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Changes in nutrient removal and flocs characteristics generated by presence of ZnO nanoparticles in activated sludge process
Chemosphere 182 (2017) 672e680
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Changes in nutrient removal and flocs characteristics generated by presence of ZnO nanoparticles in activated sludge process s, Germa n Cuevas-Rodríguez* Pabel Cervantes-Avile rez 77, Guanajuato, Gto., CP 36000, Department of Civil Engineering & Environmental Engineering, Engineering Division, University of Guanajuato, Av. Jua Mexico
h i g h l i g h t s ZnO NPs inhibited the oxygen uptake of activated sludge from 450 mg/L. The ammonia removal decreased by presence of ZnO NPs in raw and filtered wastewater. Orthophosphate removal increased by presence higher than 450 mg/L of ZnO NPs. Suspended solids in wastewater influenced in the effects of ZnO NPs on flocs size. The ZnO NPs were observed in cell membrane and internalizated in microorganisms.
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a b s t r a c t
Article history: Received 24 October 2016 Received in revised form 9 May 2017 Accepted 12 May 2017 Available online 16 May 2017
The aim of this work was to evaluate the impact generated by ZnO NPs on the activated sludge process treating raw (RWW) and filtered wastewater (FWW). It was analyzed the oxygen uptake rate, nutrient removal, flocs characteristics and the morphological interactions between activated sludge and ZnO NPs, in presence of 450e2000 mg/L. The results showed that the presence of more than 450 mg/L of ZnO NPs in raw and filtered wastewater inhibited the oxygen uptake by activated sludge. The highest inhibition was 35% in presence of 1500 mg/L in RWW. The organic matter removal was only inhibited in the presence of 450 and 900 mg/L of ZnO NPs; while ammonia removal decreased for all concentrations of ZnO NPs in both types of wastewater, around 13% for RWW and up to 9% for FWW. The orthophosphate removal improved as the concentration of ZnO NPs increased for both wastewater types, enhancing up to 8% for RWW and 17% for FWW. The flocs size of activated sludge exposed to ZnO NPs in RWW decreased as the concentration of ZnO NPs increased; while for FWW, an opposite effect was observed. The elemental mapping allowed detect the Zn inside of microorganisms, which may correspond to a toxicity mechanism in RWW and FWW. These results indicated that the changes in nutrient removal and flocs characteristics caused by the presence of ZnO NPs on the activated sludge are related to wastewater characteristics, such as suspended solids, type of substrate and concentration of ZnO NPs. © 2017 Elsevier Ltd. All rights reserved.
Handling Editor: A Adalberto Noyola Keywords: ZnO nanoparticles Activated sludge Wastewater Oxygen uptake Nanoecotoxicology Elemental mapping
1. Introduction The use of nanomaterials (NMs) in different products of everyday life is due to their unique properties, such as high surfaceto-volume ratio and a highly reactive surface (Murty et al., 2013). These properties make them excellent adsorbents, catalysts, and oxidation agents (Qu et al., 2013). ZnO nanoparticles (NPs) are one of the most produced NMs and are currently incorporated in
* Corresponding author. E-mail address: [email protected] (G. Cuevas-Rodríguez). http://dx.doi.org/10.1016/j.chemosphere.2017.05.074 0045-6535/© 2017 Elsevier Ltd. All rights reserved.
textiles, medicals, electronics, plastics, coatings, cleaning agents, pigments, cosmetics and paints (Lazareva and Keller, 2014; Musee, 2017). Current estimates put the global production of ZnO NPs at as high as 34,000 Tons per year (Keller et al., 2013). This high rate of production suggests that ZnO NPs are increasingly relevant in products being used daily. Studies investigating the life cycle of many products containing ZnO NPs found that they are released into the environment (Sun et al., 2014). ZnO NPs in the environment are transported through wastewater and freshwater streams (Quik et al., 2015), causing some to believe wastewater treatment plants (WWTPs) may have significant exposure to ZnO NPs (Gottschalk et al., 2013). The potential amount of ZnO NPs arriving
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at WWTPs has been estimated by some authors. They considered geographical factors, Inequality-Adjusted Human Development Index (IHDI) of different regions, type of applications, among others (Keller et al., 2013; Lazareva and Keller, 2014). The estimated amount of ZnO NPs arriving into WWTPs is between 0.45 and 8.19 g/person/year for cities with high population and high IHDI (Lazareva and Keller, 2014). Given the increasing amount of ZnO NPs expected in WWTPs, nanoecotoxicological studies in the activated sludge process are needed, which is the most used process in WWTPs (OECD, 2015). Current nanoecotoxicological experiments between ZnO NPs and activated sludge have normally fed synthetic wastewater (SWW) and filtered wastewater (FWW) (Liu et al., 2011; Zheng et al., 2011; Puay et al., 2015; Zhou et al., 2015). In those experiments, they were observed the inhibition of oxygen uptake for concentrations higher than 100 mg/L ZnO NPs (Liu et al., 2011; Zhou et al., 2015), and low nitrogen removal at long-term of exposure (Zheng et al., 2011; Puay et al., 2015; Tan et al., 2015). The main cause of these negative effects was the instability of ZnO NPs in the bioreactor (Liu et al., 2011; Tan et al., 2015). Several studies have demonstrated that the stability of ZnO NPs is influenced by wastewater characteristics, such as organic matter, nitrogen compounds and suspended solids (Limbach et al., 2008; Keller et al., 2010; Chaúque et al., 2014). Hence, the effects of ZnO NPs on activated sludge process depend on the wastewater characteristics. The use of SWW in these nanoecotoxicological experiments may be distant from realistic condi mez-Rivera tions as compared to using raw wastewater (RWW). Go et al. (2012) tested organic matter removal in activated sludge exposed to CeO2 NPs using SWW and RWW. These authors concluded that the use of synthetic media to model NPs removal during wastewater treatment is questionable, due to the SWW and RWW have different physicochemical characteristics which affect mez-Rivera et al., 2012). Several studies have the stability of NPs (Go reported that stability and aggregate size of NPs is influenced by water characteristics such as pH and ionic strength (Chaúque et al., 2014), concentration of organic matter, nitrogen compounds (Keller et al., 2010), and total suspended solids (TSS) concentration (SaniKast et al., 2015). Hence, the use of RWW and FWW may contribute to understanding the influence of wastewater characteristics in these nanoecotoxicological experiments. Particularly, the characteristics related to the stability of ZnO NPs such as, suspended solids, organic matter and nitrogen in wastewater. On the other hand, the fate of ZnO NPs in activated sludge reactors is mainly in sludge disposal (Musee et al., 2014; Ma et al., 2014). Hence, the interactions between flocs and ZnO NPs can be of great interest to scientist and practitioners, in order to mitigate the associated potential hazards. In this way, it is important to know the flocs characteristics and composition after exposure to ZnO NPs in a realistic wastewater matrix. Some studies performed Transmission Electron Microscopy (TEM) imaging, in order to infer these mechanisms through the location of the NPs in biological samples, assuming that electron-dense materials observed correspond to NPs (Kumar et al., 2011; Park et al., 2013). However, some undesirable artefacts must be carefully considered due to that may lead to misinterpretation of the imaging. Thus, TEM imaging can be combined with analytical or diffraction techniques to verify the chemical composition of NPs interacting with cells. In this work, we combine the High Angle Annual Dark Field (HAADF) Scanning Transmission Electronic Microscopy (STEM) imaging, with the elemental mapping by using Energy Dispersive X-ray Spectroscopy (EDS), as a novel technique to identify the chemical composition of electron-dense material in contact with the activated sludge. The aim of this work was to evaluate the impact generated by ZnO NPs on the activated sludge process treating RWW and FWW as a function of the oxygen uptake rate, nutrient removal, flocs
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characteristics and the morphological interactions between activated sludge and ZnO NPs. 2. Methods and materials 2.1. Characterization of ZnO nanoparticles The stock of ZnO NPs was purchased from I&D nanotechnology (Mexico). Characterization consisted of determining the morphology, size, elemental composition, crystal structure and ultravioletevisible spectrum. Fig. S1 in supplementary information shows the imaging and spectrum characterization. For this characterization, a ZnO NP suspension in ultrapure water was prepared and sonicated for 1 h at 200 W (Taurozzi et al., 2012). After, a drop of this suspension was placed on nickel grid; then it was dried at 120 C with plasma for a period of 5 min before observation in TEM (Jeol 2200FS HR-FE-TEM, Japan). Another drop of ZnO NP suspension was placed on a specimen mount, and was dried at 120 C with plasma for 5 min before observation using scanning electron microscopy (SEM, Jeol JSM 7401F, Japan). The ZnO NPs suspensions for experiments were prepared in ultrapure water (pH 7). The concentrations of ZnO NPs used for experiments were 450, 900, 1500 and 2000 mg/L, in order to elucidate the accumulative effect of ZnO NPs in the activated sludge process. Before each experiment, each ZnO NPs suspension was prepared and sonicated for 1 h at 200 W using a frequency of 40 kHz for immediate use (Taurozzi et al., 2012). 2.2. Raw and filtered wastewater The experiments were carried out with RWW and FWW at same time. RWW was collected from a house septic tank in Guanajuato, Mexico and was storage in lab at 4 ± 1 C, after 30 min of transportation. To get FWW, RWW was passed through 0.45 mm filters to remove suspended solids. The characterization of RWW and FWW consisted of determining total suspended solids (TSS) and volatile suspended solids (VSS) according to the 2540 D standard method (APHA, 2005). The biochemical oxygen demand (BOD5) was determined according to 5210 D standard method (APHA, 2005) by using de BOD Trak II (Hach, U.S.A.). The chemical oxygen demand (COD) total and soluble (CODs), ammonia nitrogen (NH3eN), nitrate (NO3eN), nitrite (NO2eN) and orthophosphate (PO4eP) were measured by using the method 8000, 10031, 8039, 8507 and 8114 from Hach methods, respectively. These parameters were determinated by using the spectrophotometer DR 5000 (Hach, U.S.A.). Table 1 presents the physicochemical characteristics measured in
