaminants were detected and contaminant persistence through the treatment processes were considered, and then the contaminants were prioritized. The predicted no-effect concentration of each prioritized contaminant was used to determine whether further analysis and monitoring of the contaminant was necessary. Quantitative analyses of five compounds (amantadine, atenolol, benzotriazole, diphenhydramine, and sulpiride) were performed using reference standards. Probable molecular formulae and structures were proposed for 17 contaminants, and the risks posed by the contaminants were estimated using predicted no-effect concentrations. The results provide valuable insights into how unregulated micropollutants can be identified and prioritized for monitoring in future studies.

Characterization of bacterial isolates from water reclamation systems on the basis of substrate utilization patterns and regrowth potential in reclaimed water.

Microbial regrowth causes problems during water reuse. Comprehensive understanding of the microorganisms that can regrow in reclaimed water and their substrate requirements are necessary. In this study, potential regrowth organisms were isolated from seven water reclamation plants in Japan. Based on 16S rDNA analysis, the isolates were grouped into 34 operational taxonomic units, belonging to Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes. Substrate utilization profiling using Biolog microplate™ classified the isolates into four groups. Bacteria in Cluster 1 (e.g., Methylobacterium sp. and Acinetobacter sp.) mainly utilized polymers, esters, amides, and alcohol. Isolates in Cluster 2 (e.g., Flavobacterium sp. and Microbacterium sp.) preferred to utilize polymers, carbohydrates, and esters. Isolates in Cluster 3 (e.g., Pseudomonas sp. and Acidovorax sp.) mainly utilized esters, carboxylic acids, and amino acids. Isolates in Cluster 4 (e.g., Enterobacter sp. and Rhodococcus sp.) utilized carbohydrates, esters, and amino acids. All isolates grew in reclaimed water treated by sand filtration, whereas some isolates could not grow in reclaimed water treated by coagulation and ozonation. Most bacteria in the same Biolog clusters exhibited similar growth characteristics in water samples. The potential of bacteria to regrow in reclaimed water likely depended on substrate requirement.

Dynamics of the Microbial Community and Opportunistic Pathogens after Water Stagnation in the Premise Plumbing of a Building.

In premise plumbing, microbial water quality may deteriorate under certain conditions, such as stagnation. Stagnation results in a loss of disinfectant residual, which may lead to the regrowth of microorganisms, including opportunistic pathogens. In the present study, microbial regrowth was investigated at eight faucets in a building over four seasons in one year. Water samples were obtained before and after 24 h of stagnation. In the first 100| |mL after stagnation, total cell counts measured by flow cytometry increased 14- to 220-fold with a simultaneous decrease in free chlorine from 0.17-0.36| |mg L-1 to <0.02| |mg L-1. After stagnation, total cell counts were not significantly different among seasons; however, the composition of the microbial community varied seasonally. The relative abundance of Pseudomonas spp. was dominant in winter, whereas Sphingomonas spp. were dominant in most faucets after stagnation in other seasons. Opportunistic pathogens, such as Legionella pneumophila, Mycobacterium avium, Pseudomonas aeruginosa, and Acanthamoeba spp., were below the quantification limit for real-time quantitative PCR in all samples. However, sequences related to other opportunistic pathogens, including L. feeleii, L. maceachernii, L. micdadei, M. paragordonae, M. gordonae, and M. haemophilum, were detected. These results indicate that health risks may increase after stagnation due to the regrowth of opportunistic pathogens.

Shotgun isotope array for rapid, substrate-specific detection of microorganisms in a microbial community.

The shotgun isotope array method has been proposed to be an effective new tool for use in substrate-specific microbe exploration without any prior knowledge of the community composition. Proof of concept was demonstrated by detection of acetate-degrading microorganisms in activated sludge and further verified by independent stable isotope probing (SIP).

Changes in dissolved organic matter during water treatment by sequential solid-phase extraction and unknown screening analysis.

Isolation of complex dissolved organic matter (DOM) from environmental water is a major challenge for unknown screening analysis by high-resolution mass spectrometry. In this study, DOM in process water during advanced drinking water treatment was fractionated sequentially by three solid-phase extraction (SPE) cartridges based on the polarity and charge of DOM molecules. By sequential SPE with unknown screening analysis, over 3000 DOM features were found in raw water, whereas around 2000 were obtained by a single SPE. The hydrophobic neutral (HPON) fraction contained CHO features with highest averaged molecular weight followed by hydrophobic acid (HPOA) and then hydrophilic acid (HPIA). The average degree of carbon double bond equivalents and carbon oxidation states indicated that the HPON fraction contained molecules that were more unsaturated and less oxidized than those of the HPOA and HPIA fractions. Ozone selectively decomposed (1) more unsaturated and less oxidized HPON features, (2) more unsaturated HPOA compounds, and (3) less oxidized HPIA molecules. Oxidation by-products were mostly HPON and HPIA compounds that were more oxidized than the decomposed molecules. During biological activated carbon (BAC) filtration, less oxidized HPON were preferentially removed, whereas HPOA were removed without selectivity. HPON and HPIA molecules with more oxidized character were found to be refractory to BAC treatment. HPON with more unsaturated and HPIA with more oxidized characters were decomposed by chlorine. Many types of HPIA decomposed during chlorination were the oxidation by-products of ozonation that were refractory to BAC treatment. Sequential SPE with unknown screening analysis provided previously unknown details of the molecular characteristics of DOM and its changes during advanced water treatment.

