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Article

Effect of Inorganic Coagulant and Dissolved Organic Matter on the Toxicity of Nano-Zinc Oxide to Phosphorus-Accumulating Organisms in Wastewater

by
Sen Qu
1,
Wen Zhao
1,
Yushu Wang
1,
Yuan Zhang
1,
Jinyi Liu
1 and
Yongkui Yang
1,2,*
1
School of Environmental Science and Engineering, Tianjin University, Tianjin 300350, China
2
China-Singapore Joint Center for Sustainable Water Management, Tianjin University, Tianjin 300350, China
*
Author to whom correspondence should be addressed.
Water 2025, 17(11), 1563; https://doi.org/10.3390/w17111563
Submission received: 3 April 2025 / Revised: 17 May 2025 / Accepted: 21 May 2025 / Published: 22 May 2025
(This article belongs to the Special Issue Green and Low Carbon Development of Water Treatment Technology, 2nd Edition)
Browse Figures
Versions Notes

Abstract

:
In this study, we aimed to investigate the effects of coagulants and dissolved organic matter (DOM) on the biological toxicity of nano-zinc oxide (nZnO) to key microorganisms involved in biological phosphorus removal during sewage treatment. Polyaluminum chloride and polyferric chloride were selected as coagulants, whereas fulvic acid, glucose, and aspartic acid represented the DOM. The mechanisms through which these chemicals influence nZnO toxicity were also investigated. The results show that polyaluminum chloride and polyferric chloride effectively reduced nZnO toxicity in phosphorus-accumulating organisms, demonstrating their detoxification effects. Similarly, fulvic acid and glucose mitigated nZnO toxicity, whereas aspartic acid displayed dual effects: detoxification at low concentrations and enhanced toxicity at high concentrations. These findings highlight the dual role of sewage treatment additives in enhancing traditional pollutant removal and mitigating the nanoparticle-induced inhibition of microbial biochemical processes. This study clarified the interactions between coagulant chemicals, DOM, and nanoparticles in sewage treatment, offering insights into the regulatory mechanisms that improve treatment efficacy and reduce ecological risks.

1. Introduction

Nano-zinc oxide (nZnO) has attracted considerable attention worldwide as an emerging contaminant. The global annual production of nZnO exceeded 5500 tons in 2020, ranking third among metal nanoparticles [1,2,3]. Owing to its large surface area and enhanced redox capacity, nZnO is widely used in industrial and everyday products, including coatings, skin creams, catalysts, semiconductors, pigments, and medicines [1,4,5]. Therefore, nZnO is inevitably released into surface water, municipal sewage, and industrial wastewater. In Europe and the United States, the concentrations of nZnO detected in surface water and effluent from sewage treatment plants have ranged from 0.1 to 0.3 mg/L [6,7]. Recent studies have reported that nZnO in wastewater treatment plants can reach concentrations in the mg/L range [8,9]. Moreover, the United States Environmental Protection Agency’s Targeted National Sewage Sludge Survey statistical report identified a level of 8.5 mg/g in waste-activated sludge [10].
The widespread use of nZnO has resulted in its release into the environment, raising concerns regarding its ecological toxicity [11]. Previous studies have determined the toxicological effects of nZnO on organisms that inhabit freshwater and marine environments, including algae, crustaceans, fish, and aquatic bacteria [12]. Exposure to nZnO at a ng/L concentration poses a potential risk to aquatic fungi, eventually damaging their freshwater functions [13].
Phosphorus is an essential nutrient that provides organisms with energy [14]. However, excessive phosphorus in water bodies contributes to eutrophication, leading to low-quality aquatic environments and adverse effects on human life. Therefore, efficient phosphorus removal approaches are crucial for sewage treatment systems. Enhanced biological phosphorus removal (EBPR) utilizes the unique capability of phosphorus-accumulating organisms (PAOs). Under anaerobic condition, EBPR relies on PAOs that release phosphate while taking up volatile fatty acids and storing them as polyhydroxyalkanoate. In aerobic conditions, PAOs utilize polyhydroxyalkanoate as an energy source to absorb and removal excess phosphate [15,16,17].
In EBPR systems, chemical coagulants such as polyaluminum chloride (PAC) and polyferric chloride (PFC) are broadly employed to remove suspended solids from wastewater and improve the quality of the effluent. Among these, cationic polyacrylamide could reduce the toxicity of nZnO by decreasing the production of reactive oxygen species [15]. This indicates that coagulants could simultaneously reduce nZnO toxicity and enhance phosphorus removal. Nevertheless, few studies have investigated how coagulants affect the toxicity of nZnO to PAOs, which in turn affect biological phosphorus removal. It remains uncertain whether coagulants promote or suppress the effect of nZnO on biological phosphorus removal systems, thus necessitating further investigation.
Dissolved organic matter (DOM) is one of the most ubiquitous substances in wastewater, constituting 78.1–86.5% of the total chemical oxygen demand in municipal wastewater [18]. As a highly heterogeneous mixture, DOM primarily comprises humic acids, amino acids, proteins, and polysaccharides. Owing to its multiple functional groups, studies on how DOM affects the toxicity of metal nanomaterials have been conducted [19,20]. In particular, DOM can alleviate particle toxicity through various interactions [21,22], including adsorption, dissolution, aggregation, and transformation [23,24]. For instance, humic and fulvic acids have been documented to diminish the damage induced by nZnO to the antioxidant system of Daphnia magna by adsorbing onto its surface [25]. In contrast, DOM has been observed to increase the toxicity of nZnO [20]. Nevertheless, there is a lack of research concerning the impact of DOM on the toxicity of nZnO to PAOs.
Therefore, in this study, we conducted the first attempt to investigate the effects of widely used coagulants and coexisting DOM on the toxicity of nZnO to PAOs. Specifically, we aimed to analyze the changes in the ability of PAOs to remove phosphate in the presence of nZnO exposed to coagulants (PAC and PFC) or DOM (humic substances, glucose, and amino acids) in simulated anaerobic/aerobic wastewater treatment reactors. Furthermore, we aimed to investigate the detoxifying effects of coagulants and DOM on nZnO during biological phosphorus removal. Finally, we aimed to explore the detoxification mechanisms of the coagulants and DOM at the molecular level. To this end, we analyzed the products of the complex system using UV full-wavelength scanning, x-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). This study fills a critical gap in the understanding of nanoparticle–additive interactions in wastewater systems by integrating molecular-level interactions and functional group contributions, thereby promoting practical EBPR process optimization. The findings of this study serve as a valuable reference for mitigating the impact of nZnO on phosphorus removal through optimized dosing parameter adjustments.

