Investigating the performance of three modified activated sludge processes treating municipal wastewater in organic pollutants removal and toxicity reduction

Ecotoxicology and Environmental Safety 148 (2018) 729–737

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Investigating the performance of three modified activated sludge processes treating municipal wastewater in organic pollutants removal and toxicity reduction

T

Xue Hana, Yu-Ting Zuoa,c, Yu Hua,d, Jie Zhangb, Meng-Xuan Zhoub, Mo Chena, Fei Tanga, ⁎ Wen-Qing Lua, Ai-Lin Liua, a

Key Laboratory of Environment and Health, Ministry of Education & Ministry of Environmental Protection, and State Key Laboratory of Environmental Health (Incubating), School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, PR China Wuhan Water Group Company Limited, Wuhan 430015, PR China c Wuhan Center for Disease Control and Prevention, Wuhan 430015, PR China d The Third Affiliated Hospital of Southern Medical University, Guangzhou 510000, PR China b

A R T I C L E I N F O

A B S T R A C T

Keywords: Sewage treatment Fluorescence spectroscopy Absorbance spectroscopy Caenorhabditis elegans Biotoxicity

This study investigated the treatment performance of three types of modified activated sludge processes, i.e., anoxic/oxic (A/O), anaerobic/anoxic/oxic (A2/O) and oxidation ditch process, in treating municipal wastewater by measuring physicochemical and spectroscopic parameters, and the toxicity of the influents and effluents collected from 8 full-scale municipal wastewater treatment plants (MWTPs). The relationships between spectroscopic and physicochemical parameters of the wastewater samples and the applicability of the nematode Caenorhabditis elegans (C. elegans) bioassays for the assessment of the toxic properties of municipal wastewater were also evaluated. The results indicated that the investigated MWTPs employing any of A/O, A2/O and oxidation ditch processes could effectively control the discharge of major wastewater pollutants including biochemical oxygen demand (BOD), chemical oxygen demand, nitrogen and phosphorus. The oxidation ditch process appeared to have the advantage of removing tyrosine-like substances and presented slightly better removal efficiency of tryptophan-like fluorescent (peak T) substances than the A/O and A2/O processes. Both ultraviolet absorbance at 254 nm and peak T may be used to characterize the organic load of municipal wastewater, and peak T can be adopted as a gauge of the BOD removal efficacy of municipal wastewater treatment. Using C. elegans-based oxygen consumption rate assay for monitoring municipal wastewater toxicity deserves further investigations.

1. Introduction Municipal wastewater treatment plants (MWTPs) receive domestic and industrial sewage and remove solids, organic matters and nutrients by physical, chemical and biological treatment methods to achieve a significant reduction in pollutants and ecotoxicity in the receiving surface or ground water (Morris et al., 2017). The most commonly utilized biological treatment method is an activated sludge process, which is designed to substantially remove biodegradable dissolved and colloidal organic matter (Hashimoto et al., 2014). To further treat specific wastewater constituents (nitrogen, phosphorus, etc) that can't be effectively removed by the conventional activated sludge process, certain modified activated sludge processes are used more frequently by

MWTPs (Wang et al., 2017). The anoxic/oxic (A/O), anaerobic/anoxic/oxic (A2/O) and oxidation ditch processes are the three typically used types of modified activated sludge processes. In China, these processes have been adopted in 60% of the wastewater treatment plants and treat 51% of the total volume of wastewater generated (Zhang et al., 2016). The A/O process includes anoxic and aerobic reactors. Organic carbon is removed by aerobic microorganisms; meanwhile, ammonia is converted aerobically to nitrate, which is then reduced to nitrogen gas in an anoxic reactor (Cui and Jahng, 2004). Compared with the A/O process, the A2/O process adds an anaerobic reactor prior to the anoxic and aerobic reactors and thereby provides an environment that encourages the growth of phosphorus accumulating organisms that uptake excessive

⁎ Correspondence to: Department of Occupational and Environmental Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, PR China. E-mail address: [email protected] (A.-L. Liu).

https://doi.org/10.1016/j.ecoenv.2017.11.042 Received 5 September 2017; Received in revised form 10 November 2017; Accepted 16 November 2017 0147-6513/ © 2017 Elsevier Inc. All rights reserved.

Ecotoxicology and Environmental Safety 148 (2018) 729–737

X. Han et al.

treatment due to the intoxication phenomena (Liwarska-Bizukojc et al., 2016; Xiao et al., 2015). Caenorhabditis elegans (C. elegans) is a highly attractive model for assessing aquatic toxicity. C. elegans has a short life cycle and a small body size, is easy to maintain under laboratory condition and allows for the use of high throughput techniques (Chu and Chow, 2002; TejedaBenitez and Olivero-Verbel, 2016). C. elegans has a high tolerance to pH, salinity, and water hardness and offers a wide variety of ecologically and toxicologically relevant endpoints, such as mortality, growth, and reproduction (Chu and Chow, 2002; Tejeda-Benitez and OliveroVerbel, 2016). A C. elegans bioassay with the mortality as endpoint has been used to evaluate the toxicity of municipal and industrial wastewater (Hitchcock et al., 1997). Oxygen is a key metabolite of aerobic organisms, and the rate of oxygen uptake reflects metabolic activity, health and responses to various stimuli (Schouest et al., 2009). Respirometric toxicity assay based on measuring the oxygen consumption rate (OCR) of organisms offers a sub-lethal parameter and has been used to assess the toxicity of different types of toxicants (e,g., metals, pesticides, polycyclic aromatic compounds, drinking water disinfection by-products) and industrial wastewater samples on various species including microorganisms, mammalian cells and whole animals (Kungolos, 2005; Schouest et al., 2009; Zitova et al., 2009; Zuo et al., 2017). Although C. elegans is a valuable toxicity model, to the best of our knowledge, no study has been conducted utilizing C. elegans respirometric toxicity assays for the evaluation of the municipal wastewater toxicity properties. Understanding the treatment performance of modified activated sludge processes is important for the selection and design of biological wastewater treatment technologies. This study focuses on revealing the differences in organic pollutants removal and toxicity reduction in MWTPs using A/O process, A2/O process, and oxidation ditch process, respectively. Treatment efficiency of 8 MWTPs was investigated by traditional physicochemical analysis in combination with UV–visible and EEM spectroscopy and C. elegans biotoxicity assays. The links between physicochemical and spectroscopic parameters of the wastewater samples were also assessed to determine whether the spectra analysis could be used as a monitoring tool of treatment efficiency in MWTPs. In addition, the applicability of the C. elegans-based respirometric bioassays in the assessment of the toxic properties of municipal wastewater was initially evaluated.

