Chemosphere 117 (2014) 545–551
Contents lists available at ScienceDirect
Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Characterization and biotoxicity assessment of dissolved organic matter in RO concentrate from a municipal wastewater reclamation reverse osmosis system Ying-Xue Sun a,⇑, Yue Gao a, Hong-Ying Hu b,c, Fang Tang b, Zhe Yang a a
Department of Environmental Science and Engineering, Beijing Technology and Business University, Beijing 100048, PR China Environmental Simulation and Pollution Control State Key Joint Laboratory, State Environmental Protection Key Laboratory of Microorganism Application and Risk Control, School of Environment, Tsinghua University, Beijing 100084, PR China c Shenzhen Laboratory of Microorganism Application and Risk Control, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, PR China b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
The molecular weight 1–3 kDa was
dominant in the basic and neutral DOM fractions. The hydrophilic neutrals contributed to the majority of the DOC and genotoxicity. The hydrophobic neutrals had high antiestrogenic activity per DOC of the fractions.
a r t i c l e
i n f o
Article history: Received 29 April 2014 Received in revised form 2 September 2014 Accepted 4 September 2014
Handling Editor: E. Brillas Keywords: Wastewater reclamation Reverse osmosis concentrate Dissolved organic matter fractions Genotoxicity Anti-estrogenic activity
⇑ Corresponding author. Tel.: +86 (10) 6898 4021. E-mail address: [email protected] (Y.-X. Sun). http://dx.doi.org/10.1016/j.chemosphere.2014.09.024 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.
a b s t r a c t Reverse osmosis (RO) concentrate from municipal wastewater reclamation reverse osmosis (mWRRO) system containing organic compounds may associate with toxic risk, and its discharge might pose an environmental risk. To identify a basis for the selection of feasible technology in treating RO concentrates, the characteristics and biotoxicity of different fractions of dissolved organic matter (DOM) in RO concentrates from an mWRRO system were investigated. The results indicated that the hydrophilic neutrals (HIN), hydrophobic acids (HOA) and hydrophobic bases (HOB) accounted for 96% of the dissolved organic carbon (DOC) of the total DOM in the RO concentrate. According to the SEC chromatograph detected at 254 nm wavelength of UV, the DOM with molecular weight (MW) 1–3 kDa accounted for the majority of the basic and neutral fractions. The fluorescence spectra of the excitation emission matrix (EEM) indicated that most aromatic proteins, humic/fulvic acid-like and soluble microbial by-product-like substances existed in the fractions HOA and hydrophobic neutrals (HON). The genotoxicity and antiestrogenic activity of the RO concentrate were 1795.6 ± 57.2 lg 4-NQO L 1 and 2.19 ± 0.05 mg TAM L 1, respectively. The HIN, HOA, and HOB contributed to the genotoxicity of the RO concentrate, and the HIN was with the highest genotoxic level of 1007.9 ± 94.8 lg 4-NQO L 1. The HOA, HON, and HIN lead to the total anti-estrogenic activity of the RO concentrate, and HOA occupied approximately 60% of the total, which was 1.3 ± 0.17 mg TAM L 1. Ó 2014 Elsevier Ltd. All rights reserved.
546
Y.-X. Sun et al. / Chemosphere 117 (2014) 545–551
1. Introduction Wastewater reclamation plays an important role in the development of strategies for the water resource management worldwide (Lazarova et al., 2013). Recently, the water quality requirement to reclaimed water changed from being initially focused on microbial quality to a wider and more comprehensive view of the chemical quality. With an increasing demand of high quality reclaimed water, municipal wastewater reclamation reverse osmosis (mWRRO) has been extensively and successfully applied in municipal wastewater reclamation because of its excellent separated function for organic and inorganic contaminants, as well as biological constituents (Al-Rifai et al., 2011; Subramania and Jacangeloa, 2014). However, accompanying a superb quality water for reuse, mWRRO also produces a certain quantity waste stream of RO concentrate, generally ranging from 25% to 50% of the feed, which contains high concentrations of dissolved salts and recalcitrant organics (Bellona et al., 2004; Al-Rifai et al., 2007; Dialynas et al., 2008). As the global and domestic implementation of RO technology is increasing significantly, there is an increasing concern of further disposal of RO concentrate because its discharge (e.g., via municipal