2. Materials and Methods

2.1. nZnO, Coagulants, and Other Chemicals

A 300 mg nZnO suspension (90 ± 10 nm) (98%) was added to 300 mL of water and then prepared via sonication (97.5 w) using an ultrasonic JY92-IIN homogenizer (Scientz, Ningbo, Zhejiang, China) for 0.5 h at 25 °C. The frequency of sonication was 10 s every 40 s at 20 Hz. The average particle size was 115.5 nm with a zeta potential of 17.6 mV, measured using a Malvern Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) at a fixed pH of 7.0. The main chemical coagulants used were PAC and PFC from Shanghai Merrier Biochemical Technology Co., Ltd. (Shanghai, China) and Tianjin Guangfu Science and Technology Development Co., Ltd. (Tianjin, China), respectively. Fulvic acid, glucose, and aspartic acid were purchased from Nantong Feiyu Biotechnology Co., Ltd.; Tianjin Yuanli Chemical Co., Ltd. (Tianjin, China); and Shanghai Merrier Biochemical Technology Co., Ltd., respectively. Experiments were conducted using solutions prepared with ultra-pure water (18 MΩ resistance; Elga LabWater, High Wycombe, UK).

2.2. Activation and Culture of Phosphorus-Accumulating Organisms

The PAOs used in this study were isolated using an EBPR system and were identified as Shewanella sp. in a previous study [26]. The Luria–Bertani (LB) medium (1 L) contained 10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, and 15 g of agar. The components of the synthetic wastewater were as follows (in 100 mL): 1.3992 g of CH3COONa, 183.4 mg of NH4Cl, 70.3 mg of KH2PO4, 26.4 mg of CaCl2·2H2O, 84.0 mg of MgCl2, and 0.36 mL of micro elements, with a PO43− concentration of 10 mg/L. The microelements (100 mL) in the water are listed in Table S1. The Shewanella strain was activated and cultivated in 100 mL of LB medium in a shaking incubator at 30 °C and 140 rpm for 18 h.