phosphate in aerobic reactors, and phosphorus is removed with the waste sludge (Kim et al., 2013). Thus, A2/O process can attain the effective removal of both nitrogen and phosphorus. The oxidation ditch process utilizes large round or oval ditches (channel reactors) with one or more horizontal aerators, which allows for the simultaneous removal of carbon, nitrogen, and phosphorus from sewage with long solid retention times and repetitive aerobic/anaerobic treatment phases (Terashima et al., 2016; Xu et al., 2017). Oxidation ditch technology is a good choice for small-scale and medium-scale MWTPs because of its simple infrastructure and convenient management, but for large-scale MWTPs, the A2/O and A/O technologies are more appropriate due to the lower capital investment per unit of wastewater treatment (Jin et al., 2014). Although the selection of a wastewater treatment process is a comprehensive consideration of technological, economical and environmental factors, the effective removal performance is the key point to ensure qualified effluent. Physicochemical parameters, particularly the chemical oxygen demand (COD) and biochemical oxygen demand (BOD), are traditionally used to evaluate the pollutant removal efficiency of the MWTPs. However, the frequent monitoring of COD and BOD is relatively expensive and time-consuming. Spectral measurements, including ultraviolet (UV)-visible absorbance and fluorescence signals, are cheap and rapid analyses that offer particularly promising solutions for the surrogate monitoring of COD and BOD (Henderson et al., 2009; Thomas et al., 1996). Moreover, a series of UV indices such as the absorbance at 254 nm (UV254) and the ratio of the absorbance at 250 nm to that at 365 nm (UV250/UV365) may provide insight into the nature of the organics present in municipal wastewater. UV254 is generally linked to the high molecular weight and hydrophobic (aromatic) content of natural organic matter in water (Liu et al., 2016). The UV250/UV365 ratio has been introduced to characterize the aromaticity and molecular size of organic matter. This ratio increases as the aromaticity and molecular size decrease (Uyguner and Bekbolet, 2005). A three-dimensional fluorescence excitation-emission matrix (3D-EEM) can be generated by measuring the fluorescence intensity of an aqueous sample at consecutive excitation and emission wavelengths. Thus, complex dissolved organic matter can be characterized according to its fluorescent components. Compared to the UV absorbance, the EEM provides more information regarding the organic matter fractions and their chemical characteristics, and offers at least an order of magnitude more sensitivity for the detection of organic matter (Leenheer and Croué, 2003). Characterizing the changes in organic matter during wastewater treatment can provide valuable information for the selection of the optimum operation conditions and the best biological treatment process. The use of UV–visible absorbance technology and fluorescence EEM technology for performance evaluations of MWTPs is growing as a strong complement to conventional technology that often focus only on a set of quantitative variables that typically includes COD, BOD, and nutrients. To date, however, comprehensive studies investigating the fluorescence of wastewater subjected to modified activated sludge processes, such as the A/O, A2/O and oxidation ditch processes, in full-scale MWTPs are scarce. Municipal wastewater is a complex mixture of largely unknown substances that may be hazardous to humans and aquatic organisms. Nevertheless, the current physicochemical analysis used for compliance assessment of wastewater and the above-mentioned optical approaches can detect only a limited number of chemicals, and both approaches are unable to evaluate toxic and interactive (additive, antagonistic or synergistic) toxic effects of the chemicals coexisting in wastewater. Therefore, biotoxicity assays that measure the effect of all bioavailable contaminants in wastewater are essential to complement the physicochemical and optical measures of wastewater quality and provide deeper understanding of the process performance of MWTPs. In addition, toxicity testing is vital for assessing the potential harmful effects of wastewater that is discharged into ecosystems and monitoring the toxicants present in wastewater that reduce the efficiency of biological

2. Methods and materials 2.1. Wastewater sampling Sixteen untreated influent and final-treated effluent samples were collected from eight MWTPs, namely, the Longwangzui (LWZ), Erlangmiao (ELM), Shahu (SH), Sanjintan (SJT), Nantaizihu (NTZH), Huangjiahu (HJH), Tangxunhu (TXH), and Luobuzui (LBZ) MWTPs, in Wuhan, which is the capital city of Hubei Province in Central China. Wuhan is located at the confluence of the Han and Yangtze Rivers and has a population of more than 10 million. The wastewater emission of Wuhan in 2015 reached 924 million tons, including 155 million tons of industrial wastewater and 769 million tons of domestic wastewater, and approximately 85.61% of the wastewater is treated in 26 MWTPs. The average volume of wastewater treated daily in the eight MWTPs investigated accounted for 70% of the total average volume of wastewater treated daily in all MWTPs in Wuhan city. At the eight MWTPs, the influent wastewater undergoes the following four major processes: preliminary treatment by coarse and fine screeners for the removal of coarse solids and other large materials, primary treatment by sedimentation for the removal of settleable organic and inorganic solids, secondary treatment using biological digestion by bacteria for the removal of biodegradable dissolved and colloidal organic matter and the nutrients nitrogen and phosphorus, and tertiary treatment using chlorine to kill pathogens, such as bacteria and viruses. The major 730

Ecotoxicology and Environmental Safety 148 (2018) 729–737

X. Han et al.

transferred to NGM plates seeded with E. coli OP50 and allowed to develop into age synchronous populations of L4-larval, young adult nematodes.

difference in the wastewater treatment processes performed in the eight MWTPs occurs during secondary treatment, the A2/O process is used at LWZ, ELM, and SH MWTPs, the A/O process is used at the SJT MWTP, and the oxidation ditch process is used at the NTZH, HJH, TXH, and LBZ MWTPs. The final-treated effluents of the eight MWTPs are eventually discharged into the Yangzte River. At each sampling site, the wastewater samples were collected at one-hour intervals over a continuous 24-h period, and then, equal volumes of per-hour samples were mixed to obtain a composite sample that represents the average wastewater characteristics during the composite period. The composite samples were stored at 4 ℃, and the physicochemical analyses and bioassays were conducted within three days.