Table 1 Physicochemical characteristics of raw wastewater (RWW) and filtered wastewater (FWW) used in the experiments. SD is short for standard deviation, Min is short for minimum and Max is short for maximum. Parameter
Temp. pH Conductivity TSS VSS COD CODS BOD5 NH3eN NO3eN NO2eN PO4eP
Units
C
mS/cm mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L
RWW
FWW
MineMax
Mean ± SD
MineMax
Mean ± SD
e
23.7 8.34 2857 80 ± 5 58 ± 5 468 ± 19 426 ± 18 380 ± 10 148 ± 12.1 3.6 ± 2.1 0.2 ± 0.04 84 ± 3.8
e e e e e e 387e405 337e358 136e153 4.1e6.1 0.03e0.12 46.4e54.1
23.4 8.44 2790 e e e 397 ± 9 350 ± 10 146 ± 7.2 5.5 ± 0.9 0.08 ± 0.005 50.5 ± 3.2
e 78e84 54e62 449e489 409e445 366e389 134e159 1.4e5.7 0.16e0.23 79e87
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RWW and FWW. The main nutrients of RWW and FWW were within the range reported for domestic wastewater, 250e750 mg/L for COD, 40e150 mg/L for NH3eN and 10e90 mg/L for PO4eP (Van Loosdrecht et al., 2015). 2.3. Oxygen uptake evaluation in activated sludge Oxygen uptake tests were performed in the presence of ZnO NPs and using RWW and FWW. The oxygen uptake rate (OUR) was measured every 20 min during 8 h, which is the typical hydraulic retention time in activated sludge process (Van Loosdrecht et al., 2015). The oxygen uptake in the presence of each concentration of ZnO NPs was compared with oxygen uptake in the control group to see the inhibition due to ZnO NPs after 8 h. The respirometric tests were carried out in darkness to avoid the photocatalytic activity of ZnO NPs. The respirometer used was gas-static-static (GSS), in which oxygen is transferred from gas to liquid phase. The oxygen uptake was calculated through the pressure change in the gas phase at 20 C and closed respirometric bottle (Hach BOD Trak II, U.S.A.). The respirometric bottle contained as a liquid phase 95 mL: 40 mL of activated sludge, 40 mL of wastewater (RWW or FWW) and 15 mL of the ZnO NPs suspension. Each concentration of ZnO NPs was evaluated per triplicate and in the presence of three controls without ZnO NPs. In the controls, the ZnO NPs suspension was replaced by 15 mL of ultrapure water. The activated sludge inoculum presented 3170 ± 180 mg/L of VSS, which was collected from an activated sludge pilot plant fed with synthetic wastewater s et al., 2016). The pilot plant operated as a (Cervantes-Avile sequential batch reactor with the conditions in Table S2. 2.4. Nutrient removal tests in activated sludge In nutrient removal tests, reactors with a 2 L effective volume were used, which were covered with black foil to provide darkness inside and avoid the to avoid the photocatalytic activity of ZnO NPs. The 2 L of the reactors was divided in: 0.842 L of activated sludge, 0.842 L of wastewater (RWW or FWW) and 0.316 L of ZnO NPs suspension, the same proportion as that for the oxygen uptake tests. The activated sludge seed for nutrient removal tests presented 3060 ± 100 mg/L of VSS and was collected from the same pilot plant as in the oxygen uptake tests. The aireation was continuous at 6 m3/h m3. In all nutrient removal tests, samples at zero and 8 h were collected. Then, they were filtered through 0.45 mm and analyzed for determination of CODs, NH3eN, NO3eN, NO2eN and PO4eP by using Hach methods mentioned above. The removal of CODs, NH3eN and PO4eP N in RWW and FWW were calculated as a relationship between final (C) and initial concentration (C0). In addition to these determinations, samples for calculation of sludge volumetric index (SVI) and flocs size analysis were collected in all nutrient removal tests. The flocs size was determined using 1.81 as the refractive index in static light scattering (SLS, Microtrac S3500, U.S.A.). The tests with ZnO NPs and the control were performed at the same time and per triplicate. 2.5. Morphologic interactions between ZnO NPs and activated sludge The interactions between ZnO NPs and activated sludge were studied by TEM imaging, HAADF-STEM imaging and elemental mapping. Samples for TEM and HAADF-STEM were processed as follow. They were collected at the end of nutrient removal tests. After collection, tubes of two mL were centrifuged at 13,000 rpm for 2 min, and the supernatant was replaced by 3% glutaraldehyde. Samples were suspended and left to stand for 4 h at 20 ± 1 C. After this time, samples were washed with a buffer of sodium cacodylate
(0.1 M, pH 7.4). Then, they were fixed for 1 h with 1% osmium tetroxide. After fixation, samples were washed four times with the same sodium cacodylate buffer. Next, the samples were dehydrated gradually with ethanol, from 10% to absolute ethanol. Finally, the samples were embedded in resin EPON-812 and were cut in sections of 60 nm by using an ultramicrotome. The TEM observations of all samples were performed at 80 kV in Jeol JM-1010 (Japan). After TEM imaging, same samples were used for HAADF-STEM imaging and elemental mapping. The HAADF-STEM imaging were collected with CCD camera of 1024 1024 pixels of digital resolution (Gatan). The chemical analysis was performed in STEM mode by EDS, which was synchronized with the HAADF-STEM imaging for elemental mapping in the selected area. Both HAADF-STEM imaging and elemental mapping were carried out at 300 kV in the electronic microscope FEI Titan 80-300 (Netherlands). Optical condition used was a spherical aberration Cs ¼ 1.25 mm, info. limit 1 Å and a resolution in STEM of 1.4 Å. 2.6. Statistical analysis Statistical data treatment was performed using one-way ANOVA, considering each type of wastewater per separated. Then, Dunnett tests were performed to determine the significant difference between each concentration of ZnO NPs and its respective control group. Results with p < 0.05 were considered significant. 3. Results and discussion 3.1. ZnO NPs characterization Fig. S1A and SIB (supplementary data) show nanorods as the main shape of ZnO NPs, with a size distribution between 20 and 60 nm of length and diameter between 11 and 23 nm. The presence of Zn and O was confirmed in TEM observation by three analyses of energy dispersive spectroscopy (EDS). According to the x-ray diffraction (XRD) pattern (Fig. S1E), all the diffraction peaks of ZnO NPs correspond to wurtzite with hexagonal crystal structure. The ultravioletevisible (UVevis) spectrum of the ZnO NPs showed a typical peak at 365 nm in the UV region. 3.2. Oxygen uptake of activated sludge in the presence of ZnO NPs To the knowledge of the authors, this study represents the first report of activated sludge exposed to extremely high concentrations of ZnO NPs. So, our findings correspond to situations where the concentrations of ZnO NPs that we studied can be found. Fig. 1 presents the OUR by microorganisms during treatment of RWW and FWW in the presence of ZnO NPs. The control groups using both types of wastewater presented typical values for activated sludge (Van Loosdrecht et al., 2015). In presence of ZnO NPs, OUR values for RWW were lower than in FWW. For RWW, a negative effect was observed from 450 mg/L of ZnO NPs after 2 h. In the case of FWW, the OUR had an initial adverse effect for 900 and 1500 mg/ L of ZnO NPs. However, OURs for both concentrations were recovered after 2 h. In presence of 2000 mg/L of ZnO NPs and FWW, the OUR decreased as time goes during eight hours. It means that microorganisms of activated sludge may buffer the presence of 900 and 1500 mg/L of ZnO NPs after 2 h, but can not buffer the presence of 2000 mg/L of ZnO NPs during 8 h. Hence, the presence of 2000 mg/L of ZnO NPs may gradually reduce the aerobic capability of activated sludge, which can affect the wastewater treatment process. Regarding inhibition of oxygen uptake by using both types of wastewater, it was observed for concentrations higher than 900 mg/L of ZnO NPs. i.e. For RWW, the highest inhibition was 35% for 1500 mg/L of ZnO NPs. While for FWW, the inhibition of oxygen
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3.3. CODs removal in the presence of ZnO NPs Fig. S2 shows the removal of organic matter as a relationship between the final and initial concentration of CODs (C/C0). The control groups of both wastewaters removed more than half of the initial COD concentration, which is expected due to activated sludge process removes within 50e80% of CODs (Van Loosdrecht et al., 2015). For RWW, the COD removal was affected only in the presence of 450 and 900 mg/L of ZnO NPs, which represented 13 and 15% less CODs removal than control, respectively. In the case of FWW, the removal of CODs decreased only in the presence of 450 mg/L, showing 14% less removal than control. These results showed that the COD removal by activated sludge is slightly affected by 450 and 900 mg/L ZnO NPs, regardless of the presence of TSS in wastewater. Several long-term studies reported that there were not significant the impact of ZnO NPs on the removal of CODs when was used SWW, even at too high accumulative concentrations of ZnO NPs as this study (Puay et al., 2015; Tan et al., 2015; Zhang et al., 2016; He et al., 2017). However, Musee et al. (2014) fed RWW with ZnO NPs in a simulated activated sludge WWTP during ten days and found 9% less COD removal than control in the presence of 115 mg Zn/g of sludge. According to the literature and our findings, there could be a concentration range between 100 and 900 mg/L of ZnO NPs, where the ZnO NPs may cause inhibition in the CODs removal by activated sludge. 3.4. Ammonia removal in the presence of ZnO NPs
Fig. 1. Oxygen uptake rate (OUR) in the two types of wastewater used. A) Raw wastewater (RWW), B) Filtered wastewater (FWW), C) Inhibition of the oxygen uptake in the presence of ZnO nanoparticles (NPs) by using RWW and FWW. Mark (asterisk) represents no significant difference when compared with control.
uptake was from 13 to 27% in presence from 450 to 2000 mg/L of ZnO NPs. Physicochemical characteristics of the wastewater showed that main difference between RWW and FWW was the presence of suspended solids. It means that the presence of suspended solids in RWW may promote the negative effects of ZnO NPs in the oxygen uptake by activated sludge process. Chaúque et al. (2014) tested the stability of ZnO NPs in RWW and found that suspended solids easily adsorb the ZnO NPs forming heteroaggregates, which may have relationship with the toxic effect of ZnO NPs to aerobic microorganisms.