sewer) may pose an environmental risk (Khan et al., 2009; Greenlee et al., 2009; Subramania and Jacangeloa, 2014). The organic matter in the RO concentrate of the mWRRO process comprises not only the concentrated dissolved organic matter (DOM) from the secondary effluent but also certain chemical agents injected during the mWRRO process, and new organic pollutants are also formed during the mWRRO process (Linge et al., 2013). Non-oxidizing biocide (Kathon biocide) injected in mWRRO elicits high genotoxic in vitro and contributes to a higher genotoxicity of the RO concentrate (Tang et al., 2013a). New disinfection byproducts, such as trihalomethanes, haloacetic acids, haloacetonitriles, and N-nitrosodimethylamine, are also formed during the pre-chlorination in the mWRRO process to protect membranes from bio-fouling; these byproducts are likely rejected as RO concentrate (Megan et al., 2008; Agus et al., 2009; Linge et al., 2013). Moreover, the mechanism of RO membrane separation for organic matter involves not only a simple physical process of separating solutes from solvents in terms of size difference but also the interactions between the organic matter and the membrane itself; furthermore, large, branched, poly-functional and sterically complex molecules are better retained by various membranes (Reinhard et al., 1986; Kastelan-Kunst et al., 1997; Hu et al., 2003). Thus, the characteristics of DOM in the RO concentrate from the mWRRO process cannot be simply inferred using the investigated characteristics of the secondary treated effluent. Studies have examined the treatment methods for organic matter in the RO concentrate; however, studies have mainly investigated the removal efficiencies of DOC and UV254, as well as pharmaceutically active compounds and disinfection by-products (Benner et al., 2008; Dialynas et al., 2008; Westerhoff et al., 2009; Radjenovic et al., 2011; Bagastyo et al., 2013; Justo et al., 2013; Lu et al., 2013). An in-depth study of the characteristics of DOM in RO concentrate should be conducted to develop effective treatment methods. Bagastyo et al. (2011) studied the DOM molecular weight fractions of the RO concentrate through physicochemical characterization and fluorescence excitation-emission scans. Zhou et al. (2011) evaluated the effects of different advanced oxidation processes on the DOM in the RO concentrate by the conventional parameters (COD, DOC and UV254), molecular weight distributions and acute ecotoxicity assayed by luminescent marine bacteria. However, related investigation regarding different polarity fractions, such as the hydrophobic and hydrophilic fractions of DOM in the RO concentrate has not yet been reported.
RO membrane rejection is closely associated with DOM compounds grouped by the dissociation potential (charge or neutral), hydrophobicity and molecular size (Hu et al., 2003; Bellona et al., 2004). Moreover, some specific fractions of DOM in wastewater are associated with the haloacetic acid or trihalomethane formation potential and anti-estrogenic activity (Zhang et al., 2009; Tang et al., 2013b). Therefore, investigating the specific fractions (such as hydrophobic and hydrophilic fractions) of DOM in the RO concentrate is important to create treatable and feasible technology for RO concentrate treatment. The DOM in RO concentrates from mWRRO processes are associated with endocrine disrupting compounds, pharmaceutically active compounds and disinfection by-products. Thus, the toxic risk of DOM in the RO concentrate cannot be ignored. Studies have evaluated the removal levels of the acute toxicity, genotoxicity and retinoic acid receptor (RAR) activity in reclaimed municipal effluent treated by different wastewater reclamation technologies, including RO systems (Zha and Wang, 2005; Cao et al., 2009; Macova et al., 2011; Tang et al., 2013a). However, the DOM fractions and the characterization of anti-estrogenic activity of the RO concentrate have not been reported. Therefore, this study investigated the characteristics and biotoxicity of the DOM fractions of the RO concentrate from an mWRRO process. The DOM of the RO concentrate was divided into six fractions. Each DOM fraction was subsequently characterized by size exclusion chromatography to identify the molecular weight distributions and excitation emission matrix fluorescence spectroscopy to identify the organic components. The general biotoxicity of the DOM fractions of the RO concentrate were evaluated by genotoxicity and anti-estrogenic activity.