2.5. Lethal toxicity test A lethal toxicity test was performed in 96-well plates. In total, 5 μL of K medium (32 mM KCl and 51 mM NaCl) containing approximately 30 L4-larvae were transferred into each well containing 95 μL of the wastewater sample. The K medium, which was suitable for C. elegans culture, was used as a control. The plates were incubated at 20 °C for 24 h, and then the numbers of live and dead C. elegans were determined by the absence of touch-provoked movement upon stimulation with a platinum wire under a dissecting microscope. Each wastewater sample was analyzed in four replicates.

2.2. Conventional wastewater parameters analyses The chemical parameters, i.e., 5-day BOD and COD, were determined in the influent and effluent samples. Other physicochemical parameters, such as ammonia nitrogen, total nitrogen, total phosphorus, and total suspended solids (TSS) were also determined in the effluent samples. All wastewater parameters were analyzed using the standard methods (Ministry of Enviromental Protection of the People's Republic of China, 2002).

2.6. OCR assay The wastewater samples were filtered through microfiltration membrane (0.45 µm), and the OCR assay was performed as described by Yu-ting et al. (Zuo et al., 2017). After 24 h of exposure to wastewater sample, young adult C. elegans were transferred to 96-well plates with the corresponding wastewater sample. The C. elegans were similarly treated with the K medium as a positive control (100% respiration). Duplicate wells with the corresponding wastewater samples but no nematodes were established concurrently as a negative control (0% respiration). The oxygen probe MitoXpress® Xtra (Luxcel Biosciences, Cork, Ireland) was added to each well in the 96-well plates, which were then read using a BioTek Synergy 2 microplate reader. The obtained time-fluorescence intensity profile of each well was normalized to the signal at time zero and then analyzed using a linear regression to determine the slope. The relative respiration rate (R) of C. elegans was calculated as follows: R = (Ss-Sn)/Sp × 100%, where Ss, Sn and Sp is the slope of the sample, the negative control, and the positive control, respectively. Each wastewater sample was analyzed in three replicates.

2.3. Measurements of UV–visible and EEM spectroscopy Before performing optical measurements, the wastewater samples were filtered through a microfiltration membrane (0.45 µm). A UV–visible spectrophotometer (PerkinElmer Lambda 35, America) was used to collect the absorbance spectra between 200 and 900 nm using 1-cm-path quartz cuvettes. A zero absorbance standard was established using ultrapure Milli-Q water. The baseline offset was corrected by subtracting the average absorbance at 700–800 nm from each spectrum (Green and Blough, 1994). An EEM was generated for each sample using a fluorescence spectrophotometer (Hitachi F-4600 FL, Japan) with 1-cm-path quartz cuvettes. The excitation and emission were scanned simultaneously at wavelengths ranging from 200 to 400 nm and 250–500 nm respectively at 5 nm intervals, with a 5-nm slit width at a 1200 nm/min scan rate. Then, the Milli-Q water EEM was subtracted from the sample data to remove the Raman signal. To monitor the instrument stability, the Raman peak intensity of Milli-Q water at 348 nm excitation wavelength was repeatedly analyzed throughout the experimental period. Information regarding the fluorescence peak was extracted using the peak-picking method which identifies the intensity and excitation and emission wavelengths of the peaks by searching for the maximum fluorescence intensity value. The software Origin 8.0 was used to plot the EEM. The inner filter effect (IFE) is an influential factor in the analysis of the fluorescence EEM spectra of organic matter from wastewater samples. To date, various methods have been suggested for correcting the IFE, but these methods have disadvantages; thus, retaining the data in an uncorrected state is an alternative approach (Henderson et al., 2009). Various studies analyzing the organic matter in wastewater using an EEM have not performed an IFE correction (Janhom et al., 2009; Louvet et al., 2013). Hence, a correction for the IFE was not applied to the dataset used in the present study.

2.7. Statistical analyses All analyses were conducted using SPSS Statistics 20.0 software. Spearman correlation coefficients were calculated to examine the relationship between the chemical parameters and the optical parameters. The results of the OCR assay are presented as the mean ± standard deviation. The respiration rates of the C. elegans following the different treatments were compared using Games-Howell test. Differences were considered significant at p < 0.05. 3. Results and discussion 3.1. The general characterization of wastewater samples The conventional physicochemical characteristics of the wastewater samples are shown in Table 1. The seven measured parameters applied to all effluent samples complied with the Chinese effluent discharge standard limits (Standard A of Level Ⅰ) (Ministry of Enviromental Protection of the People's Republic of China, 2002). The COD removal efficiency was 82.50%, 69.23–90% and 71.74–88.89% using the A/O, A2/O and oxidation ditch processes, respectively. As shown in Fig. 1a, although the COD removal efficiency varied among MWTPs using the same treatment process, no substantial difference existed among the A/ O, A2/O and oxidation ditch processes in the average removal efficiency (82.50%, 78.57% versus 82.49%) (A/O process: N = 1; A2/O process: N = 3; oxidation ditch process: N = 4). The efficiency of these three processes was approximately 81.19% on average, which is similar to the finding reported in a previous review that analyzed the COD removal efficiency of wastewater treatment processes in China (Jin

2.4. Preparation of test organisms for bioassays The nematode C. elegans (strain Bristol N2) and Escherichia coli (E. coli) strain OP50 were obtained from the Caenorhabditis Genomics Center (Minneapolis, MN, USA). The C. elegans were cultured at 20 °C on nematode growth media (NGM) plates seeded with E. coli OP50. Gravid hermaphrodites were treated with a bleaching buffer (0.45 M NaOH, 2% HClO), and the liberated eggs were hatched overnight at 20 °C in M9 solution (3 g/L KH2PO4, 6 g/L Na2HPO4, 5 g/L NaCl, 1 M MgSO4) and allowed to starve as L1 larvae, which were then collected, 731

Ecotoxicology and Environmental Safety 148 (2018) 729–737

X. Han et al.

removal in the MWTPs using the A2/O, oxidation ditch and A/O processes was 95.38–99.16%, 96.74–98.20% and 89.58%, respectively (Fig. 1b). Notably, all three treatment processes were very effective for BOD removal, and the performance of the A2/O and oxidation ditch processes was slightly better than that of the A/O process. In the present study, the MWTP using the A2/O process, the oxidation ditch process or the A/O process could effectively control the discharge of certain key pollutants, such as BOD, COD, ammonia nitrogen, total nitrogen, total phosphorus, and TSS.