Fig. 2A presents the ammonia removal experiments. Ammonia removal for controls were 72 and 63% for RWW and FWW, respectively. These values corresponded to the typical ammonia removal for activated sludge within 50 and 80% (Van Loosdrecht et al., 2015). However, it was inhibited in the presence of ZnO NPs for both, RWW and FWW. For RWW, the ammonia removal was inhibited 13%, while for FWW removal was also inhibited in a range from 6 to 9% for all concentrations of ZnO NPs. The measurements of nitrite and nitrate helped to elucidate the ammonia oxidation by microorganisms in the presence of ZnO NPs (Fig. 2B and C). For RWW, the nitrite concentration was higher than control in the presence of more than 450 mg/L of ZnO NPs, while nitrate formation was higher than the control for 1500 and 2000 mg/L of ZnO NPs. For experiments with FWW, the concentration of nitrite decreases as the concentration of ZnO NPs increases. The nitrate formation using FWW was lower than the control and similar in the presence of all concentrations of ZnO NPs tested. The ammonia oxidation study showed that nitrite and nitrate production decreased mainly in experiments with FWW. This negative effect corresponds with the findings of Liu et al. (2011), who used concentrations of ZnO NPs from 16 up to 802 mg Zn/L and evaluated the nitrification process in autotrophic nitrifying bacteria using SWW. They found that ammonia oxidation was inhibited from 13 mg Zn/L, while nitrite oxidation was hindered from 476 mg Zn/L (Liu et al., 2011). This suggests that our findings may correspond to similar effects at lower concentrations in experiments with SWW. Related to the toxicity of ZnO NPs over nitrifying bacteria, it has been reported that ZnO NPs inhibited the metabolism of autotrophic bacteria involved in ammonia and nitrite oxidation (Zheng et al., 2014; Xu et al., 2016). Hence, the ZnO NPs in wastewater without suspended solids such as FWW, may cause the damage to microorganisms involved in the nitrification process, mainly developed by nitrosomonas and nitrobacter. Considering that for both, RWW and FWW, the ammonia removal in presence of ZnO NPs was inhibited, the nitrification analysis indicated that the presence of suspended solids in RWW can buffer the adverse effect of ZnO NPs on the nitrification process.
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The nitrification process analysis suggests that the presence of ZnO NPs may damage the biological ammonia oxidation for FWW. However, in the case of RWW, this process was not affected. Hence, it is possible that the ZnO NPs may adsorb the ammonia present in RWW. This adsorption of nitrogen compounds in NPs was reported for other NPs such as TiO2 (Lin et al., 2012) and CeO2 (Limbach et al., 2008). 3.5. Orthophosphate removal in the presence of ZnO NPs The orthophosphate removal was enhanced in the presence of ZnO NPs for both types of wastewater (Fig. 3). Control groups of RWW and FWW presented typical orthophosphate removal for activated sludge 78 and 82%, respectively (Van Loosdrecht et al., 2015). In the case of RWW, orthophosphate removal was improved for all concentrations of ZnO NPs. The highest removal was observed for 450 and 900 mg/L of ZnO NPs, with 12 and 14% higher removal than control, respectively. Using FWW, orthophosphate removal was similar to control in presence of 450 mg/L ZnO NPs, but was higher than control as the concentration of ZnO NPs increased, achieving 17% more removal than control in the presence of 2000 mg/L of ZnO NPs. These results are consistent with studies for long term experiments feeding SWW with lower concentrations of ZnO NPs than our study, l mg/L during 92 days and 10 mg/L during 81days (Tan et al., 2015). However, Zheng et al. (2011) reported that exopolyphosphatase (PPX) and polyphosphate kinase (PPK), which are key enzymes involved in phosphate removal, had less activity of as increase the concentration of ZnO NPs. A recent study by Hu et al. (2017) determined that phosphorous removal was significantly deteriorated after long term exposure of 2, 6 and 10 mg/L of ZnO NPs in SWW, due to the decrease of the microbial diversity. Thus, the phosphate removal in the presence of ZnO NPs and real wastewater could be due to the crystallization and precipitation of phosphate compounds. Ma et al. (2014) reported the formation of Zn3(PO4)2 in an activated sludge reactor as a stable compound in sludge from a pilot WWTP. Briefly, the formation of Zn3(PO4)2 from ZnO NPs in aqueous solution involves the hydroxylation on the surface of ZnO NPs that induce the release of Zn2þ from NPs. After Zn2þ release, PO4eP replaces OH and amorphous zinc phosphate crystallizes (Lv et al., 2012). The improvement on phosphate removal must be considered as a positive effect caused by the presence of ZnO NPs, which is related to the
Fig. 2. Ammonia removal at the end of the experiments using raw wastewater (RWW) and filtered wastewater (FWW). A) The relation between final (C) and initial (C0) concentration of ammonia (NH3eN), B) Final concentration of nitrites (NO2eN) and C) Final concentration of nitrates (NO3eN). Mark (asterisk) represents no significant difference when compared with control.
It can be attributed to the formation of heteroaggregates in RWW, which may reduce the amount of ZnO NPs in contact with microorganisms. However, the nitrite and nitrate concentrations were higher than control in experiments using RWW with more than 900 mg/L of ZnO NPs. The over production of nitrite and nitrate can be attributed to hydroxylamine (NH2eOH) formation (Wunderlin et al., 2012), which is the first step of ammonia oxidation. The NH2eOH formation may increase due to the over production of OH generated on the surface of ZnO NPs (Lv et al., 2012). Then, the nitrite and nitrate concentrations may also increase.
Fig. 3. The relation between final (C) and initial (C0) concentration of orthophosphate (PO4eP) in activated sludge exposed to ZnO nanoparticles (NPs) in raw wastewater (RWW) and filtered wastewater (FWW). Mark (asterisk) represents no significant difference when compared with control.
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concentration of ZnO NPs and wastewater characteristics. 3.6. Flocs size and settling of activated sludge exposed to ZnO NPs The flocs size of controls for RWW and FWW was mainly between 100 and 1000 mm (Fig. 4), which is the normal size range for steady flocs. In experiments using RWW, the flocs size was gradually reducing as the concentration of ZnO NPs increased. Hence, in the presence of 2000 mg/L of ZnO NPs the smallest sized flocs of 200 mm were detected. For FWW experiments, the main effect was on flocs smaller than 100 mm, which were 25% for 450 and 900 mg/ L, 12% for 1500 mg/L and finally 3% for 2000 mg/L of ZnO NPs. This distribution indicates that the presence of ZnO NPs in FWW promoted the formation of larger flocs than in the control. Hou et al. (2015) studied flocs stability in activated sludge using SWW with CuO NPs and reported that flocs presented less flocculation ability due to over production of loosely bound EPS (LB-EPS). The over production of LB-EPS may explain the smaller flocs found using RWW with ZnO NPs. In recent studies, it has reported that the increasing on flocs size exposed to ZnO NPs are related to the double layer theory (Xu and Li, 2016; Peng et al., 2017). It means that the stability of ZnO NPs in FWW may have relation with the increasing size on flocs of activated sludge. Fig. S3 shows through SVI values that ZnO NPs enhanced the sludge settling for both types of wastewater. The high compaction of sludge in the presence of ZnO NPs may be related to a high density of flocs due to biosorption of ZnO NPs by activated sludge (Kiser et al., 2010; Zheng et al., 2011; Chaúque et al., 2014). These
Fig. 4. Flocs size distribution of activated sludge exposed to ZnO nanoparticles (NPs) during treatment of A) Raw wastewater (RWW) and B) Filtered wastewater (FWW).