2. Materials and methods 2.1. Sampling and water quality analysis The RO concentrate water samples were collected at a wastewater reclamation plant in Beijing utilizing an mWRRO process, which has a treatment capacity of 21 000 m3 d 1. The influent of the mWRRO process was the secondary treatment effluent from a sequencing batch bioreactor system treating the municipal wastewater. This mWRRO system uses microfiltration (MF) with polyvinylidene fluoride hollow fiber as the pretreatment. The RO membrane is a cross-linking aromatic polyamide composite membrane, and the RO recovery ratio (RO permeate volume per RO influent volume) was 60–70%. Several chemical agents, such as chlorine, chemical cleaning, antiscalant, reductant and non-oxidizing biocide, were added to protect the RO membrane from chemical and biological damage. The schematic diagram of the mWRRO and chemicals adding points were shown in Fig. SM-1. In the secondary treatment effluent, 1.36–1.70 mg Cl2 L 1 chlorine is added continuously. However, the residual chlorine is low than 0.5 mg Cl2 L 1 before the water pumped to the MF membrane. The MF effluent is stored temporarily in the middle water tank, in which 3 mg L 1 antiscalant and appropriate amount of reductant are added sequentially. More details of the chemical agents’ dosages along the mWRRO process have been described in the previous study (Tang et al., 2013a). The RO concentrate water samples were transported to the laboratory and were filtered immediately by glass fiber (0.45 lm) to eliminate suspended solids. After filtration, the samples were stored at 4 °C to minimize the changes in the constituents in the water. The conventional water quality parameters were analyzed according to the standard methods within 24 h after sampling (APHA, 1998). The concentration of DOC was detected using a
Y.-X. Sun et al. / Chemosphere 117 (2014) 545–551 Table 1 Water quality characteristics of the studied mWRRO system. Parameter 1
TDS (mg L ) DOC (mg L 1) UV254 (cm 1) TN (mg L 1) NH3–N (mg L 1) TP (mg L 1)
Secondary effluent
RO permeate
RO concentrate
877–1390 5.8–12.4 0.11–0.12 17.1–28 0.5–5.6 0.5–3.6
4–28.5 0.2–0.6 0–0.01 1–2.2 – –
3210–4980 18–46 0.36–0.45 53.7–84.8 5.5–14 11.2–24.1
TOC-5000A analyzer (Shimadzu, Japan). UV absorbance was measured using a UV-2401PC UV–VIS recording spectrophotometer (Shimadzu, Japan). The water samples were obtained monthly from March 2013 to July 2013 to perform the primary properties of the secondary effluent, RO permeate and RO concentrate (Table 1). Also, triplicate RO concentrate water samples were employed in the DOM fractionated process and subsequent analyses, then the mean values were calculated. 2.2. Fractionation method The RO concentrate water sample was fractionated to six fractions of hydrophilic acids (HIA), hydrophilic bases (HIB), hydrophilic neutrals (HIN), hydrophobic acids (HOA), hydrophobic bases (HOB) and hydrophobic neutrals (HON). The fractionation was conducted through selected nonionic macroporous resins: Supelite XAD-8 (20–60 meshes) resin, cation-exchange Dowex Marathon MSC (H) (20–50 meshes) resin, and anion-exchange Duolite A-7 (free base) resin. The method was based on the procedure described in the references (Tang et al., 2013b). All of the six fractions were the diluted to the original sample volume of 1 L with ultrapure water and stored at 4 °C until further analysis. 2.3. Size exclusion chromatography The molecular weight (MW) distributions of the six DOM fractions were obtained through high-performance size exclusion chromatography (SEC) using a HPLC (LC20A, Shimadzu, Japan) system with a SPD-M20A UV detector and two connected columns (a TSK-GEL G3000PWXL column followed by a TSK-GEL G2500PWXL column). The determination of the relationship between the MW and the chromatography retention time was conducted through MW standard materials: polyethylene glycol (330, 700, 1050, 5250, 10 225, and 30 000 Da) and acetone. 2.4. Fluorescence spectra Excitation emission matrix (EEM) fluorescence spectra of the six fractionated water samples were recorded on a fluorescence spectrophotometer (F-2500, Hitachi, Japan). Three-dimensional spectra were obtained by measuring the emission spectra from 240 to 600 nm repeatedly at the excitation wavelengths from 220 to 450 nm. The three-dimensional plots and contour maps were produced using the OriginPro7.5 program. All the contour maps were plotted using the same scale ranges of the fluorescence intensities and the number of contours. The EEM spectra were divided into five regions (I–V), associated with tyrosine-like aromatic protein (Region I), tryptophan-like aromatic protein (Region II), fulvic acid-like (Region III), soluble microbial by-product-like (Region IV), or humic acid-like organic compounds (Region V) (Chen et al., 2003). 2.5. Sample concentration The RO concentrate and the fractionated water samples were concentrated by Oasis HLB resin cartridges (Waters Corporation,
547
America) before performing the genotoxicity and anti-estrogenic activity assays. Each water sample (500 mL) was acidified to a pH of 2.0 with 2 M H2SO4 and then passed through the Oasis HLB resin cartridge that was washed previously with 10 mL of methanol and 10 mL of ultrapure water. Organics retained on the cartridge were eluted with acetone and completely dried under a nitrogen flow. Next, the dry residues were dissolved in 1 mL and 0.5 mL volumes of dimethylsulfoxide (DMSO) to obtain 500- and 1000-fold concentration (wastewater volume per the extract volume), respectively, for the genotoxicity and anti-estrogenic activity analysis.
2.6. Genotoxicity assay The SOS/umu assay for genotoxicity was conducted with Salmonella typhimurium TA1535/pSK1002 without S9 activation in accordance with ISO 13829 (2000). The genotoxicity evaluation method was conducted according to previously described methods (Wang et al., 2007). The DMSO solutions with different concentrations of 4-NQO (4-nitroquinoline-Noxide) were used as positive controls to obtain the dose–response curve of 4-NQO. The genotoxicity of the water samples was standardized to an equivalent 4-NQO concentration.