Table 1 General characterization of wastewater samples. MWTP name

Samples

pH

BOD (mg/L)

COD (mg/ L)

ANc (mg/ L)

TNd (mg/L)

TPe (mg/ L)

TSSf (mg/ L)

SJT

Ua Tb Ua Tb Ua Tb Ua Tb Ua Tb Ua Tb Ua Tb Ua Tb

7.53 7.40 7.47 7.32 7.70 7.77 7.48 7.67 7.67 7.79 7.60 7.67 7.56 7.90 7.54 7.62

57.80 6.02 27.70 1.28 23.80 0.20 50.30 1.01 56.00 1.56 53.30 1.13 56.70 1.85 44.50 0.80

120 21 52 16 68 16 220 22 100 14 120 20 92 26 144 16

– 2.60 – 0.20 – 0.30 – 1.10 – 0.60 – 0.30 – 0.50 – 0.30

– 11.30 – 5.44 – 8.30 – 4.09 – 5.78 – 14.90 – 10.20 – 13.30

– 0.32 – 0.63 – 0.26 – 0.69 – 0.40 – 1.00 – 0.26 – 0.66

– 7 – 7 – 1 – 9 – 7 – 8 – 9 – 4

SH LWZ ELM NTZH LBJ HJH TXH

3.2. UV–visible spectral characteristics Fig. 2 shows UV–visible spectra of the influent and effluent samples in the range of 200–500 nm (absorbance values of all samples are close to zero in the range of 500–900 nm). The continuous UV–visible absorbance spectrum is obviously different between the influents and effluents. In the untreated influents, the key features of the spectra included a rapid decrease in the range of 200 < λ < 230 nm and a gradual decrease in absorbance at λ > 230 nm (Fig. 2a). In the final treated effluents, as the wavelength increased, the absorbance values slightly increased and reached a maximum at approximately 205 nm, followed by a sharp decrease close to 0 (Fig. 2b). Thus, as shown in Fig. 2c, between wavelength 216 nm and 240 nm, the absorbance curves of the influent samples cross the corresponding curves of the effluents, indicating that the absorbance values of the wastewater increase at lower wavelengths but decrease at higher wavelengths after treatment. This phenomenon may be explained by the following facts. A UV absorbance of municipal wastewater below 230 nm is influenced by various non-aromatic functional groups (i.e., carboxylic acids, esters, amides, etc.) and inorganics (i.e., nitrate, sulfate, phosphate, etc.), but

–: The parameter was not measured. a Untreated influent sample. b Final-treated effluent sample. c Ammonia nitrogen. d Total nitrogen. e Total phosphorus. f Total suspended solids.

et al., 2014). The similarity in the removal efficiency of the investigated processes may be attributed to the high COD removal achieved by the primary wastewater treatment process (Jin et al., 2014). The BOD

Fig. 1. COD, BOD, UV254 and peak T intensity removal efficiency in MWTPs using the A/O, A2/O and oxidation ditch processes. Numbers in the bars represent the different MWTPs: 1SJT; 2-SH; 3-LWZ; 4-ELM; 5-HJH; 6-LBJ; 7-NTZH; and 8-TXH.

732

Ecotoxicology and Environmental Safety 148 (2018) 729–737

X. Han et al.

Fig. 2. UV–visible spectra of untreated influent samples (a) and final-treated effluent samples (b); (c) shows an example of UV–visible spectra of influent and effluent samples obtained from the same SJT wastewater treatment plant.

related to the influent characteristics, such as the UV254 value. For example, the UV254 values were 0.09/0.06, 0.36/0.04, and 0.37/ 0.06 cm−1 for the influent/effluent collected from the SH, LWZ and ELM MWTPs (using the A2/O process) respectively. The UV254 values of the three effluents are similar, and the SH influent presented a low UV254 value that is much smaller than the ones found in the LWZ and ELM influents. The very low UV254 of the influent samples suggests there is a relatively small amount of removable organic contaminants that therefore results in a removal effect that is not obviously observed after the treatment. Due to the obvious variation in the UV254 removal efficiency using the same treatment process, the UV254 removal efficiency could not be compared among the three treatment processes in this study. In addition, no significant correlations existed between the UV254 removal and COD removal (r = 0.36, n = 8; p = 0.39) and BOD removal (r = 0.62, n = 8; p = 0.10). Therefore, the measurement of the UV254 removal provided limited information regarding the COD and BOD removal. The UV254 removal appears to be unsuitable for the evaluation of organic matter removal efficiency by wastewater treatment processes. The UV250/UV365 ratio was applied to obtain additional information regarding the total aromaticity and average molecular weights of the organic matter in the wastewater. The UV250/UV365 ratio of all wastewater samples increased after treatment (data no shown), which indicated a decrease in the aromaticity and molecular size of the organic matter. The increase in the UV250/UV365 ratio coincided with the decrease in the UV254values in the treated effluents; both of which suggest the degradation/removal of organic matters, particularly aromatic compounds.