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results are contradictory from Puay et al. (2015), where a sequential batch reactor of activated sludge fed with SWW and ZnO NPs was studied. They found the activated sludge had a poor ability to settle due to the over production of EPS. Hence, the settling of activated sludge with ZnO NPs using RWW and FWW may provide conditions to remove the ZnO NPs through sludge disposal in WWTPs. The likely implications of changes in flocs in the WWTP could compromise the wastewater treatment process, as well as the availability of ZnO NPs in the biosolids once they are released in the environment. 3.7. Interactions between ZnO NPs and activated sludge Fig. 5 presents the interactions between ZnO NPs and microorganisms involved in activated sludge process after treatment of RWW and FWW. The black color corresponds to the electrodense material. For both types of wastewater three main patterns were found: aggregate formation, attachment of electrodense material to cell membrane and internalization of electron-dense material in cells. For RWW, electrodense particles were observed forming large aggregates bound to an organic material present in RWW (Fig. 5A). These aggregates are consistent with the description of heteroaggregates suggested by Sani-Kast et al. (2015) and Dale et al. (2015). The aggregates observed in FWW were smaller than in RWW (Fig. 5D). The main behavior showed in RWW was the presence of particles attached to cell membrane (Fig. 5B). However, for RWW with 2000 mg/L of ZnO NPs, electrodense particles were also found inside the cell's surrounding organelles (Fig. 5C). For FWW in the presence of ZnO NPs, the presence of electrodense material internalized in cells was found (Fig. 5E and F). As it can be seen in Fig. 5F, the presence of electron-dense material across the cell membrane suggests that the internalization process occurs without mechanical damage to the cell membrane. Fig. 6 presents the elemental mapping developed in activated sludge exposed to 2000 mg/L of ZnO NPs in RWW. It can be seen that in the normal mode of TEM, the electron-dense material was shown in black color (Fig. 6A). However, when HAADF-STEM was applied the electrodense material was observed in white color due to the Z-contrast (Fig. 6B). According to the mapping of Zn-K and Zn-L in the sample, the abundance and distribution of electrondense material observed in TEM image correspond to Zn. The Zn and O distributions suggest that material internalized inside the cells could be the ZnO NPs. However, it is important to consider that a fraction of ZnO NPs can be soluble and pass inside the cell by diffusion and form Zn complexes (Liu et al., 2011). These results are consistent with previous studies which showed a high abundance of electron-dense material inside the cells from activated sludge s without membrane damage (Kumar et al., 2011; Cervantes-Avile et al., 2016). Therefore, the internalization of electron-dense material could be attributed to passive diffusion. The passive diffusion or facilitated transport across an intact membrane cell is related with the type of bacteria and the stability of NPs (Liu et al., 2016). Thus, the internalization observed could be related to the compounds adsorbed by ZnO NPs in RWW and FWW. The effects induced by internalization of ZnO NPs may alter the performance of aerobic microorganisms. The internalization of ZnO NPs was not observed for all microorganisms. This suggests that characteristics of microorganism, such as thickness and composition of cell membrane, metabolism, electrostatic forces, among others, may also be related to the internalization of ZnO NPs. According with information compiled by Sirelkhatim et al. (2015), the major ability of ZnO NPs as an antibacterial agent is due to the generation of reactive oxygen species (ROS), which cause cell death by atrophying metabolic processes of bacteria or damage on the cell membrane structure. In our experiments, the
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Fig. 5. Transmission electron microscopy (TEM) imaging of activated sludge after treatment of raw wastewater (RWW) with different concentration of ZnO nanoparticles (NPs): A) 450 mg/L, B) 900 mg/L, C) 2000 mg/L, and after treatment of filtered wastewater (FWW) in presence of ZnO NPs: D) 450 mg/L, E) 900 mg/L and F) 2000 mg/L.
Fig. 6. Activated sludge exposed to 2000 mg/L of ZnO NPs in RWW. A) Conventional transmission electron microscopy (TEM) imaging and B) Elemental mapping (ZneO) using high angle annular dark field (HAADF) detector in scanning transmission electron microscopy (STEM) mode.
physiochemical characteristics of real wastewater may promote the toxicity of ZnO NPs in microorganisms involved in activated sludge reactors, which may repercussions in biological nutrient removal and flocs characteristics. 4. Conclusions In this work the nutrient removal and the flocs characteristics of the activated sludge exposed to ZnO NPs by using RWW and FWW was evaluated. The presence of more than 450 mg/L of ZnO NPs in RWW and FWW inhibited the oxygen uptake by microorganisms in
the activated sludge process. The organic matter removal decreased in the presence of 450 and 900 mg/L of ZnO NPs for RWW and only for 450 mg/L of ZnO for FWW. The study of the nitrification process indicated that microorganisms did not efficiently develop ammonia oxidation when FWW with more than 450 mg/L of ZnO NPs was used. The orthophosphate removal in activated sludge was improved in the presence of concentrations higher than 450 mg/L of ZnO NPs in RWW and FWW. The settling rate of activated sludge was enhanced by the presence of ZnO NPs in both wastewater types. The concentration of ZnO NPs altered the flocs size of activated sludge of opposite way for two types of wastewater,
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decreasing for RWW and increasing in FWW. The negative effects of ZnO NPs on the activated sludge process using real wastewater are related to the formation of heteroaggregates between suspended solids and NPs, the attachment of NPs to the cell membrane and their internalization. Thereby, wastewater characteristics, such as suspended solids, type of substrate and concentrations of ZnO NPs have a direct influence in flocs characteristics and nutrient removal by the activated sludge process. Acknowledgements This work was supported by the National Council of Science and Technology of Mexico (CONACYT; 175089, 2013), the National Council for Scientific and Technological Development of Brazil (CNPq; 44, 2012), Research Direction and Support to Postgraduate of the University of Guanajuato (DAIP; 599, 2015), and CONACYT through Ph.D. Scholarship (No. 359919). The authors would like to thank Lourdes Palma for technical support in the Microscopy Unit rez for at the Neurobiology Institute e UNAM, to Eng. Eneidy Pe technical support and the Mexican Center of Renewable Energies S.C. supported by the University of Guanajuato, Guanajuato State Government and Salamanca municipality (RENIECYT: 2011/15369). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.chemosphere.2017.05.074. References APHA, 2005. Standard Methods for the Examination of Water and Wastewater, 21th ed. American Public Health Association Inc., Washington, DC, ISBN 0875530478. s, P., Brito, E.M., Duran, R., Martínez, A.B., Cuevas-Rodríguez, G., Cervantes-Avile 2016. Effect of ZnO nanoparticles in the oxygen uptake during aerobic wastewater treatment. J. Nanopart. Res. 18, 1e12. http://dx.doi.org/10.1007/s11051016-3481-3. Chaúque, E.F.C., Zvimba, J.N., Ngila, J.C., Musee, N., 2014. Stability studies of commercial ZnO engineered nanoparticles in domestic wastewater. Phys. Chem. Earth Pt. A/B/C 67, 140e144. http://dx.doi.org/10.1016/j.pce.2013.09.011. Dale, A.L., Lowry, G.V., Casman, E.A., 2015. Stream dynamics and chemical transformations control the environmental fate of silver and zinc oxide nanoparticles in a watershed-scale model. Environ. Sci. Technol. 49, 7285e7293. http:// dx.doi.org/10.1021/acs.est.5b01205. mez-Rivera, F., Field, J.A., Brown, D., Sierra-Alvarez, R., 2012. Fate of cerium diGo oxide (CeO2) nanoparticles in municipal wastewater during activated sludge treatment. Bioresour. Technol. 108, 300e304. http://dx.doi.org/10.1016/ j.biortech.2011.12.113. Gottschalk, F., Sun, T., Nowack, B., 2013. Environmental concentrations of engineered nanomaterials: review of modeling and analytical studies. Environ. Pollut. 181, 287e300. http://dx.doi.org/10.1016/j.envpol.2013.06.003. He, Q., Yuan, Z., Zhang, J., Zhang, S., Zhang, W., Zou, Z., Wang, H., 2017. Insight into the impact of ZnO nanoparticles on aerobic granular sludge under shock loading. Chemosphere 173, 411e416. http://dx.doi.org/10.1016/j.chemosphere.2017.01.085. Hou, J., Miao, L., Wang, C., Wang, P., Ao, Y., Lv, B., 2015. Effect of CuO nanoparticles on the production and composition of extracellular polymeric substances and physicochemical stability of activated sludge flocs. Bioresour. Technol. 176, 65e70. http://dx.doi.org/10.1016/j.biortech.2014. Hu, Z., Lu, X., Sun, P., Hu, Z., Wang, R., Lou, C., Han, J., 2017. Understanding the performance of microbial community induced by ZnO nanoparticles in enhanced biological phosphorus removal system and its recoverability. Bioresour. Technol. 225, 279e285. http://dx.doi.org/10.1016/j.biortech.2016.11.080. Keller, A., Wang, H., Zhou, D., Lenihan, H.S., Cherr, G., Cardinale, B.J., Miller, R., Ji, Z., 2010. Stability and aggregation of metal oxide nanoparticles in natural aqueous matrices. Environ. Sci. Technol. 44, 1962e1967. http://dx.doi.org/10.1021/ es902987d. Keller, A.A., McFerran, S., Lazareva, A., Suh, S., 2013. Global life cycle releases of engineered nanomaterials. J. Nanopart. Res. 15, 1e17. http://dx.doi.org/10.1007/ s11051-013-1692-4. Kiser, M.A., Ryu, H., Jang, H., Hristovski, K., Westerhoff, P., 2010. Biosorption of nanoparticles to heterotrophic wastewater biomass. Water Res. 44, 4105e4114. http://dx.doi.org/10.1016/j.watres.2010.05.036. Kumar, A., Pandey, A.K., Singh, S.S., Shanker, R., Dhawan, A., 2011. Cellular uptake and mutagenic potential of metal oxide nanoparticles in bacterial cells. Chemosphere 83, 1124e1132. http://dx.doi.org/10.1016/j.chemosphere.2011.01.025.