2.3. Toxicity Exposure Experiment

For the toxicity experiment, the amount of biomass was maintained at an optical density (OD600) of 1.1–1.2 measured using a HACH DR6000 spectrophotometer (Loveland, CO, USA). Synthetic wastewater (5 mL), a bacterial solution (12 mL), a certain volume of coagulant, DOM, and the nZnO suspension were added to a flask to a final volume of 120 mL. Each cycle was initiated with 2 h of anaerobic treatment preceding 4 h of aeration while maintaining the operational logic of the EBPR. During the first anaerobic phase, pure nitrogen gas was bubbled through the conical flasks. In the subsequent aerobic phase, the flasks were placed in a constant temperature shaker at 180 rpm and 30 °C for 4 h. The PO43− concentration in the supernatant was analyzed.
The toxic effects were quantified based on the inhibition rate of PO43− removal by the PAOs as follows:
Inhibition   Rate   ( % ) = R c o n t − R e x p / R c o n t × 100 %
where Rcont represents the removal rate of PO43− without the addition of nZnO, coagulants, or DOM, and Rexp represents the removal rate of PO43− after exposure to different concentrations of nZnO, coagulant, or DOM.
The detoxification (antagonistic) effects of different additives on the PO43− removal rate were determined as follows:
Detoxification (%) = Isum − Icom
where Isum is the PO43− removal inhibition rate based on the summed inhibition of individual nZnO and coagulant or DOM, and Icom is the PO43− removal inhibition rate based on the combined inhibition of nZnO and coagulant or DOM.

2.4. Analysis

The PO43− concentration in the wastewater was analyzed using the standard Mo–Sb colorimetric method employing a Hach DR6000 UV spectrophotometer (Loveland, CO, USA) at a wavelength of 700 nm. The UV–Vis spectra of the aqueous samples of the nZnO–coagulant and nZnO–DOM treatments were analyzed using a Hach DR6000 UV spectrophotometer (Loveland, CO, USA) at 1 nm/s in the 190–900 nm range. The coagulant/DOM concentration was chosen to reflect both the experimental occurrence and sensitivity of the analytical method for fully investigating the mechanism of the interaction between nZnO and coagulants/DOM. Solid samples were prepared through freeze-drying (FD-1C-50; Biocool, Beijing, China). Additionally, XRD (D8-ADVANCE Bruker, Billerica, MA, USA) was used to determine the crystalline structures of PAC–nZnO composites with Cu-Ka radiation (40 kV, 150 mA) within 10–80° in 2θ with samples prepared through freeze-drying the reaction products prior to measurement. The functional groups of the compounds in the freeze-dried DOM (fluvic acid/glucose/aspartic acid)–nZnO composites were identified using FTIR (IRAffinity-1S, Shimadzu Corporation, Kyoto, Japan) using samples prepared via the KBr pellet method after freeze-drying the reaction products.

3. Results

3.1. Effects of Individual nZnO, Coagulant, and DOM on PO43− Removal by PAOs

A comparison of phosphorus removal efficiencies among different biological phosphorus removal systems is presented in Table 1. The EBPR system demonstrates a moderate level of phosphorus removal efficiency. As shown in Figure 1, different individual concentrations of nZnO inhibited PO43− removal by PAOs. Initially, no distinct changes are observed at nZnO concentrations of 0.5 and 1.0 mg/L. As the nZnO concentration increases to 1.5 mg/L, the PO43− removal efficiency of the PAOs decreases significantly by 42.9% compared with that in the absence of nZnO. The inhibition of PO43− removal by nZnO rises nearly sixfold (from 7.7% to 42.9%) as the nZnO concentration increases from 0.5 to 1.5 mg/L. The Zn2+ released from nZnO and the reactive oxygen species induced by nZnO may be the main contributors to the observed toxicity [27]. At a concentration of 2.5 mg/L nZnO, phosphorus removal by PAOs is entirely suppressed, resulting in a 17.2% increase in the phosphorus concentration. Exposure to toxic nanomaterials typically induces intracellular oxidative stress, which damages the cell membrane and may even lead to cell death, promoting the excessive release of phosphorus [28,29]. The toxicity of nZnO in wastewater systems is mediated by its ionic strength and speciation [23]. Elevated ionic strength can induce nanomaterial aggregation, reducing the reactive surface area, while simultaneously enhancing Zn2+ dissolution through chloride complexation. This in turn increases bioavailability and oxidative stress in microorganisms [1,27,30]. Similarly, for 10 mg/L of nZnO, the aerobic phosphorus uptake (72.9 mg/L) is lower than the anaerobic release (73.2 mg/L), resulting in no net phosphorus removal [27]. However, in the sequencing batch reactor system, the presence of nZnO decreases phosphorous removal by 8.2% when exposed to 60 mg/L of nZnO, which shows weaker efficiency in phosphorus removal [1,30]. This discrepancy in toxicity may have arisen from the differential responses of individual bacterial strains and microbial consortia in activated sludge systems. The small size and high specific surface area of nZnO facilitate its penetration through the cell walls of single bacteria [31], whereas the protective effect of extracellular polymeric substances can mitigate the toxicity of nZnO to the microbial community in sludge.