the UV absorbance at higher wavelength is mainly influenced by organic compounds (Her et al., 2008). Nitrate absorbs light at 200 < λ < 230 nm with a maximum absorbance at 205 nm (van den Broeke et al., 2006). Thus incomplete denitrification during the wastewater treatment may contribute to the increases in the absorbance values of the effluents at the lower wavelengths. The decreases in absorbance of effluents at the higher wavelengths may be due to the degradation of organic matter. Previous studies have found good correlations between the UV absorbance values of municipal wastewater at 254 and 295 nm wavelength and the concentrations of COD and between the UV absorbance values of farm slurry effluent at 280 nm wavelength and the concentrations of BOD (Brookman, 1997; Matsché and Stumwöhrer, 1996). The relationships between the absorbance (254, 280 and 295 nm) and COD and BOD were also assessed in the present study to determine the proper UV absorbance wavelength that can be used as a surrogate variable for an integrated assessment of the changes in the wastewater quality. Strong correlations between UV254, UV 280 and UV295 and COD (r = 0.88, 0.86 and 0.83, respectively) and BOD (r = 0.82, 0.81 and 0.80, respectively) were observed, which suggested that UV254 was the most robust proxy for COD or BOD. Many UV254 absorbing organics (e.g., aromatic compounds such as polycyclic aromatic hydrocarbons) in wastewater are harmful to both humans and the environment, and the removal of these contaminants by a wastewater treatment process is desirable. The UV254 removal was determined to compare the removal efficiency of different treatment processes. The UV254 removal efficiency was 73.50%, 32.06–87.66% and 49.47–80.15% using the A/O, A2/O, and oxidation ditch processes, respectively. As shown in Fig. 1c, there is obvious variation in the UV254 removal among MWTPs using the same process (A2/O process or oxidation ditch process). This difference in removal efficiency may be 733

Ecotoxicology and Environmental Safety 148 (2018) 729–737

X. Han et al.

Fig. 3. Fluorescence EEMs of wastewater collected from the NTZH MWTP. (a) Untreated influent sample. (b) Final-treated effluent sample. (1) and (2) Tryptophan-like fluorescence; (3) Tyrosine-like fluorescence; (4) Humic-like fluorescence. Contour interval of (a) and (b) is 80 and 20, respectively.

cause of this phenomenon remains uncertain. In this study, the changes in the fluorescent signatures of the wastewater after treatment are obvious (Fig. 4). Peak A could not be identified in the influent samples. Considering the possible overlapping of peak A and intense peak T2, a four-time dilution of each influent sample was conducted, and peak A was still undetected, which is suggestive of a low concentration of microbial humic-like substances in the investigated influents. In contrast to peak A, peaks T1 and T2 were observed in all influents and peak B was observed in some influents. Previous studies have also indicated that peak T was present in most municipal wastewater, and peak A showed a lower prevalence than peak T (Carstea et al., 2016). The wastewater treatment processes had a great influence on the individual fluorescent components in the influents. First, the fluorescence of all peaks found in the influents decreased in intensity after treatment, and the peak fluorescence was entirely removed for peaks T1, T2 and B in 6 of 8 samples, 1 of 8 samples and 2 of 3 samples respectively, which suggested that certain tryptophan-like compounds (peak T1) and tyrosine-like compounds (peak B) were easily removed by the wastewater treatment process. Notably, peak T2 existed a 5–15 nm shift toward the longer emission wavelength (red shift) in the treated effluent samples than in the corresponding influent samples, which could have been caused by formational changes that permit vibrational energy losses of the promoted electrons, an increase in the number of aromatic rings condensed in a straight chain and conjugated double bonds (Wu et al., 2003). This phenomenon implies that certain tryptophan-like substances with more complex structure may have contributed to peak T2. The conventional municipal wastewater treatment process has been reported to have a low removal efficiency for certain compounds with complex structure (Kimura et al., 2005), thus unsurprisingly, the peak T2 fluorescent components appeared to be more difficult to remove than those of peak T1. Second, two new fluorescence peaks could be observed in the effluents compared to the corresponding influents. A new peak A was generated in 5 of 8 samples and a new peak B was generated in 2 of 5 samples. The presence of new fluorescent peaks in the effluents suggested that new substances were produced during the wastewater treatment process, which might have been derived from the degradation of the original components in the influents and living and dead microorganisms used to treat the wastewater and their exudates. The results of the present study indicated that peak T was a ubiquitous peak observed in the influents that was sensitive to the wastewater treatment process; therefore, peak T may be applied to the monitoring of municipal wastewater quality and further the control of treatment processes. We further explored the effect of different types of treatment processes on the EEM fluorescence spectra of wastewater. First, the removal efficiency of fluorescent substances using various treatment processes was compared. The removal of the peak T intensity was

3.3. 3D-EEM spectral characteristics Based on the commonly detected fluorescence peak location, as shown in Fig. 3, four distinct peaks were identified from the 3D-EEMs of the wastewater samples as follows: peak A, humic-like fluorescence (λex: 240–245 nm, λem: 400–405 nm); peak B, tyrosine-like fluorescence (λex: 220–225 nm, λem: 295–310 nm); and peak T, tryptophanlike fluorescence (peaks T1 and T2, λex: 275–290 nm, λem:320–340 nm and λex: 225–235 nm, λem: 335–355 nm, respectively) (Henderson et al., 2009). Numerous compounds may have contributed to the fluorescence peaks (Carstea et al., 2016). The humic-like fluorescence at peak A in the wastewater is mainly related to microbial sources (Murphy et al., 2011). Peaks T and B (both of which represent proteinlike compounds) in wastewater environments are a result of a mixture of cellular materials and cellular metabolites and materials derived from human activities (Bridgeman et al., 2013; Yu et al., 2014). Fig. 4 illustrates the general fluorescence characteristics of the wastewater samples. Peak T (peak T1 or peak T2) was observed in all influents and effluents, and was commonly more intense than peaks A and B. The peak T2 fluorescence intensities in the influents were always greater than those of peak T1 (the ratio of peak T2:T1 ranged from 1.22 to 2.53). This finding is consistent with an early observation of the average T2/T1 ratio (1.87) in sewage (Hudson et al., 2008), but the

Fig. 4. Stack columns of fluorescence peaks observed in wastewater sampled in each MWTP. The left and right stack columns in each sampling site represent the results of observations of influent and effluent samples, respectively. A/O process is used at the SJT MWTP; the A2/O process is used at the SH, LWZ, and ELM MWTPs; and the oxidation ditch process is used at the HJH, LBJ, NTZH, and TXH MWTPs.