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Changes in nutrient removal and flocs characteristics generated by presence of ZnO nanoparticles in activated sludge process s, Germa n Cuevas-Rodríguez* Pabel Cervantes-Avile rez 77, Guanajuato, Gto., CP 36000, Department of Civil Engineering & Environmental Engineering, Engineering Division, University of Guanajuato, Av. Jua Mexico
h i g h l i g h t s ZnO NPs inhibited the oxygen uptake of activated sludge from 450 mg/L. The ammonia removal decreased by presence of ZnO NPs in raw and filtered wastewater. Orthophosphate removal increased by presence higher than 450 mg/L of ZnO NPs. Suspended solids in wastewater influenced in the effects of ZnO NPs on flocs size. The ZnO NPs were observed in cell membrane and internalizated in microorganisms.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 October 2016 Received in revised form 9 May 2017 Accepted 12 May 2017 Available online 16 May 2017
The aim of this work was to evaluate the impact generated by ZnO NPs on the activated sludge process treating raw (RWW) and filtered wastewater (FWW). It was analyzed the oxygen uptake rate, nutrient removal, flocs characteristics and the morphological interactions between activated sludge and ZnO NPs, in presence of 450e2000 mg/L. The results showed that the presence of more than 450 mg/L of ZnO NPs in raw and filtered wastewater inhibited the oxygen uptake by activated sludge. The highest inhibition was 35% in presence of 1500 mg/L in RWW. The organic matter removal was only inhibited in the presence of 450 and 900 mg/L of ZnO NPs; while ammonia removal decreased for all concentrations of ZnO NPs in both types of wastewater, around 13% for RWW and up to 9% for FWW. The orthophosphate removal improved as the concentration of ZnO NPs increased for both wastewater types, enhancing up to 8% for RWW and 17% for FWW. The flocs size of activated sludge exposed to ZnO NPs in RWW decreased as the concentration of ZnO NPs increased; while for FWW, an opposite effect was observed. The elemental mapping allowed detect the Zn inside of microorganisms, which may correspond to a toxicity mechanism in RWW and FWW. These results indicated that the changes in nutrient removal and flocs characteristics caused by the presence of ZnO NPs on the activated sludge are related to wastewater characteristics, such as suspended solids, type of substrate and concentration of ZnO NPs. © 2017 Elsevier Ltd. All rights reserved.
Handling Editor: A Adalberto Noyola Keywords: ZnO nanoparticles Activated sludge Wastewater Oxygen uptake Nanoecotoxicology Elemental mapping
1. Introduction The use of nanomaterials (NMs) in different products of everyday life is due to their unique properties, such as high surfaceto-volume ratio and a highly reactive surface (Murty et al., 2013). These properties make them excellent adsorbents, catalysts, and oxidation agents (Qu et al., 2013). ZnO nanoparticles (NPs) are one of the most produced NMs and are currently incorporated in
* Corresponding author. E-mail address: [email protected] (G. Cuevas-Rodríguez). http://dx.doi.org/10.1016/j.chemosphere.2017.05.074 0045-6535/© 2017 Elsevier Ltd. All rights reserved.
textiles, medicals, electronics, plastics, coatings, cleaning agents, pigments, cosmetics and paints (Lazareva and Keller, 2014; Musee, 2017). Current estimates put the global production of ZnO NPs at as high as 34,000 Tons per year (Keller et al., 2013). This high rate of production suggests that ZnO NPs are increasingly relevant in products being used daily. Studies investigating the life cycle of many products containing ZnO NPs found that they are released into the environment (Sun et al., 2014). ZnO NPs in the environment are transported through wastewater and freshwater streams (Quik et al., 2015), causing some to believe wastewater treatment plants (WWTPs) may have significant exposure to ZnO NPs (Gottschalk et al., 2013). The potential amount of ZnO NPs arriving
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at WWTPs has been estimated by some authors. They considered geographical factors, Inequality-Adjusted Human Development Index (IHDI) of different regions, type of applications, among others (Keller et al., 2013; Lazareva and Keller, 2014). The estimated amount of ZnO NPs arriving into WWTPs is between 0.45 and 8.19 g/person/year for cities with high population and high IHDI (Lazareva and Keller, 2014). Given the increasing amount of ZnO NPs expected in WWTPs, nanoecotoxicological studies in the activated sludge process are needed, which is the most used process in WWTPs (OECD, 2015). Current nanoecotoxicological experiments between ZnO NPs and activated sludge have normally fed synthetic wastewater (SWW) and filtered wastewater (FWW) (Liu et al., 2011; Zheng et al., 2011; Puay et al., 2015; Zhou et al., 2015). In those experiments, they were observed the inhibition of oxygen uptake for concentrations higher than 100 mg/L ZnO NPs (Liu et al., 2011; Zhou et al., 2015), and low nitrogen removal at long-term of exposure (Zheng et al., 2011; Puay et al., 2015; Tan et al., 2015). The main cause of these negative effects was the instability of ZnO NPs in the bioreactor (Liu et al., 2011; Tan et al., 2015). Several studies have demonstrated that the stability of ZnO NPs is influenced by wastewater characteristics, such as organic matter, nitrogen compounds and suspended solids (Limbach et al., 2008; Keller et al., 2010; Chaúque et al., 2014). Hence, the effects of ZnO NPs on activated sludge process depend on the wastewater characteristics. The use of SWW in these nanoecotoxicological experiments may be distant from realistic condi mez-Rivera tions as compared to using raw wastewater (RWW). Go et al. (2012) tested organic matter removal in activated sludge exposed to CeO2 NPs using SWW and RWW. These authors concluded that the use of synthetic media to model NPs removal during wastewater treatment is questionable, due to the SWW and RWW have different physicochemical characteristics which affect mez-Rivera et al., 2012). Several studies have the stability of NPs (Go reported that stability and aggregate size of NPs is influenced by water characteristics such as pH and ionic strength (Chaúque et al., 2014), concentration of organic matter, nitrogen compounds (Keller et al., 2010), and total suspended solids (TSS) concentration (SaniKast et al., 2015). Hence, the use of RWW and FWW may contribute to understanding the influence of wastewater characteristics in these nanoecotoxicological experiments. Particularly, the characteristics related to the stability of ZnO NPs such as, suspended solids, organic matter and nitrogen in wastewater. On the other hand, the fate of ZnO NPs in activated sludge reactors is mainly in sludge disposal (Musee et al., 2014; Ma et al., 2014). Hence, the interactions between flocs and ZnO NPs can be of great interest to scientist and practitioners, in order to mitigate the associated potential hazards. In this way, it is important to know the flocs characteristics and composition after exposure to ZnO NPs in a realistic wastewater matrix. Some studies performed Transmission Electron Microscopy (TEM) imaging, in order to infer these mechanisms through the location of the NPs in biological samples, assuming that electron-dense materials observed correspond to NPs (Kumar et al., 2011; Park et al., 2013). However, some undesirable artefacts must be carefully considered due to that may lead to misinterpretation of the imaging. Thus, TEM imaging can be combined with analytical or diffraction techniques to verify the chemical composition of NPs interacting with cells. In this work, we combine the High Angle Annual Dark Field (HAADF) Scanning Transmission Electronic Microscopy (STEM) imaging, with the elemental mapping by using Energy Dispersive X-ray Spectroscopy (EDS), as a novel technique to identify the chemical composition of electron-dense material in contact with the activated sludge. The aim of this work was to evaluate the impact generated by ZnO NPs on the activated sludge process treating RWW and FWW as a function of the oxygen uptake rate, nutrient removal, flocs
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characteristics and the morphological interactions between activated sludge and ZnO NPs. 2. Methods and materials 2.1. Characterization of ZnO nanoparticles The stock of ZnO NPs was purchased from I&D nanotechnology (Mexico). Characterization consisted of determining the morphology, size, elemental composition, crystal structure and ultravioletevisible spectrum. Fig. S1 in supplementary information shows the imaging and spectrum characterization. For this characterization, a ZnO NP suspension in ultrapure water was prepared and sonicated for 1 h at 200 W (Taurozzi et al., 2012). After, a drop of this suspension was placed on nickel grid; then it was dried at 120 C with plasma for a period of 5 min before observation in TEM (Jeol 2200FS HR-FE-TEM, Japan). Another drop of ZnO NP suspension was placed on a specimen mount, and was dried at 120 C with plasma for 5 min before observation using scanning electron microscopy (SEM, Jeol JSM 7401F, Japan). The ZnO NPs suspensions for experiments were prepared in ultrapure water (pH 7). The concentrations of ZnO NPs used for experiments were 450, 900, 1500 and 2000 mg/L, in order to elucidate the accumulative effect of ZnO NPs in the activated sludge process. Before each experiment, each ZnO NPs suspension was prepared and sonicated for 1 h at 200 W using a frequency of 40 kHz for immediate use (Taurozzi et al., 2012). 2.2. Raw and filtered wastewater The experiments were carried out with RWW and FWW at same time. RWW was collected from a house septic tank in Guanajuato, Mexico and was storage in lab at 4 ± 1 C, after 30 min of transportation. To get FWW, RWW was passed through 0.45 mm filters to remove suspended solids. The characterization of RWW and FWW consisted of determining total suspended solids (TSS) and volatile suspended solids (VSS) according to the 2540 D standard method (APHA, 2005). The biochemical oxygen demand (BOD5) was determined according to 5210 D standard method (APHA, 2005) by using de BOD Trak II (Hach, U.S.A.). The chemical oxygen demand (COD) total and soluble (CODs), ammonia nitrogen (NH3eN), nitrate (NO3eN), nitrite (NO2eN) and orthophosphate (PO4eP) were measured by using the method 8000, 10031, 8039, 8507 and 8114 from Hach methods, respectively. These parameters were determinated by using the spectrophotometer DR 5000 (Hach, U.S.A.). Table 1 presents the physicochemical characteristics measured in