2.7. Anti-estrogenic activity assay The ability of the concentrated sample to inhibit the b-galactosidase activity of E2 was measured to determine the anti-estrogenic activity of the concentrated sample according to the yeast two-hybrid assay (Shiraishi et al., 2001; Jung et al., 2004). An anti-estrogenic standard chemical tamoxifen (TAM) was used. The inhibitions of the concentrated sample and TAM to the b-galactosidase activity induced by E2 were measured, and then the dose–response curves of the sample and standard TAM were generated (Tang et al., 2013b). The anti-estrogenic activity of the concentrated sample was given as a tamoxifen (TAM) equivalent, and the anti-estrogenic activity values of the samples before concentration were subsequently obtained.
3. Results and discussion 3.1. DOC and UV254 distribution of the DOM fractions The DOM of the RO concentrate was fractionated into six fractions: HIA, HIB, HIN, HOA, HOB and HON (Fig. 1). The DOC ratio of the hydrophilic fractions HIA, HIB, and HIN (55%) was higher than that of the hydrophobic fractions HOA, HOB, and HON (45%) in the RO concentrate. However, the HIN fraction accounted for 97% of the DOC of the hydrophilic solutes, and was the most abundant fraction accounting for 53% of the DOC of the total DOM. The HOA was the second abundant fraction occupying 23% DOC of the total DOM. Additionally, the HIN, HOA and HOB were absolutely the main fractions qualified by DOC, and accounted for 96% of the total DOM in the RO concentrate. Because that the electrostatic repulsion and/or hydrophobic interactions of DOM are connected with RO membrane fouling (Antony et al., 2012), some fractions in the secondary effluent might deposit on the RO membrane. Furthermore, the UV254 ratio of the hydrophilic fractions (53%) was higher than that of the hydrophobic fractions in the RO concentrate. the UV254 of the HIN was the highest and accounted for 37% of the total fractions, and the HOA was in the second place. As SUVA (UV254/DOC) is often used for estimating the aromaticity of water, the fractions HON, HIA, and HIB appeared to have higher intensities of SUVA than that of the HOA and HIN.
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Y.-X. Sun et al. / Chemosphere 117 (2014) 545–551
Molecular Weight (Da)
60 10000 65000
HIN
40
HOA
30 20
HON HIA
10
HIB 0
UV254 Absorbance Intensity (mV)
Percentage of UV254 (%)
50
0
10
HOB 20
30
40
50
60000
12000
The components of the DOM fractions in the RO concentrate were characterized by EEM fluorescence spectroscopy. Fig. 3 shows the EEM contour maps of the six DOM fractions, which exhibited distinct differences of the EEM pattern. To clarify the differences of the fluorescence intensity amount of the six fractions at the different regions, the EEM volumes of each region for each fraction were calculated according to Chen et al. (2003). The results indicated that the EEM volume of the HOA and HON were evidently higher than those of the other fractions in every EEM region (Fig. SM-2). The HOA occupied the highest components of tyrosine-like aromatic protein (Region I), humic/fulvic acid-like (Regions III and V) and soluble microbial by-product-like (Region IV) substances in the RO concentrate, followed by the HON. However, HON had the highest EEM volume in Region II, which associated with tryptophan-like aromatic protein. The HIN had the lowest EEM volume in both Regions I and II among the six fractions, such phenomenon coincided with their SUVA distribution to some extent. Comparing to SUVA, the EEM volume can provide more information about the DOM constituents. In terms of the aromaticity
HIN
10000
HON
8000
HOA
6000
HIA
4000
HIB
2000
HOB
0 30
32
34
36
38
Retention Time (min)
Fig. 1. Percentage of UV254 vs. percentage of DOC of the DOM fractions in the RO concentrate. HOA, HOB and HON are hydrophobic acids, bases and neutrals, and HIA, HIB and HIN are hydrophilic acids, bases and neutrals, respectively.
3.3. Fluorescence spectroscopy of the DOM fractions
HOA > HON > HIA > HIB > HOB. Furthermore, DOM with the MW rang of 3–10 kDa lain in the HOA, HIA and HIN of the RO concentrate, the other fractions existing in the secondary effluent (Tang et al., 2014) might deposite on the RO membrane. Additionally, DOM with MW < 1 kDa only presented in the HIN, and such phenomenon accorded with the tendency that hydrophilic fraction accounting for the most of DOM with low MW in the secondary effluent (Tang et al., 2014).