734

Ecotoxicology and Environmental Safety 148 (2018) 729–737

X. Han et al.

81.42%, 51.89–83.43% (average 71.78%), and 81.83–85.83% (average 83.65%) using the A/O, A2/O, and oxidation ditch processes, respectively (Fig. 1d). It should be noted that the removal of the peak T intensity could be underestimated if the IFE that reduces the fluorescence of the influent samples is taken into account. Although only eight MWTPs (one MWTP employs the A/O process, and three and four MWTPs employ the A2/O and oxidation ditch processes, respectively) were investigated in this study, which makes it difficult to fully assess the removal efficiency of the treatment process, the oxidation ditch process appears to have a slightly better performance in the removal of tryptophan-like fluorescent substances using the A/O or A2/O process. Second, the elimination and generation of individual fluorescent components in the treated wastewater was analyzed. Peak B was not found in the wastewater treated with the oxidation ditch process; moreover, peak B presented in influents could be completely removed using this type of treatment (Fig. 4). While a new peak B was generated in wastewater treated with the A/O or A2/O processes (e.g., SJT and LWZ effluents using A/O and A2/O processes, respectively), peak B in the SH influents was not entirely removed by the A2/O treatment. These results suggest that the oxidation ditch process has the advantage of removing tyrosine-like substances from municipal wastewater compared with the A/O and A2/O processes. Because peak T was observed in all municipal wastewater samples, to assess the capability of fluorescence spectroscopy to act as a monitoring tool, the correlations between the fluorescence peak T and BOD and COD were evaluated. The peak T fluorescence intensity was highly correlated with both BOD (r = 0.83, n = 16; p < 0.01) and COD (r = 0.86, n = 16; p < 0.01). A similar strong correlation between peak T and both BOD and COD was also reported by a previous study (Cohen et al., 2014). The correlations with both BOD and COD highlight the significant contribution of the tryptophan-like matter to both the biologically degradable organic matter and the chemically oxidizable organic matter in the wastewater samples. Moreover, a statistically significant relationship was also observed between the peak T removal and the BOD removal (r = 0.74, n = 8, p = 0.037). Thus, the fluorescence of the tryptophan-like matter can be used as an indicator of both BOD and COD, and may serve as a marker of the BOD removal efficacy of municipal wastewater treatment.

Fig. 5. The effect of wastewater samples on C. elegans respiration. All data were normalized and are shown as a percentage of the positive respiratory control (100%). Error bars represent the standard deviation of three replicates. Statistical significance was calculated using Games-Howell's comparison with a positive control. N = not statistically significant (p > 0.05). *p < 0.05, **p < 0.01.

0.016, p = 0.007, p = 0.010, and p = 0.021, respectively). An inhibition of respiration in C. elegans was observed following the exposure to the SJT (18.4% decrease, p > 0.05 versus positive control) and TXH (31.2% decrease, p > 0.05 versus positive control) effluents, while a 0.07 to 3.3- fold increase in respiratory rate was found in the C. elegans treated with effluents from the other MWTPs; the increased respiratory rate caused by the LWZ, HJH, ELM, and LBJ effluents were significantly different from that in the control (p = 0.002, p = 0.011, p = 0.039, and p = 0.001, respectively). Regarding influent and effluent samples that were collected on the same day from the same MWTP, the effluent exposure resulted in either a higher or lower respiratory rate than the corresponding influent exposure in C. elegans. In general, there is no consistent effect (stimulated breathing or inhibited breathing) was observed in C. elegans after exposure to either influents or effluents. Additionally, no consistent change (increased respiratory rate or reduced respiratory rate) was found by comparing the respiratory rate of C. elegans exposed to effluents to that of the corresponding influents. These results imply that the effects of municipal wastewater on the respiratory function of C. elegans are very complex, which may be mainly related to the nature of the wastewater. In this study, the respiratory inhibition effect in C. elegans caused by exposure to wastewater samples could have been caused by the presence of many toxic chemicals in the wastewater. In fact, this effect has also been observed in C. elegans exposed to HJH influent, SJT effluent and TXH effluents. However, the remaining 13 investigated wastewater samples produced different degrees of enhanced effect on the respiration rate of C. elegans. A similar stimulating effect of wastewater on respiratory function or other functional indicators of the exposed organisms have also been observed in previous studies. A study conducted by Zitova et al. showed that the respiration rates of model organisms (Artemia salina and Jurkat cell) were enhanced after exposure to several treated industrial discharges (Zitova et al., 2009). Grande et al. found that exposure to 9 of 29 effluent waters derived from purification plants stimulated the bacterial luminescence whose output reflected changes in the metabolic activity of bacteria (Grande et al., 2007). These stimulation phenomena resulting from environmental exposure may be partly explained by hormesis, which is a dose-response concept that is characterized by a low-dose stimulation and a high-dose inhibition. Physiological reactions such as an increase in protein synthesis and energy expenditure in the presence of toxic substances, which will eventually be reflected by an increase in the rate of respiration, enable the organisms to survive following low sub-lethal exposures (Ramesh

3.4. Ecotoxicological effects of municipal wastewaters on C. elegans 3.4.1. Survival The lethal toxicity tests showed that a 24 h exposure to any of the unfiltered raw municipal wastewater samples (8 influents and 8 effluents) lead to negligible mortality in C. elegans, which is suggestive of mild acute toxicity from the wastewater. Thus, lethality was not sufficiently sensitive to discern the toxic impacts of the municipal wastewater, including the influents and effluents. 3.4.2. Respiration Respiration is a critical biological process that sustains the lives of aerobic organisms. Monitoring the changes in the respiratory function of organisms has been demonstrated to provide a reliable analysis of the toxic effects of a broad range of toxicants that have different mechanisms of action (Schouest et al., 2009). In the present study, the respiratory rate of nematodes cultured in K medium (positive control) represented the normal respiratory function under physiological conditions. Fig. 5 shows an altered respiratory rate in C. elegans after a 24 h-exposure to municipal wastewaters. Exposure to both the influents and effluents exposure could result in either an enhanced or attenuated respiratory activity in C. elegans. The HJH influent caused a 13% decrease in the respiratory rate (p > 0.05 versus positive control), while influents from other MWTPs resulted in a approximately 0.2 to 9.8- fold increase in the respiratory rate, and the increased respiratory rates induced by the LWZ, SJT, ELM, SH, and LBJ influent samples were significantly different from that induced by the control (p = 0.002, p = 735