Table 1 Physicochemical characteristics of raw wastewater (RWW) and filtered wastewater (FWW) used in the experiments. SD is short for standard deviation, Min is short for minimum and Max is short for maximum. Parameter
Temp. pH Conductivity TSS VSS COD CODS BOD5 NH3eN NO3eN NO2eN PO4eP
Units
C
mS/cm mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L
RWW
FWW
MineMax
Mean ± SD
MineMax
Mean ± SD
e
23.7 8.34 2857 80 ± 5 58 ± 5 468 ± 19 426 ± 18 380 ± 10 148 ± 12.1 3.6 ± 2.1 0.2 ± 0.04 84 ± 3.8
e e e e e e 387e405 337e358 136e153 4.1e6.1 0.03e0.12 46.4e54.1
23.4 8.44 2790 e e e 397 ± 9 350 ± 10 146 ± 7.2 5.5 ± 0.9 0.08 ± 0.005 50.5 ± 3.2
e 78e84 54e62 449e489 409e445 366e389 134e159 1.4e5.7 0.16e0.23 79e87
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RWW and FWW. The main nutrients of RWW and FWW were within the range reported for domestic wastewater, 250e750 mg/L for COD, 40e150 mg/L for NH3eN and 10e90 mg/L for PO4eP (Van Loosdrecht et al., 2015). 2.3. Oxygen uptake evaluation in activated sludge Oxygen uptake tests were performed in the presence of ZnO NPs and using RWW and FWW. The oxygen uptake rate (OUR) was measured every 20 min during 8 h, which is the typical hydraulic retention time in activated sludge process (Van Loosdrecht et al., 2015). The oxygen uptake in the presence of each concentration of ZnO NPs was compared with oxygen uptake in the control group to see the inhibition due to ZnO NPs after 8 h. The respirometric tests were carried out in darkness to avoid the photocatalytic activity of ZnO NPs. The respirometer used was gas-static-static (GSS), in which oxygen is transferred from gas to liquid phase. The oxygen uptake was calculated through the pressure change in the gas phase at 20 C and closed respirometric bottle (Hach BOD Trak II, U.S.A.). The respirometric bottle contained as a liquid phase 95 mL: 40 mL of activated sludge, 40 mL of wastewater (RWW or FWW) and 15 mL of the ZnO NPs suspension. Each concentration of ZnO NPs was evaluated per triplicate and in the presence of three controls without ZnO NPs. In the controls, the ZnO NPs suspension was replaced by 15 mL of ultrapure water. The activated sludge inoculum presented 3170 ± 180 mg/L of VSS, which was collected from an activated sludge pilot plant fed with synthetic wastewater s et al., 2016). The pilot plant operated as a (Cervantes-Avile sequential batch reactor with the conditions in Table S2. 2.4. Nutrient removal tests in activated sludge In nutrient removal tests, reactors with a 2 L effective volume were used, which were covered with black foil to provide darkness inside and avoid the to avoid the photocatalytic activity of ZnO NPs. The 2 L of the reactors was divided in: 0.842 L of activated sludge, 0.842 L of wastewater (RWW or FWW) and 0.316 L of ZnO NPs suspension, the same proportion as that for the oxygen uptake tests. The activated sludge seed for nutrient removal tests presented 3060 ± 100 mg/L of VSS and was collected from the same pilot plant as in the oxygen uptake tests. The aireation was continuous at 6 m3/h m3. In all nutrient removal tests, samples at zero and 8 h were collected. Then, they were filtered through 0.45 mm and analyzed for determination of CODs, NH3eN, NO3eN, NO2eN and PO4eP by using Hach methods mentioned above. The removal of CODs, NH3eN and PO4eP N in RWW and FWW were calculated as a relationship between final (C) and initial concentration (C0). In addition to these determinations, samples for calculation of sludge volumetric index (SVI) and flocs size analysis were collected in all nutrient removal tests. The flocs size was determined using 1.81 as the refractive index in static light scattering (SLS, Microtrac S3500, U.S.A.). The tests with ZnO NPs and the control were performed at the same time and per triplicate. 2.5. Morphologic interactions between ZnO NPs and activated sludge The interactions between ZnO NPs and activated sludge were studied by TEM imaging, HAADF-STEM imaging and elemental mapping. Samples for TEM and HAADF-STEM were processed as follow. They were collected at the end of nutrient removal tests. After collection, tubes of two mL were centrifuged at 13,000 rpm for 2 min, and the supernatant was replaced by 3% glutaraldehyde. Samples were suspended and left to stand for 4 h at 20 ± 1 C. After this time, samples were washed with a buffer of sodium cacodylate
(0.1 M, pH 7.4). Then, they were fixed for 1 h with 1% osmium tetroxide. After fixation, samples were washed four times with the same sodium cacodylate buffer. Next, the samples were dehydrated gradually with ethanol, from 10% to absolute ethanol. Finally, the samples were embedded in resin EPON-812 and were cut in sections of 60 nm by using an ultramicrotome. The TEM observations of all samples were performed at 80 kV in Jeol JM-1010 (Japan). After TEM imaging, same samples were used for HAADF-STEM imaging and elemental mapping. The HAADF-STEM imaging were collected with CCD camera of 1024 1024 pixels of digital resolution (Gatan). The chemical analysis was performed in STEM mode by EDS, which was synchronized with the HAADF-STEM imaging for elemental mapping in the selected area. Both HAADF-STEM imaging and elemental mapping were carried out at 300 kV in the electronic microscope FEI Titan 80-300 (Netherlands). Optical condition used was a spherical aberration Cs ¼ 1.25 mm, info. limit 1 Å and a resolution in STEM of 1.4 Å. 2.6. Statistical analysis Statistical data treatment was performed using one-way ANOVA, considering each type of wastewater per separated. Then, Dunnett tests were performed to determine the significant difference between each concentration of ZnO NPs and its respective control group. Results with p < 0.05 were considered significant. 3. Results and discussion 3.1. ZnO NPs characterization Fig. S1A and SIB (supplementary data) show nanorods as the main shape of ZnO NPs, with a size distribution between 20 and 60 nm of length and diameter between 11 and 23 nm. The presence of Zn and O was confirmed in TEM observation by three analyses of energy dispersive spectroscopy (EDS). According to the x-ray diffraction (XRD) pattern (Fig. S1E), all the diffraction peaks of ZnO NPs correspond to wurtzite with hexagonal crystal structure. The ultravioletevisible (UVevis) spectrum of the ZnO NPs showed a typical peak at 365 nm in the UV region. 3.2. Oxygen uptake of activated sludge in the presence of ZnO NPs To the knowledge of the authors, this study represents the first report of activated sludge exposed to extremely high concentrations of ZnO NPs. So, our findings correspond to situations where the concentrations of ZnO NPs that we studied can be found. Fig. 1 presents the OUR by microorganisms during treatment of RWW and FWW in the presence of ZnO NPs. The control groups using both types of wastewater presented typical values for activated sludge (Van Loosdrecht et al., 2015). In presence of ZnO NPs, OUR values for RWW were lower than in FWW. For RWW, a negative effect was observed from 450 mg/L of ZnO NPs after 2 h. In the case of FWW, the OUR had an initial adverse effect for 900 and 1500 mg/ L of ZnO NPs. However, OURs for both concentrations were recovered after 2 h. In presence of 2000 mg/L of ZnO NPs and FWW, the OUR decreased as time goes during eight hours. It means that microorganisms of activated sludge may buffer the presence of 900 and 1500 mg/L of ZnO NPs after 2 h, but can not buffer the presence of 2000 mg/L of ZnO NPs during 8 h. Hence, the presence of 2000 mg/L of ZnO NPs may gradually reduce the aerobic capability of activated sludge, which can affect the wastewater treatment process. Regarding inhibition of oxygen uptake by using both types of wastewater, it was observed for concentrations higher than 900 mg/L of ZnO NPs. i.e. For RWW, the highest inhibition was 35% for 1500 mg/L of ZnO NPs. While for FWW, the inhibition of oxygen
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3.3. CODs removal in the presence of ZnO NPs Fig. S2 shows the removal of organic matter as a relationship between the final and initial concentration of CODs (C/C0). The control groups of both wastewaters removed more than half of the initial COD concentration, which is expected due to activated sludge process removes within 50e80% of CODs (Van Loosdrecht et al., 2015). For RWW, the COD removal was affected only in the presence of 450 and 900 mg/L of ZnO NPs, which represented 13 and 15% less CODs removal than control, respectively. In the case of FWW, the removal of CODs decreased only in the presence of 450 mg/L, showing 14% less removal than control. These results showed that the COD removal by activated sludge is slightly affected by 450 and 900 mg/L ZnO NPs, regardless of the presence of TSS in wastewater. Several long-term studies reported that there were not significant the impact of ZnO NPs on the removal of CODs when was used SWW, even at too high accumulative concentrations of ZnO NPs as this study (Puay et al., 2015; Tan et al., 2015; Zhang et al., 2016; He et al., 2017). However, Musee et al. (2014) fed RWW with ZnO NPs in a simulated activated sludge WWTP during ten days and found 9% less COD removal than control in the presence of 115 mg Zn/g of sludge. According to the literature and our findings, there could be a concentration range between 100 and 900 mg/L of ZnO NPs, where the ZnO NPs may cause inhibition in the CODs removal by activated sludge. 3.4. Ammonia removal in the presence of ZnO NPs
Fig. 1. Oxygen uptake rate (OUR) in the two types of wastewater used. A) Raw wastewater (RWW), B) Filtered wastewater (FWW), C) Inhibition of the oxygen uptake in the presence of ZnO nanoparticles (NPs) by using RWW and FWW. Mark (asterisk) represents no significant difference when compared with control.
uptake was from 13 to 27% in presence from 450 to 2000 mg/L of ZnO NPs. Physicochemical characteristics of the wastewater showed that main difference between RWW and FWW was the presence of suspended solids. It means that the presence of suspended solids in RWW may promote the negative effects of ZnO NPs in the oxygen uptake by activated sludge process. Chaúque et al. (2014) tested the stability of ZnO NPs in RWW and found that suspended solids easily adsorb the ZnO NPs forming heteroaggregates, which may have relationship with the toxic effect of ZnO NPs to aerobic microorganisms.