3-10 kDa (a)
16000
60
3.2. MW distribution of the DOM fractions
1000
(b)
45
HIA HOA
HIB HOB
HIN HON
40 35 30 25 20 15 10 5 0
Contents lists available at ScienceDirect
Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Characterization and biotoxicity assessment of dissolved organic matter in RO concentrate from a municipal wastewater reclamation reverse osmosis system Ying-Xue Sun a,⇑, Yue Gao a, Hong-Ying Hu b,c, Fang Tang b, Zhe Yang a a
Department of Environmental Science and Engineering, Beijing Technology and Business University, Beijing 100048, PR China Environmental Simulation and Pollution Control State Key Joint Laboratory, State Environmental Protection Key Laboratory of Microorganism Application and Risk Control, School of Environment, Tsinghua University, Beijing 100084, PR China c Shenzhen Laboratory of Microorganism Application and Risk Control, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, PR China b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
The molecular weight 1–3 kDa was
dominant in the basic and neutral DOM fractions. The hydrophilic neutrals contributed to the majority of the DOC and genotoxicity. The hydrophobic neutrals had high antiestrogenic activity per DOC of the fractions.
a r t i c l e
i n f o
Article history: Received 29 April 2014 Received in revised form 2 September 2014 Accepted 4 September 2014
Handling Editor: E. Brillas Keywords: Wastewater reclamation Reverse osmosis concentrate Dissolved organic matter fractions Genotoxicity Anti-estrogenic activity
⇑ Corresponding author. Tel.: +86 (10) 6898 4021. E-mail address: [email protected] (Y.-X. Sun). http://dx.doi.org/10.1016/j.chemosphere.2014.09.024 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.
a b s t r a c t Reverse osmosis (RO) concentrate from municipal wastewater reclamation reverse osmosis (mWRRO) system containing organic compounds may associate with toxic risk, and its discharge might pose an environmental risk. To identify a basis for the selection of feasible technology in treating RO concentrates, the characteristics and biotoxicity of different fractions of dissolved organic matter (DOM) in RO concentrates from an mWRRO system were investigated. The results indicated that the hydrophilic neutrals (HIN), hydrophobic acids (HOA) and hydrophobic bases (HOB) accounted for 96% of the dissolved organic carbon (DOC) of the total DOM in the RO concentrate. According to the SEC chromatograph detected at 254 nm wavelength of UV, the DOM with molecular weight (MW) 1–3 kDa accounted for the majority of the basic and neutral fractions. The fluorescence spectra of the excitation emission matrix (EEM) indicated that most aromatic proteins, humic/fulvic acid-like and soluble microbial by-product-like substances existed in the fractions HOA and hydrophobic neutrals (HON). The genotoxicity and antiestrogenic activity of the RO concentrate were 1795.6 ± 57.2 lg 4-NQO L 1 and 2.19 ± 0.05 mg TAM L 1, respectively. The HIN, HOA, and HOB contributed to the genotoxicity of the RO concentrate, and the HIN was with the highest genotoxic level of 1007.9 ± 94.8 lg 4-NQO L 1. The HOA, HON, and HIN lead to the total anti-estrogenic activity of the RO concentrate, and HOA occupied approximately 60% of the total, which was 1.3 ± 0.17 mg TAM L 1. Ó 2014 Elsevier Ltd. All rights reserved.
546
Y.-X. Sun et al. / Chemosphere 117 (2014) 545–551
1. Introduction Wastewater reclamation plays an important role in the development of strategies for the water resource management worldwide (Lazarova et al., 2013). Recently, the water quality requirement to reclaimed water changed from being initially focused on microbial quality to a wider and more comprehensive view of the chemical quality. With an increasing demand of high quality reclaimed water, municipal wastewater reclamation reverse osmosis (mWRRO) has been extensively and successfully applied in municipal wastewater reclamation because of its excellent separated function for organic and inorganic contaminants, as well as biological constituents (Al-Rifai et al., 2011; Subramania and Jacangeloa, 2014). However, accompanying a superb quality water for reuse, mWRRO also produces a certain quantity waste stream of RO concentrate, generally ranging from 25% to 50% of the feed, which contains high concentrations of dissolved salts and recalcitrant organics (Bellona et al., 2004; Al-Rifai et al., 2007; Dialynas et al., 2008). As the global and domestic implementation of RO technology is increasing significantly, there is an increasing concern of further disposal of RO