Ecotoxicology and Environmental Safety 148 (2018) 729–737

X. Han et al.

Author declaration

and David, 2009). Because the stimulatory response of hormesis is modest, and the a maximum usually does not exceed the control value by more than twofold (Calabrese, 2008). The respiration rates that are above a 2-fold increase in C. elegans exposed to certain wastewater, such as LWZ, ELM, and LBJ wastewater, are difficult to explain using the hormesis response. The increase in the measured parameter indicated the presence of elements of disturbance in the analyzed samples; however, the reasons underlying this increase are uncertain. Based on the current understanding of changes in respiratory rates in C. elegans exposed to wastewater, the toxic impacts on respiratory rate in this study by the influents and effluents are difficult to discern, and thus could not be used to compare the toxicity reduction efficiency among the A/O, A2/O and oxidation ditch processes. Altogether, 81.25% (13/16) of the wastewater samples induces a greater than 20% change (stimulation or inhibition) in the respiration rate of C. elegans compared to the value observed in the control, which implies that the C. elegans-based OCR assay is sensitive enough to detect the biological effects of wastewater. The sensitivity of the test is particularly important for screening the biological toxicity of wastewater. Undoubtedly, before using the C. elegans-based OCR assay to assess the toxicity of wastewater, the causes and toxicological and ecological significance of the enhanced respiration in C. elegans exposed to wastewater should be determined.

We wish to confirm that there are no known conflicts of interest associated with this publication. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. References Bridgeman, J., Baker, A., Carliell-Marquet, C., Carstea, E., 2013. Determination of changes in wastewater quality through a treatment works using fluorescence spectroscopy. Environ. Technol. 34, 3069–3077. Brookman, S.K.E., 1997. Estimation of biochemical oxygen demand in slurry and effluents using ultra-violet spectrophotometry. Water Res. 31, 372–374. Calabrese, E.J., 2008. Hormesis: why it is important to toxicology and toxicologists. Environ. Toxicol. Chem. 27, 1451–1474. Carstea, E.M., Bridgeman, J., Baker, A., Reynolds, D.M., 2016. Fluorescence spectroscopy for wastewater monitoring: a review. Water Res. 95, 205–219. Chu, K.W., Chow, K.L., 2002. Synergistic toxicity of multiple heavy metals is revealed by a biological assay using a nematode and its transgenic derivative. Aquat. Toxicol. 61, 53–64. Cohen, E., Levy, G.J., Borisover, M., 2014. Fluorescent components of organic matter in wastewater: efficacy and selectivity of the water treatment. Water Res. 55, 323–334. Cui, R., Jahng, D., 2004. Nitrogen control in AO process with recirculation of solubilized excess sludge. Water Res. 38, 1159–1172. Grande, R., Di Pietro, S., Di Campli, E., Di Bartolomeo, S., Filareto, B., Cellini, L., 2007. Bio-toxicological assays to test water and sediment quality. J. Environ. Sci. Health A Tox. Hazard. Subst. Environ. Eng. 42, 33–38. Green, S.A., Blough, N.V., 1994. Optical absorption and fluorescence properties of chromophoric dissolved organic matter in natural waters. Limnol. Oceanogr. 39, 1903–1916. Hashimoto, K., Matsuda, M., Inoue, D., Ike, M., 2014. Bacterial community dynamics in a full-scale municipal wastewater treatment plant employing conventional activated sludge process. J. Biosci. Bioeng. 118, 64–71. Henderson, R.K., Baker, A., Murphy, K.R., Hambly, A., Stuetz, R.M., Khan, S.J., 2009. Fluorescence as a potential monitoring tool for recycled water systems: a review. Water Res. 43, 863–881. Her, N.G., Amy, G., Sohn, J., Gunten, U., 2008. UV Absorbance Ratio Index with Size Exclusion Chromatography (URI-SEC) as an NOM Property Indicator. J. Water Supply.: Res. Technol. –AQUA 57, 35–44. Hitchcock, D.R., Black, M.C., Williams, P.L., 1997. Investigations into using the nematode Caenorhabditis elegans for municipal and industrial wastewater toxicity testing. Arch. Environ. Contam. Toxicol. 33, 252–260. Hudson, N., Baker, A., Ward, D., Reynolds, D.M., Brunsdon, C., Carliell-Marquet, C., Browning, S., 2008. Can fluorescence spectrometry be used as a surrogate for the Biochemical Oxygen Demand (BOD) test in water quality assessment? An example from South West England. Sci. Total Environ. 391, 149–158. Janhom, T., Wattanachira, S., Pavasant, P., 2009. Characterization of brewery wastewater with spectrofluorometry analysis. J. Environ. Manag. 90, 1184–1190. Jin, L., Zhang, G., Tian, H., 2014. Current state of sewage treatment in China. Water Res. 66, 85–98. Kim, B.C., Kim, S., Shin, T., Kim, H., Sang, B.I., 2013. Comparison of the bacterial communities in anaerobic, anoxic, and oxic chambers of a pilot A(2)O process using pyrosequencing analysis. Curr. Microbiol. 66, 555–565. Kimura, K., Hara, H., Watanabe, Y., 2005. Removal of pharmaceutical compounds by submerged membrane bioreactors (MBRs). Desalination 178, 135–140. Kungolos, A., 2005. Evaluation of toxic properties of industrial wastewater using on-line respirometry. J. Environ. Sci. Health A Tox. Hazard. Subst. Environ. Eng. 40, 869–880. Leenheer, J.A., Croué, J.P., 2003. Characterizing aquatic dissolved organic matter. Environ. Sci. Technol. 37, 18A–26A. Liu, Y., Duan, J., Li, W., Beecham, S., Mulcahy, D., 2016. Effects of organic matter removal from a wastewater secondary effluent by aluminum sulfate coagulation on haloacetic acids formation. Environ. Eng. Sci. 33, 484–493. Liwarska-Bizukojc, E., Slezak, R., Klink, M., 2016. Study on wastewater toxicity using ToxTrak™ method. Environ. Sci. Pollut. Res. Int. 23, 9105–9113. Louvet, J.N., Homeky, B., Casellas, M., Pons, M.N., Dagot, C., 2013. Monitoring of slaughterhouse wastewater biodegradation in a SBR using fluorescence and UV–Visible absorbance. Chemosphere 91, 648–655. Matsché, N., Stumwöhrer, K., 1996. UV absorption as control-parameter for biological treatment plants. Water Sci. Technol. 33, 211–218. Ministry of Enviromental Protection of the People’s Republic of China, 2002. Pollutant Discharge Standard of Municipal Wastewater Treatment Plants. Morris, L., Colombo, V., Hassell, K., Kellar, C., Leahy, P., Long, S.M., Myers, J.H., Pettigrove, V., 2017. Municipal wastewater effluent licensing: a global perspective and recommendations for best practice. Sci. Total Environ. 580, 1327–1339. Murphy, K.R., Hambly, A., Singh, S., Henderson, R.K., Baker, A., Stuetz, R., Khan, S.J., 2011. Organic matter fluorescence in municipal water recycling schemes: toward a unified PARAFAC model. Environ. Sci. Technol. 45, 2909–2916. Ramesh, H., David, M., 2009. Respiratory performance and behavioral responses of the