Orbitrap mass spectrometry for the molecular characterization of water resource recovery from polluted surface water using membrane bioreactor.

The increasing organic contamination of surface water hinders the conventional tap water treatment process. Membrane bioreactors (MBRs) are a promising alternative technology for recovering water from polluted surface water. In this study, the composition changes of dissolved organic matters (DOMs) in MBR and ultraviolet/ozone (UV/O3)-MBR systems for polluted surface water treatment were investigated using Orbitrap mass spectrometry analysis with unknown screening. The intense DOM ions within a mass-to-charge ratio range of 100-500 was detected, and 2340 molecular formulae from 5743 peaks were assigned to the two systems. The most abundant components were formulae with C, H, O, N, and CHO only classes. The highest formulae decrease including CHO, CHON, CHOS, and CHONS were attributed to the bio-carrier used in both systems. Results showed that bioprocess was the main contributor in the DOM reduction, and the integration of UV/O3 into the MBR improved the DOM composition changes. Biodegradable components with low O/C ratio in the CHO and CHON classes remarkably increased in the UV/O3-MBR system. The integration of UV/O3 as a polishing step in the recirculation stream of MBR system was effective in improve the DOM removal.

Optimized Cultivation and Syntrophic Relationship of Anaerobic Benzene-Degrading Enrichment Cultures under Methanogenic Conditions.

Current challenges in the anaerobic bioremediation of benzene are the lack of capable cultures and limited knowledge on the biodegradation pathway. Under methanogenic conditions, benzene may be mineralized by syntrophic interactions between microorganisms, which are poorly understood. The present study developed an optimized formula for anoxic medium to successfully promote the growth of the putative benzene degrader Deltaproteobacterium Hasda-A and enhance the benzene degradation activity of methanogenic enrichment cultures. Within 70| |d of incubation, the benzene degradation activity and relative abundance of Hasda-A in cultures in the new defined medium increased from 0.5 to >3| |mg L-1 d-1 and from 2.5% to >17%, respectively. Together with Hasda-A, we found a strong positive relationship between the abundances of superphylum OD1 bacteria, three methanogens (Methanoregula, Methanolinea, and Methanosaeta) and benzene degradation activity. The syntrophic relationship between these microbial taxa and Hasda-A was then demonstrated in a correlation analysis of longitudinal data. The involvement of methanogenesis in anaerobic benzene mineralization was confirmed by inhibition experiments. The high benzene degradation activity and growth of Hasda-A were quickly recovered in successive dilutions of enrichment cultures, proving the feasibility of using the medium developed in the present study to produce highly capable cultures. The present results will facilitate practical applications in bioremediation and research on the molecular mechanisms underlying benzene activation and syntrophic interactions in benzene mineralization.

Characterization of lower Phong river dissolved organic matters and formations of unknown chlorine dioxide and chlorine disinfection by-products by Orbitrap mass spectrometry.

Dissolved organic matter (DOM) have been reported as precursors of disinfection byproducts (DBPs) and its molecular characteristics are rarely investigated due to its complexity. In this study, changes in the characteristics of DOM were investigated in the lower Phong River in Thailand in dry season and after the first rain in rainy season, using a non-targeted analysis with Orbitrap mass spectrometry. The river was rich with CHO features dominated by lignin-like molecules, while lipid-like molecules increase after domestic wastewater discharges. Wastewater discharge released DOM with higher molecular weight (MW) that was less oxygenated (low O/C) and less oxidized (low carbon oxidation state [Cos]). A lake affected by anthropogenic activities contributed more oxidized DOM into the river, while surface runoff carried DOM that is more oxygenated (high O/C), less hydrogenated (low H/C), and more oxidized (high Cos) to the stream. Water treatment further modified DOM to be lower MW. Approximately three hundred Cl-containing features (CHOCl) detected upstream were also found downstream. Disinfection by chlorine (Cl2) or chlorine dioxide (ClO2) formed both CHO and CHOCl DBPs. Low chlorine dosage applied to upstream and downstream samples resulted in many common unknown DBPs while increasing chlorine dosage resulted in more unique DBPs. At the same dosage, Cl2 reacted with DOM more than ClO2, including more oxidized molecules that are refractory to ClO2. Both Cl2 and ClO2 produced chlorinated and non-chlorinated DBPs, and some DBPs were commonly found by both disinfections. Cl2-produced DBPs were more unsaturated (higher [DBE-O]/C) and oxidized (higher Cos) than ClO2-DBPs.

Non-target screening of dissolved organic matter in raw water, coagulated water, and chlorinated water by Orbitrap mass spectrometry.