concentrate because its discharge (e.g., via municipal sewer) may pose an environmental risk (Khan et al., 2009; Greenlee et al., 2009; Subramania and Jacangeloa, 2014). The organic matter in the RO concentrate of the mWRRO process comprises not only the concentrated dissolved organic matter (DOM) from the secondary effluent but also certain chemical agents injected during the mWRRO process, and new organic pollutants are also formed during the mWRRO process (Linge et al., 2013). Non-oxidizing biocide (Kathon biocide) injected in mWRRO elicits high genotoxic in vitro and contributes to a higher genotoxicity of the RO concentrate (Tang et al., 2013a). New disinfection byproducts, such as trihalomethanes, haloacetic acids, haloacetonitriles, and N-nitrosodimethylamine, are also formed during the pre-chlorination in the mWRRO process to protect membranes from bio-fouling; these byproducts are likely rejected as RO concentrate (Megan et al., 2008; Agus et al., 2009; Linge et al., 2013). Moreover, the mechanism of RO membrane separation for organic matter involves not only a simple physical process of separating solutes from solvents in terms of size difference but also the interactions between the organic matter and the membrane itself; furthermore, large, branched, poly-functional and sterically complex molecules are better retained by various membranes (Reinhard et al., 1986; Kastelan-Kunst et al., 1997; Hu et al., 2003). Thus, the characteristics of DOM in the RO concentrate from the mWRRO process cannot be simply inferred using the investigated characteristics of the secondary treated effluent. Studies have examined the treatment methods for organic matter in the RO concentrate; however, studies have mainly investigated the removal efficiencies of DOC and UV254, as well as pharmaceutically active compounds and disinfection by-products (Benner et al., 2008; Dialynas et al., 2008; Westerhoff et al., 2009; Radjenovic et al., 2011; Bagastyo et al., 2013; Justo et al., 2013; Lu et al., 2013). An in-depth study of the characteristics of DOM in RO concentrate should be conducted to develop effective treatment methods. Bagastyo et al. (2011) studied the DOM molecular weight fractions of the RO concentrate through physicochemical characterization and fluorescence excitation-emission scans. Zhou et al. (2011) evaluated the effects of different advanced oxidation processes on the DOM in the RO concentrate by the conventional parameters (COD, DOC and UV254), molecular weight distributions and acute ecotoxicity assayed by luminescent marine bacteria. However, related investigation regarding different polarity fractions, such as the hydrophobic and hydrophilic fractions of DOM in the RO concentrate has not yet been reported.
RO membrane rejection is closely associated with DOM compounds grouped by the dissociation potential (charge or neutral), hydrophobicity and molecular size (Hu et al., 2003; Bellona et al., 2004). Moreover, some specific fractions of DOM in wastewater are associated with the haloacetic acid or trihalomethane formation potential and anti-estrogenic activity (Zhang et al., 2009; Tang et al., 2013b). Therefore, investigating the specific fractions (such as hydrophobic and hydrophilic fractions) of DOM in the RO concentrate is important to create treatable and feasible technology for RO concentrate treatment. The DOM in RO concentrates from mWRRO processes are associated with endocrine disrupting compounds, pharmaceutically active compounds and disinfection by-products. Thus, the toxic risk of DOM in the RO concentrate cannot be ignored. Studies have evaluated the removal levels of the acute toxicity, genotoxicity and retinoic acid receptor (RAR) activity in reclaimed municipal effluent treated by different wastewater reclamation technologies, including RO systems (Zha and Wang, 2005; Cao et al., 2009; Macova et al., 2011; Tang et al., 2013a). However, the DOM fractions and the characterization of anti-estrogenic activity of the RO concentrate have not been reported. Therefore, this study investigated the characteristics and biotoxicity of the DOM fractions of the RO concentrate from an mWRRO process. The DOM of the RO concentrate was divided into six fractions. Each DOM fraction was subsequently characterized by size exclusion chromatography to identify the molecular weight distributions and excitation emission matrix fluorescence spectroscopy to identify the organic components. The general biotoxicity of the DOM fractions of the RO concentrate were evaluated by genotoxicity and anti-estrogenic activity.