Fig. 2A presents the ammonia removal experiments. Ammonia removal for controls were 72 and 63% for RWW and FWW, respectively. These values corresponded to the typical ammonia removal for activated sludge within 50 and 80% (Van Loosdrecht et al., 2015). However, it was inhibited in the presence of ZnO NPs for both, RWW and FWW. For RWW, the ammonia removal was inhibited 13%, while for FWW removal was also inhibited in a range from 6 to 9% for all concentrations of ZnO NPs. The measurements of nitrite and nitrate helped to elucidate the ammonia oxidation by microorganisms in the presence of ZnO NPs (Fig. 2B and C). For RWW, the nitrite concentration was higher than control in the presence of more than 450 mg/L of ZnO NPs, while nitrate formation was higher than the control for 1500 and 2000 mg/L of ZnO NPs. For experiments with FWW, the concentration of nitrite decreases as the concentration of ZnO NPs increases. The nitrate formation using FWW was lower than the control and similar in the presence of all concentrations of ZnO NPs tested. The ammonia oxidation study showed that nitrite and nitrate production decreased mainly in experiments with FWW. This negative effect corresponds with the findings of Liu et al. (2011), who used concentrations of ZnO NPs from 16 up to 802 mg Zn/L and evaluated the nitrification process in autotrophic nitrifying bacteria using SWW. They found that ammonia oxidation was inhibited from 13 mg Zn/L, while nitrite oxidation was hindered from 476 mg Zn/L (Liu et al., 2011). This suggests that our findings may correspond to similar effects at lower concentrations in experiments with SWW. Related to the toxicity of ZnO NPs over nitrifying bacteria, it has been reported that ZnO NPs inhibited the metabolism of autotrophic bacteria involved in ammonia and nitrite oxidation (Zheng et al., 2014; Xu et al., 2016). Hence, the ZnO NPs in wastewater without suspended solids such as FWW, may cause the damage to microorganisms involved in the nitrification process, mainly developed by nitrosomonas and nitrobacter. Considering that for both, RWW and FWW, the ammonia removal in presence of ZnO NPs was inhibited, the nitrification analysis indicated that the presence of suspended solids in RWW can buffer the adverse effect of ZnO NPs on the nitrification process.
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The nitrification process analysis suggests that the presence of ZnO NPs may damage the biological ammonia oxidation for FWW. However, in the case of RWW, this process was not affected. Hence, it is possible that the ZnO NPs may adsorb the ammonia present in RWW. This adsorption of nitrogen compounds in NPs was reported for other NPs such as TiO2 (Lin et al., 2012) and CeO2 (Limbach et al., 2008). 3.5. Orthophosphate removal in the presence of ZnO NPs The orthophosphate removal was enhanced in the presence of ZnO NPs for both types of wastewater (Fig. 3). Control groups of RWW and FWW presented typical orthophosphate removal for activated sludge 78 and 82%, respectively (Van Loosdrecht et al., 2015). In the case of RWW, orthophosphate removal was improved for all concentrations of ZnO NPs. The highest removal was observed for 450 and 900 mg/L of ZnO NPs, with 12 and 14% higher removal than control, respectively. Using FWW, orthophosphate removal was similar to control in presence of 450 mg/L ZnO NPs, but was higher than control as the concentration of ZnO NPs increased, achieving 17% more removal than control in the presence of 2000 mg/L of ZnO NPs. These results are consistent with studies for long term experiments feeding SWW with lower concentrations of ZnO NPs than our study, l mg/L during 92 days and 10 mg/L during 81days (Tan et al., 2015). However, Zheng et al. (2011) reported that exopolyphosphatase (PPX) and polyphosphate kinase (PPK), which are key enzymes involved in phosphate removal, had less activity of as increase the concentration of ZnO NPs. A recent study by Hu et al. (2017) determined that phosphorous removal was significantly deteriorated after long term exposure of 2, 6 and 10 mg/L of ZnO NPs in SWW, due to the decrease of the microbial diversity. Thus, the phosphate removal in the presence of ZnO NPs and real wastewater could be due to the crystallization and precipitation of phosphate compounds. Ma et al. (2014) reported the formation of Zn3(PO4)2 in an activated sludge reactor as a stable compound in sludge from a pilot WWTP. Briefly, the formation of Zn3(PO4)2 from ZnO NPs in aqueous solution involves the hydroxylation on the surface of ZnO NPs that induce the release of Zn2þ from NPs. After Zn2þ release, PO4eP replaces OH and amorphous zinc phosphate crystallizes (Lv et al., 2012). The improvement on phosphate removal must be considered as a positive effect caused by the presence of ZnO NPs, which is related to the
Fig. 2. Ammonia removal at the end of the experiments using raw wastewater (RWW) and filtered wastewater (FWW). A) The relation between final (C) and initial (C0) concentration of ammonia (NH3eN), B) Final concentration of nitrites (NO2eN) and C) Final concentration of nitrates (NO3eN). Mark (asterisk) represents no significant difference when compared with control.
It can be attributed to the formation of heteroaggregates in RWW, which may reduce the amount of ZnO NPs in contact with microorganisms. However, the nitrite and nitrate concentrations were higher than control in experiments using RWW with more than 900 mg/L of ZnO NPs. The over production of nitrite and nitrate can be attributed to hydroxylamine (NH2eOH) formation (Wunderlin et al., 2012), which is the first step of ammonia oxidation. The NH2eOH formation may increase due to the over production of OH generated on the surface of ZnO NPs (Lv et al., 2012). Then, the nitrite and nitrate concentrations may also increase.
Fig. 3. The relation between final (C) and initial (C0) concentration of orthophosphate (PO4eP) in activated sludge exposed to ZnO nanoparticles (NPs) in raw wastewater (RWW) and filtered wastewater (FWW). Mark (asterisk) represents no significant difference when compared with control.
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concentration of ZnO NPs and wastewater characteristics. 3.6. Flocs size and settling of activated sludge exposed to ZnO NPs The flocs size of controls for RWW and FWW was mainly between 100 and 1000 mm (Fig. 4), which is the normal size range for steady flocs. In experiments using RWW, the flocs size was gradually reducing as the concentration of ZnO NPs increased. Hence, in the presence of 2000 mg/L of ZnO NPs the smallest sized flocs of 200 mm were detected. For FWW experiments, the main effect was on flocs smaller than 100 mm, which were 25% for 450 and 900 mg/ L, 12% for 1500 mg/L and finally 3% for 2000 mg/L of ZnO NPs. This distribution indicates that the presence of ZnO NPs in FWW promoted the formation of larger flocs than in the control. Hou et al. (2015) studied flocs stability in activated sludge using SWW with CuO NPs and reported that flocs presented less flocculation ability due to over production of loosely bound EPS (LB-EPS). The over production of LB-EPS may explain the smaller flocs found using RWW with ZnO NPs. In recent studies, it has reported that the increasing on flocs size exposed to ZnO NPs are related to the double layer theory (Xu and Li, 2016; Peng et al., 2017). It means that the stability of ZnO NPs in FWW may have relation with the increasing size on flocs of activated sludge. Fig. S3 shows through SVI values that ZnO NPs enhanced the sludge settling for both types of wastewater. The high compaction of sludge in the presence of ZnO NPs may be related to a high density of flocs due to biosorption of ZnO NPs by activated sludge (Kiser et al., 2010; Zheng et al., 2011; Chaúque et al., 2014). These
Fig. 4. Flocs size distribution of activated sludge exposed to ZnO nanoparticles (NPs) during treatment of A) Raw wastewater (RWW) and B) Filtered wastewater (FWW).