4. Conclusion In this study, a combined physical-chemical, optical and toxicological analysis was performed to characterize municipal wastewater influent and effluent samples to evaluate the pollutant removal performance of three types of modified activated sludge processes and understand how the spectral signal of wastewater relates to BOD and COD. We also investigated whether C. elegans-based methods are suitable for monitoring wastewater toxicity. Collectively, the results suggested that: (1) common MWTPs employing any of the A/O, A2/O and oxidation ditch processes is effective in controlling the discharge of important municipal wastewater pollutants, including BOD, COD, ammonia nitrogen, total nitrogen, total phosphorus, and TSS. The BOD removal performance using the A2/O and oxidation ditch processes is slightly better than that using the A/O process; (2) Tryptophan-like substances are the main components in the dissolved organic matter pool in the investigated municipal wastewater influents. Humic-like substances in municipal wastewater effluents could be generated using the A/O, A2/O and oxidation ditch processes, and tyrosine-like substances could be generated using both the A/O process and the A2/O process. The oxidation ditch process has the advantage of removing tyrosine-like substances from municipal wastewater and presents a slightly better removal efficiency for tryptophan-like fluorescent substances than the A/O and A2/O processes; (3) Both UV254 and peak T may be used to characterize the organic load of municipal wastewater. Peak T can also be adopted as a gauge of the BOD removal efficacy in municipal wastewater treatment; (4) Mortality is not a suitable endpoint in C. elegans for measuring the acute toxicity of municipal wastewater, while the respiratory rate in C. elegans can provide a sensitive effect indicator of wastewater exposure. Using a C. elegans-based OCR assay for toxicity monitoring of municipal wastewater deserves further investigation.

Acknowledgements The work was supported by the National High Technology Research and Development Program of China (863 Program, Grant No. 2013AA065204), and the Fundamental Research Funds for the Central Universities (HUST: No. 2015TS103).

736

Ecotoxicology and Environmental Safety 148 (2018) 729–737

X. Han et al. freshwater fish, Cyprinus carpio (Linnaeus) under sublethal chlorpyrifos exposure. J. Basic Clin. Physiol. Pharmacol. 20, 127–139. Schouest, K., Zitova, A., Spillane, C., Papkovsky, D., 2009. Toxicological assessment of chemicals using Caenorhabditis elegans and optical oxygen respirometry. Environ. Toxicol. Chem. 28, 791–799. Tejeda-Benitez, L., Olivero-Verbel, J., 2016. Caenorhabditis elegans, a Biological Model for Research in Toxicology. Rev. Environ. Contam. Toxicol. 237, 1–35. Terashima, M., Yama, A., Sato, M., Yumoto, I., Kamagata, Y., Kato, S., 2016. Culturedependent and -independent identification of polyphosphate-accumulating Dechloromonas spp. predominating in a full-scale oxidation ditch wastewater treatment plant. Microbes Environ. 31, 449–455. Thomas, O., Theraulaz, F., Agnel, C., Suryani, S., 1996. Advanced UV examination of wastewater. Environ. Technol. 17, 251–261. Uyguner, C.S., Bekbolet, M., 2005. Implementation of spectroscopic parameters for practical monitoring of natural organic matter. Desalination 176, 47–55. van den Broeke, J., Langergraber, G., Weingartner, A., 2006. On-line and in-situ UV/vis spectroscopy for multi-parameter measurements: a brief review. Spectrosc. Eur. 18, 15–18. Wang, J.H., Zhang, T.Y., Dao, G.H., Xu, X.Q., Wang, X.X., Hu, H., 2017. Microalgae-based advanced municipal wastewater treatment for reuse in water bodies. Appl. Microbiol. Biotechnol. 101, 2659–2675. Wu, F.C., Evans, R.D., Dillon, P.J., 2003. Separation and characterization of NOM by

high-performance liquid chromatography and on-line three-dimensional excitation emission matrix fluorescence detection. Environ. Sci. Technol. 37, 3687–3693. Xiao, Y., De Araujo, C., Sze, C.C., Stuckey, D.C., 2015. Toxicity measurement in biological wastewater treatment processes: a review. J. Hazard. Mater. 286, 15–29. Xu, D., Liu, S., Chen, Q., Ni, J., 2017. Microbial community compositions in different functional zones of Carrousel oxidation ditch system for domestic wastewater treatment. AMB Express 7, 40. Yu, H., Song, Y., Liu, R., Pan, H., Xiang, L., Feng, Q., 2014. Identifying changes in dissolved organic matter content and characteristics by fluorescence spectroscopy coupled with self-organizing map and classification and regression tree analysis during wastewater treatment. Chemosphere 113, 79–86. Zhang, Q.H., Yang, W.N., Ngo, H.H., Guo, W.S., Jin, P.K., Dzakpasu, M., Yang, S.J., Wang, Q., Wang, X.C., Ao, D., 2016. Current status of urban wastewater treatment plants in China. Environ. Int. 92–93, 11–22. Zitova, A., O'Mahony, F.C., Cross, M., Davenport, J., Papkovsky, D.B., 2009. Toxicological profiling of chemical and environmental samples using panels of test organisms and optical oxygen respirometry. Environ. Toxicol. 24, 116–127. Zuo, Y.T., Hu, Y., Lu, W.W., Cao, J.J., Wang, F., Han, X., Lu, W.Q., Liu, A.L., 2017. Toxicity of 2,6-dichloro-1,4-benzoquinone and five regulated drinking water disinfection by-products for the Caenorhabditis elegans nematode. J. Hazard. Mater. 321, 456–463.

737