2. Materials and methods 2.1. Sampling and water quality analysis The RO concentrate water samples were collected at a wastewater reclamation plant in Beijing utilizing an mWRRO process, which has a treatment capacity of 21 000 m3 d 1. The influent of the mWRRO process was the secondary treatment effluent from a sequencing batch bioreactor system treating the municipal wastewater. This mWRRO system uses microfiltration (MF) with polyvinylidene fluoride hollow fiber as the pretreatment. The RO membrane is a cross-linking aromatic polyamide composite membrane, and the RO recovery ratio (RO permeate volume per RO influent volume) was 60–70%. Several chemical agents, such as chlorine, chemical cleaning, antiscalant, reductant and non-oxidizing biocide, were added to protect the RO membrane from chemical and biological damage. The schematic diagram of the mWRRO and chemicals adding points were shown in Fig. SM-1. In the secondary treatment effluent, 1.36–1.70 mg Cl2 L 1 chlorine is added continuously. However, the residual chlorine is low than 0.5 mg Cl2 L 1 before the water pumped to the MF membrane. The MF effluent is stored temporarily in the middle water tank, in which 3 mg L 1 antiscalant and appropriate amount of reductant are added sequentially. More details of the chemical agents’ dosages along the mWRRO process have been described in the previous study (Tang et al., 2013a). The RO concentrate water samples were transported to the laboratory and were filtered immediately by glass fiber (0.45 lm) to eliminate suspended solids. After filtration, the samples were stored at 4 °C to minimize the changes in the constituents in the water. The conventional water quality parameters were analyzed according to the standard methods within 24 h after sampling (APHA, 1998). The concentration of DOC was detected using a
Y.-X. Sun et al. / Chemosphere 117 (2014) 545–551 Table 1 Water quality characteristics of the studied mWRRO system. Parameter 1
TDS (mg L ) DOC (mg L 1) UV254 (cm 1) TN (mg L 1) NH3–N (mg L 1) TP (mg L 1)
Secondary effluent
RO permeate
RO concentrate
877–1390 5.8–12.4 0.11–0.12 17.1–28 0.5–5.6 0.5–3.6
4–28.5 0.2–0.6 0–0.01 1–2.2 – –
3210–4980 18–46 0.36–0.45 53.7–84.8 5.5–14 11.2–24.1
TOC-5000A analyzer (Shimadzu, Japan). UV absorbance was measured using a UV-2401PC UV–VIS recording spectrophotometer (Shimadzu, Japan). The water samples were obtained monthly from March 2013 to July 2013 to perform the primary properties of the secondary effluent, RO permeate and RO concentrate (Table 1). Also, triplicate RO concentrate water samples were employed in the DOM fractionated process and subsequent analyses, then the mean values were calculated. 2.2. Fractionation method The RO concentrate water sample was fractionated to six fractions of hydrophilic acids (HIA), hydrophilic bases (HIB), hydrophilic neutrals (HIN), hydrophobic acids (HOA), hydrophobic bases (HOB) and hydrophobic neutrals (HON). The fractionation was conducted through selected nonionic macroporous resins: Supelite XAD-8 (20–60 meshes) resin, cation-exchange Dowex Marathon MSC (H) (20–50 meshes) resin, and anion-exchange Duolite A-7 (free base) resin. The method was based on the procedure described in the references (Tang et al., 2013b). All of the six fractions were the diluted to the original sample volume of 1 L with ultrapure water and stored at 4 °C until further analysis. 2.3. Size exclusion chromatography The molecular weight (MW) distributions of the six DOM fractions were obtained through high-performance size exclusion chromatography (SEC) using a HPLC (LC20A, Shimadzu, Japan) system with a SPD-M20A UV detector and two connected columns (a TSK-GEL G3000PWXL column followed by a TSK-GEL G2500PWXL column). The determination of the relationship between the MW and the chromatography retention time was conducted through MW standard materials: polyethylene glycol (330, 700, 1050, 5250, 10 225, and 30 000 Da) and acetone. 2.4. Fluorescence spectra Excitation emission matrix (EEM) fluorescence spectra of the six fractionated water samples were recorded on a fluorescence spectrophotometer (F-2500, Hitachi, Japan). Three-dimensional spectra were obtained by measuring the emission spectra from 240 to 600 nm repeatedly at the excitation wavelengths from 220 to 450 nm. The three-dimensional plots and contour maps were produced using the OriginPro7.5 program. All the contour maps were plotted using the same scale ranges of the fluorescence intensities and the number of contours. The EEM spectra were divided into five regions (I–V), associated with tyrosine-like aromatic protein (Region I), tryptophan-like aromatic protein (Region II), fulvic acid-like (Region III), soluble microbial by-product-like (Region IV), or humic acid-like organic compounds (Region V) (Chen et al., 2003). 2.5. Sample concentration The RO concentrate and the fractionated water samples were concentrated by Oasis HLB resin cartridges (Waters Corporation,
547
America) before performing the genotoxicity and anti-estrogenic activity assays. Each water sample (500 mL) was acidified to a pH of 2.0 with 2 M H2SO4 and then passed through the Oasis HLB resin cartridge that was washed previously with 10 mL of methanol and 10 mL of ultrapure water. Organics retained on the cartridge were eluted with acetone and completely dried under a nitrogen flow. Next, the dry residues were dissolved in 1 mL and 0.5 mL volumes of dimethylsulfoxide (DMSO) to obtain 500- and 1000-fold concentration (wastewater volume per the extract volume), respectively, for the genotoxicity and anti-estrogenic activity analysis.
2.6. Genotoxicity assay The SOS/umu assay for genotoxicity was conducted with Salmonella typhimurium TA1535/pSK1002 without S9 activation in accordance with ISO 13829 (2000). The genotoxicity evaluation method was conducted according to previously described methods (Wang et al., 2007). The DMSO solutions with different concentrations of 4-NQO (4-nitroquinoline-Noxide) were used as positive controls to obtain the dose–response curve of 4-NQO. The genotoxicity of the water samples was standardized to an equivalent 4-NQO concentration.