3.2. Effects of Coagulants on Inhibition of PO43− Removal by PAOs Due to nZnO Toxicity

We found that PAC had significant detoxifying effects on the inhibition of PO43− removal due to nZnO toxicity, particularly for nZnO at higher concentrations (Figure 2a). When nZnO was applied at 1.25 mg/L, PAC concentrations ranging from 3 to 50 mg/L had no significant effect on PO43− removal (p > 0.05). At 1.80 mg/L of nZnO, the detoxification of the inhibition of PO43− removal significantly increased from 6.2% to 51.3% as the PAC concentration increased from 3 to 50 mg/L. Detoxification further increased at 2.50 mg/L nZnO. At a PAC concentration of 50 mg/L, detoxification reached 95.5%. Simultaneously, PO43− removal only reached 11.2% in the presence of individual PAC (50 mg/L) (Figure S1). In a previous study, PAC was used to effectively remove nTiO2 from water, thus reducing the toxicity of the metal nanoparticles [42]. At a coagulant concentration of 25 mg/L, the nCuO removal efficiency of PAC-CA was 88.8% [43]. Notably, PFC exhibited a similar detoxifying effect on the inhibition of PO43− removal (Figure 2b). Researchers have reported that Fe coagulants can bind to and chelate heavy metal ions [44,45]. As the Fe3+/Cu2+ molar ratio increased to 8:1, the removal rate of Cu2+ increased to 66%, thereby attenuating the biotoxicity of the nanoparticles to organisms [46].
The detoxification effect of the coagulant may be due to the direct removal of nZnO through the addition of the coagulant, which inhibits the dissolution of nano-oxides, thereby reducing the toxicity of nZnO. The hydroxide colloids formed through the hydrolysis of PAC/PFC have a large specific surface area and surface charge, which enables the adsorption of nanoparticles [43]. This process promotes the coalescence of nZnO into larger flocs, thereby facilitating their subsequent removal [47]. Studies have reported that in traditional water treatment during coagulation and sedimentation processes, the efficiency of ferric chloride in removing nanosilver from raw water is 92% [48].

3.3. Effects of DOM on Inhibition of PO43− Removal by PAOs Due to nZnO Toxicity

Figure 3a shows the detoxifying effect of fulvic acid on nZnO for PO43− removal. With an increase in the nZnO concentration from 1.80 to 2.50 mg/L, fulvic acid generally exhibits a significant detoxifying effect on PO43− removal. The adsorption of negatively charged DOM components onto nZnO can shift its zeta potential from positive to negative, destabilizing colloids via aggregation and thereby mitigating toxicity [25,49]. As the concentration of humic acid increases from 0 to 10 mg/L, the zeta potential of the flocs formed by PAC and copper oxide nanoparticles decreases from 1.85 to −2.87 mV, which is conducive to the removal of nanometal oxides [43]. At 1.80 mg/L of nZnO, the highest detoxification rate reaches 31.7% at a fulvic acid concentration of 8 mg/L, whereas at 2.50 mg/L of nZnO, the detoxification rate reaches 89.4% at a fulvic acid concentration of 5 mg/L. Higher nZnO concentrations have a stronger inhibitory effect on phosphorus removal by the PAOs, thus presenting a more significant DOM detoxification effect. Fulvic acid decreases the antibacterial activity of nZnO against Bacillus subtilis in freshwater, which may be due to the formation of coordination compounds between Zn2+ and fulvic acid [50]. Overall, DOM promotes bacterial metabolism, thereby weakening the inhibition of phosphorus removal by PAOs due to nZnO toxicity [51,52,53] and alleviating the phosphorus toxicity of nZnO. The optimal concentrations of fluvic acid, glucose, and aspartic acid are 5, 100 and 20 mg/L, respectively. However, dynamic adjustments are necessary, depending on the DOM composition and nZnO concentration.
Glucose exhibited a similar effect on detoxification (Figure 3b). However, its detoxification capacity was significantly lower than that of fulvic acid. At a nZnO concentration of 1.80 mg/L, the highest detoxification rate was 22.3% at a glucose concentration of 100 mg/L. When the nZnO concentration reached 2.50 mg/L, a detoxification rate of 37.7% was observed at a glucose concentration of 100 mg/L, indicating that adding glucose at an appropriate concentration can reduce the biological toxicity of nZnO in real wastewater treatment. Notably, DOM, including proteins and polysaccharides, contains functional groups such as carbonyl, carboxyl, amino, and sulfhydryl groups [54,55,56]. Monosaccharides can participate in redox reactions with metal ions, thereby altering the toxic potential of other chemicals, such as nanoparticles [57,58,59], which may explain why glucose mitigates the toxicity of nZnO.
Aspartic acid had a similar effect on detoxification (Figure 3c). However, its detoxification capacity was significantly lower than those of fulvic acid and glucose. At 2.50 mg/L of nZnO, detoxification of up to 11.2% was observed for 20 mg/L of aspartic acid. However, as the aspartic acid concentration increased to 50 and 100 mg/L, the detoxification rate decreased significantly, followed by a further increase in the toxicity of nZnO for PO43− removal. In the EBPR system, as the substrate for anaerobic digestion was converted from casein hydrolysate to a mixture of amino acids, the phosphorus removal rate increased from 50% to 95%, indicating that the addition of amino acids significantly enhanced phosphorus removal [60]. This may be attributable to a lower phosphorus release/carbon uptake ratio with aspartic acid (0.24–0.29 mol P/mol C) [61]. Less energy consumption in the anaerobic phase results in extra carbon being available for phosphate uptake in the aerobic phase, thereby enhancing the phosphate removal efficiency [61,62]. However, the uptake of amino acids by PAOs during anaerobic conditions may produce NH4+-N through hydrolysis and the subsequent accumulation of NO2−-N during nitrification [60]. The increase in aspartic acid concentration may contribute to higher NO2-N accumulation, which has been demonstrated to inhibit the viability of PAOs [63], thus affecting their phosphorus removal capacity. This may explain why the detoxification efficacy of aspartic acid was compromised rather than enhanced with increasing amino acid concentrations.