This study aimed to classify the possible molecular formulas of precursors for disinfection by-products (DBPs) in raw, coagulated, and chlorinated water samples from the U-Tapao Canal, Songkhla, Thailand. The molecular formulas of DBPs in chlorinated water were investigated. Polyaluminum chloride (PACl) was employed as a coagulant. Orbitrap Fourier transform-mass spectrometry was able to estimate the composition of dissolved organic matter (DOM) with the carbon (C), hydrogen (H), oxygen (O), nitrogen (N), and sulfur (S) elements contained and DBPs at the molecular level. The molecular formulas of the DOM in the raw water primarily consisted of CHO and CHON when extracted by lichrolut EN. The CHO and CHON species were rich in lignin-, tannin-, and condensed aromatic-like substances. The DOM with high-molecular-weight from 300 to 500 Da were preferentially removed by coagulation. The PACl coagulation decreased the abundances of lignin-, tannin-, and condensed aromatic-like substances in the CHO formulas, while lignin- and condensed aromatic-like substances in the CHON formulas remained. The remaining precursors corresponded to CHON molecules in the coagulated water, which may result in the formation of some chlorine (Cl)-containing molecules. Several DBPs among the CHOCl and CHONCl species were produced in the chlorinated water through the addition reaction of chlorine. New chlorinated N-DBPs of 21 formulas were detected.

Abundance and diversity of ammonia-oxidizing archaea and bacteria on biological activated carbon in a pilot-scale drinking water treatment plant with different treatment processes.

The effects of different placements of rapid sand filtration on nitrification performance of BAC treatment in a pilot-scale plant were evaluated. In this plant, rapid sand filtration was placed after ozonation-BAC treatment in Process (A), while it preceded ozonation-BAC treatment in Process (B). Analysis of amoA genes of ammonia-oxidizing archaea (AOA) and bacteria (AOB) combined with nitrification potential test was conducted. BAC from Process (A) demonstrated slightly higher nitrification potential at every sampling occasion. This might be due to higher abundances of AOB on BAC from Process (A) than those on BAC from Process (B). However, AOA rather than AOB could be predominant ammonia-oxidizers in BAC treatment regardless of the position of rapid sand filtration. The highest nitrification potential was observed for BAC from both processes in February when the highest abundances of AOA-amoA and AOB-amoA genes were detected. Since rapid sand filtration was placed after BAC treatment in Process (A), residual aluminum concentration in BAC influent was higher in Process (A). However, adverse effects of aluminum on nitrification activity were not observed. These results suggest that factors other than aluminum concentration in different treatment processes could possibly have some influence on abundances of ammonia-oxidizing microorganisms on BAC.

Evaluating the mobile heavy metal pool in soak-away sediment, road dust and soil through sequential extraction and isotopic exchange.

Selective sequential dissolution (SSD) and isotopic dilution are two most commonly applied techniques for the measurement of mobile fraction of heavy metal present in the urban environment. This work examined the compliance between SSD proposed by the Community Bureau of Reference (BCR) and isotopic dilution technique (IDT) for determination of mobile pool of heavy metal contained in soakaway sediment, road dust, and soil sample. Heavy metals (Cu, Zn, Cd and Pb) were fractionated using the three-stage BCR protocol, while isotopically exchangeable metal concentrations (E-value) were investigated through isotopic tracers (¹¹¹Cd, 65Cu, ²°7Pb and 66Zn). In general, total contamination level, E-value and BCR exchangeable fractions of different samples followed the similar order of road dust > soakaway sediment > soil. Results revealed that the E-value exceeded the BCR exchangeable fraction in all samples. In addition, the first three fractions of BCR which have potential to become mobile under certain environmental conditions were collectively termed as "potential mobile pool" and compared with E-value. It was concluded that metal extracted by weak acid underestimates the exchangeable fraction while the potential mobile pool extracted by three reagents overestimates the real mobile forms of heavy metals. However, better mobility characteristics of heavy metals can be assessed by coupling information obtained through sequential extraction and isotopic exchange.

Comparison of metal (Zn and Cu) complexation characteristics of DOM in urban runoff, domestic wastewater and secondary effluent.

This study was aimed at comparing the Zn and Cu binding parameters with dissolved organic matter (DOM) in road runoff, wastewater treatment plant (WWTP) effluent and influent. Conditional stability constant (K') and binding site concentration ([L]) are important to predict free or labile metal concentration and toxicity in the water environment. The values of K' and [L] of three DOMs were determined by metal titration and Scatchard linearization. The Zn and Cu titration data for DOMs in WWTP effluent and influent fitted to a two-ligand model, while DOM in road runoff fitted to a single-ligand model. The order of the values of K' was WWTP influent > WWTP effluent > road runoff both for Zn and Cu. Total ambient binding site concentration ([L(T)]) was lower in DOM of road runoff (1.4-1.5 µM) than those in DOM of WWTP effluent (1.6-2.3 µM) and influent (17-18 µM), suggesting lower metal complexation capacity in DOM of road runoff. DOM in WWTP influent was expected to bind to both Zn and Cu more strongly than that of the effluent DOM.