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results are contradictory from Puay et al. (2015), where a sequential batch reactor of activated sludge fed with SWW and ZnO NPs was studied. They found the activated sludge had a poor ability to settle due to the over production of EPS. Hence, the settling of activated sludge with ZnO NPs using RWW and FWW may provide conditions to remove the ZnO NPs through sludge disposal in WWTPs. The likely implications of changes in flocs in the WWTP could compromise the wastewater treatment process, as well as the availability of ZnO NPs in the biosolids once they are released in the environment. 3.7. Interactions between ZnO NPs and activated sludge Fig. 5 presents the interactions between ZnO NPs and microorganisms involved in activated sludge process after treatment of RWW and FWW. The black color corresponds to the electrodense material. For both types of wastewater three main patterns were found: aggregate formation, attachment of electrodense material to cell membrane and internalization of electron-dense material in cells. For RWW, electrodense particles were observed forming large aggregates bound to an organic material present in RWW (Fig. 5A). These aggregates are consistent with the description of heteroaggregates suggested by Sani-Kast et al. (2015) and Dale et al. (2015). The aggregates observed in FWW were smaller than in RWW (Fig. 5D). The main behavior showed in RWW was the presence of particles attached to cell membrane (Fig. 5B). However, for RWW with 2000 mg/L of ZnO NPs, electrodense particles were also found inside the cell's surrounding organelles (Fig. 5C). For FWW in the presence of ZnO NPs, the presence of electrodense material internalized in cells was found (Fig. 5E and F). As it can be seen in Fig. 5F, the presence of electron-dense material across the cell membrane suggests that the internalization process occurs without mechanical damage to the cell membrane. Fig. 6 presents the elemental mapping developed in activated sludge exposed to 2000 mg/L of ZnO NPs in RWW. It can be seen that in the normal mode of TEM, the electron-dense material was shown in black color (Fig. 6A). However, when HAADF-STEM was applied the electrodense material was observed in white color due to the Z-contrast (Fig. 6B). According to the mapping of Zn-K and Zn-L in the sample, the abundance and distribution of electrondense material observed in TEM image correspond to Zn. The Zn and O distributions suggest that material internalized inside the cells could be the ZnO NPs. However, it is important to consider that a fraction of ZnO NPs can be soluble and pass inside the cell by diffusion and form Zn complexes (Liu et al., 2011). These results are consistent with previous studies which showed a high abundance of electron-dense material inside the cells from activated sludge s without membrane damage (Kumar et al., 2011; Cervantes-Avile et al., 2016). Therefore, the internalization of electron-dense material could be attributed to passive diffusion. The passive diffusion or facilitated transport across an intact membrane cell is related with the type of bacteria and the stability of NPs (Liu et al., 2016). Thus, the internalization observed could be related to the compounds adsorbed by ZnO NPs in RWW and FWW. The effects induced by internalization of ZnO NPs may alter the performance of aerobic microorganisms. The internalization of ZnO NPs was not observed for all microorganisms. This suggests that characteristics of microorganism, such as thickness and composition of cell membrane, metabolism, electrostatic forces, among others, may also be related to the internalization of ZnO NPs. According with information compiled by Sirelkhatim et al. (2015), the major ability of ZnO NPs as an antibacterial agent is due to the generation of reactive oxygen species (ROS), which cause cell death by atrophying metabolic processes of bacteria or damage on the cell membrane structure. In our experiments, the
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Fig. 5. Transmission electron microscopy (TEM) imaging of activated sludge after treatment of raw wastewater (RWW) with different concentration of ZnO nanoparticles (NPs): A) 450 mg/L, B) 900 mg/L, C) 2000 mg/L, and after treatment of filtered wastewater (FWW) in presence of ZnO NPs: D) 450 mg/L, E) 900 mg/L and F) 2000 mg/L.
Fig. 6. Activated sludge exposed to 2000 mg/L of ZnO NPs in RWW. A) Conventional transmission electron microscopy (TEM) imaging and B) Elemental mapping (ZneO) using high angle annular dark field (HAADF) detector in scanning transmission electron microscopy (STEM) mode.
physiochemical characteristics of real wastewater may promote the toxicity of ZnO NPs in microorganisms involved in activated sludge reactors, which may repercussions in biological nutrient removal and flocs characteristics. 4. Conclusions In this work the nutrient removal and the flocs characteristics of the activated sludge exposed to ZnO NPs by using RWW and FWW was evaluated. The presence of more than 450 mg/L of ZnO NPs in RWW and FWW inhibited the oxygen uptake by microorganisms in
the activated sludge process. The organic matter removal decreased in the presence of 450 and 900 mg/L of ZnO NPs for RWW and only for 450 mg/L of ZnO for FWW. The study of the nitrification process indicated that microorganisms did not efficiently develop ammonia oxidation when FWW with more than 450 mg/L of ZnO NPs was used. The orthophosphate removal in activated sludge was improved in the presence of concentrations higher than 450 mg/L of ZnO NPs in RWW and FWW. The settling rate of activated sludge was enhanced by the presence of ZnO NPs in both wastewater types. The concentration of ZnO NPs altered the flocs size of activated sludge of opposite way for two types of wastewater,
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decreasing for RWW and increasing in FWW. The negative effects of ZnO NPs on the activated sludge process using real wastewater are related to the formation of heteroaggregates between suspended solids and NPs, the attachment of NPs to the cell membrane and their internalization. Thereby, wastewater characteristics, such as suspended solids, type of substrate and concentrations of ZnO NPs have a direct influence in flocs characteristics and nutrient removal by the activated sludge process. Acknowledgements This work was supported by the National Council of Science and Technology of Mexico (CONACYT; 175089, 2013), the National Council for Scientific and Technological Development of Brazil (CNPq; 44, 2012), Research Direction and Support to Postgraduate of the University of Guanajuato (DAIP; 599, 2015), and CONACYT through Ph.D. Scholarship (No. 359919). The authors would like to thank Lourdes Palma for technical support in the Microscopy Unit rez for at the Neurobiology Institute e UNAM, to Eng. Eneidy Pe technical support and the Mexican Center of Renewable Energies S.C. supported by the University of Guanajuato, Guanajuato State Government and Salamanca municipality (RENIECYT: 2011/15369). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.chemosphere.2017.05.074. References APHA, 2005. Standard Methods for the Examination of Water and Wastewater, 21th ed. American Public Health Association Inc., Washington, DC, ISBN 0875530478. s, P., Brito, E.M., Duran, R., Martínez, A.B., Cuevas-Rodríguez, G., Cervantes-Avile 2016. Effect of ZnO nanoparticles in the oxygen uptake during aerobic wastewater treatment. J. Nanopart. Res. 18, 1e12. http://dx.doi.org/10.1007/s11051016-3481-3. Chaúque, E.F.C., Zvimba, J.N., Ngila, J.C., Musee, N., 2014. Stability studies of commercial ZnO engineered nanoparticles in domestic wastewater. Phys. Chem. Earth Pt. A/B/C 67, 140e144. http://dx.doi.org/10.1016/j.pce.2013.09.011. Dale, A.L., Lowry, G.V., Casman, E.A., 2015. Stream dynamics and chemical transformations control the environmental fate of silver and zinc oxide nanoparticles in a watershed-scale model. Environ. Sci. Technol. 49, 7285e7293. http:// dx.doi.org/10.1021/acs.est.5b01205. mez-Rivera, F., Field, J.A., Brown, D., Sierra-Alvarez, R., 2012. Fate of cerium diGo oxide (CeO2) nanoparticles in municipal wastewater during activated sludge treatment. Bioresour. Technol. 108, 300e304. http://dx.doi.org/10.1016/ j.biortech.2011.12.113. Gottschalk, F., Sun, T., Nowack, B., 2013. Environmental concentrations of engineered nanomaterials: review of modeling and analytical studies. Environ. Pollut. 181, 287e300. http://dx.doi.org/10.1016/j.envpol.2013.06.003. He, Q., Yuan, Z., Zhang, J., Zhang, S., Zhang, W., Zou, Z., Wang, H., 2017. Insight into the impact of ZnO nanoparticles on aerobic granular sludge under shock loading. Chemosphere 173, 411e416. http://dx.doi.org/10.1016/j.chemosphere.2017.01.085. Hou, J., Miao, L., Wang, C., Wang, P., Ao, Y., Lv, B., 2015. Effect of CuO nanoparticles on the production and composition of extracellular polymeric substances and physicochemical stability of activated sludge flocs. Bioresour. Technol. 176, 65e70. http://dx.doi.org/10.1016/j.biortech.2014. Hu, Z., Lu, X., Sun, P., Hu, Z., Wang, R., Lou, C., Han, J., 2017. Understanding the performance of microbial community induced by ZnO nanoparticles in enhanced biological phosphorus removal system and its recoverability. Bioresour. Technol. 225, 279e285. http://dx.doi.org/10.1016/j.biortech.2016.11.080. Keller, A., Wang, H., Zhou, D., Lenihan, H.S., Cherr, G., Cardinale, B.J., Miller, R., Ji, Z., 2010. Stability and aggregation of metal oxide nanoparticles in natural aqueous matrices. Environ. Sci. Technol. 44, 1962e1967. http://dx.doi.org/10.1021/ es902987d. Keller, A.A., McFerran, S., Lazareva, A., Suh, S., 2013. Global life cycle releases of engineered nanomaterials. J. Nanopart. Res. 15, 1e17. http://dx.doi.org/10.1007/ s11051-013-1692-4. Kiser, M.A., Ryu, H., Jang, H., Hristovski, K., Westerhoff, P., 2010. Biosorption of nanoparticles to heterotrophic wastewater biomass. Water Res. 44, 4105e4114. http://dx.doi.org/10.1016/j.watres.2010.05.036. Kumar, A., Pandey, A.K., Singh, S.S., Shanker, R., Dhawan, A., 2011. Cellular uptake and mutagenic potential of metal oxide nanoparticles in bacterial cells. Chemosphere 83, 1124e1132. http://dx.doi.org/10.1016/j.chemosphere.2011.01.025.
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