2.7. Anti-estrogenic activity assay The ability of the concentrated sample to inhibit the b-galactosidase activity of E2 was measured to determine the anti-estrogenic activity of the concentrated sample according to the yeast two-hybrid assay (Shiraishi et al., 2001; Jung et al., 2004). An anti-estrogenic standard chemical tamoxifen (TAM) was used. The inhibitions of the concentrated sample and TAM to the b-galactosidase activity induced by E2 were measured, and then the dose–response curves of the sample and standard TAM were generated (Tang et al., 2013b). The anti-estrogenic activity of the concentrated sample was given as a tamoxifen (TAM) equivalent, and the anti-estrogenic activity values of the samples before concentration were subsequently obtained.
3. Results and discussion 3.1. DOC and UV254 distribution of the DOM fractions The DOM of the RO concentrate was fractionated into six fractions: HIA, HIB, HIN, HOA, HOB and HON (Fig. 1). The DOC ratio of the hydrophilic fractions HIA, HIB, and HIN (55%) was higher than that of the hydrophobic fractions HOA, HOB, and HON (45%) in the RO concentrate. However, the HIN fraction accounted for 97% of the DOC of the hydrophilic solutes, and was the most abundant fraction accounting for 53% of the DOC of the total DOM. The HOA was the second abundant fraction occupying 23% DOC of the total DOM. Additionally, the HIN, HOA and HOB were absolutely the main fractions qualified by DOC, and accounted for 96% of the total DOM in the RO concentrate. Because that the electrostatic repulsion and/or hydrophobic interactions of DOM are connected with RO membrane fouling (Antony et al., 2012), some fractions in the secondary effluent might deposit on the RO membrane. Furthermore, the UV254 ratio of the hydrophilic fractions (53%) was higher than that of the hydrophobic fractions in the RO concentrate. the UV254 of the HIN was the highest and accounted for 37% of the total fractions, and the HOA was in the second place. As SUVA (UV254/DOC) is often used for estimating the aromaticity of water, the fractions HON, HIA, and HIB appeared to have higher intensities of SUVA than that of the HOA and HIN.
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Molecular Weight (Da)
60 10000 65000
HIN
40
HOA
30 20
HON HIA
10
HIB 0
UV254 Absorbance Intensity (mV)
Percentage of UV254 (%)
50
0
10
HOB 20
30
40
50
60000
12000
The components of the DOM fractions in the RO concentrate were characterized by EEM fluorescence spectroscopy. Fig. 3 shows the EEM contour maps of the six DOM fractions, which exhibited distinct differences of the EEM pattern. To clarify the differences of the fluorescence intensity amount of the six fractions at the different regions, the EEM volumes of each region for each fraction were calculated according to Chen et al. (2003). The results indicated that the EEM volume of the HOA and HON were evidently higher than those of the other fractions in every EEM region (Fig. SM-2). The HOA occupied the highest components of tyrosine-like aromatic protein (Region I), humic/fulvic acid-like (Regions III and V) and soluble microbial by-product-like (Region IV) substances in the RO concentrate, followed by the HON. However, HON had the highest EEM volume in Region II, which associated with tryptophan-like aromatic protein. The HIN had the lowest EEM volume in both Regions I and II among the six fractions, such phenomenon coincided with their SUVA distribution to some extent. Comparing to SUVA, the EEM volume can provide more information about the DOM constituents. In terms of the aromaticity
HIN
10000
HON
8000
HOA
6000
HIA
4000
HIB
2000
HOB
0 30
32
34
36
38
Retention Time (min)
Fig. 1. Percentage of UV254 vs. percentage of DOC of the DOM fractions in the RO concentrate. HOA, HOB and HON are hydrophobic acids, bases and neutrals, and HIA, HIB and HIN are hydrophilic acids, bases and neutrals, respectively.
3.3. Fluorescence spectroscopy of the DOM fractions
HOA > HON > HIA > HIB > HOB. Furthermore, DOM with the MW rang of 3–10 kDa lain in the HOA, HIA and HIN of the RO concentrate, the other fractions existing in the secondary effluent (Tang et al., 2014) might deposite on the RO membrane. Additionally, DOM with MW < 1 kDa only presented in the HIN, and such phenomenon accorded with the tendency that hydrophilic fraction accounting for the most of DOM with low MW in the secondary effluent (Tang et al., 2014).
3-10 kDa (a)
16000
60
3.2. MW distribution of the DOM fractions
1000
(b)
45
HIA HOA
HIB HOB
HIN HON
40 35 30 25 20 15 10 5 0