3.4. Complex Toxicity Mechanism

3.4.1. Compound Reaction Identification

(1)
PAC and PFC
As shown in Figure 4a, for PAC (50 mg/L) and nZnO (10 mg/L), the difference between the UV absorbance of the nZnO–PAC composite and the sum of the absorbances of individual PAC and nZnO samples was not significant. At 500 mg/L of PAC (Figure 4b), the UV absorption of the nZnO–PAC composite was similar to that of individual PAC. At 50 mg/L of nZnO (Figure 4c), the characteristic peak wavelength (376 nm) of the nZnO–PAC composite shifted by 4 nm compared with the peak of the individual nZnO system. The absorbance of the nZnO–PAC composite increased by 35% compared with the sum of the absorbances of individual PAC and nZnO samples. With increasing concentrations of PAC (50 mg/L) and nZnO (500 mg/L) (Figure 4d), the UV characteristic peak of the nZnO–PAC composite nearly disappeared compared with that of nZnO, indicating a significant reaction. The observed changes in UV absorbance and peak shifts across different nZnO–PAC concentration ratios can be attributed to concentration-dependent interactions between PAC and nZnO, which altered the physicochemical properties and aggregation states of the composite system [27,45].
As shown in Figure 4e, compared with the UV–Vis spectra of individual PFC (50 mg/L) and nZnO (10 mg/L) samples, the UV absorption peaks and troughs of the nZnO–PFC composite at 302 and 260 nm disappeared. A similar phenomenon was observed when the PFC concentration was increased to 150 mg/L (Figure 4f). At 50 mg/L of nZnO (Figure 4g), the characteristic absorbances of the composite at 260 and 376 nm were higher than the sum of the absorbances of individual PFC and nZnO samples, increasing by 42% and 100%, respectively (Figure 4g). With PFC and nZnO doses of 150 mg/L and 50 mg/L, respectively (Figure 4h), the UV absorbance of the nZnO–PFC composite was significantly lower than the sum of the absorbances of individual PFC and nZnO samples, indicating a strong reaction between PFC and nZnO.
(2)
Fulvic acid, glucose, and aspartic acid
As shown in Figure 5a, at 5 mg/L of fulvic acid and 10 mg/L of nZnO, the UV absorbance of the nZnO–fulvic acid composite at 376 nm shifted by 4 nm and increased more than twice compared with the absorbance of individual nZnO. When the fulvic acid concentration was increased to 20 mg/L (Figure 5b), the absorbance of the composite approached the sum of the absorbances of the individual compounds. At 50 mg/L of nZnO (Figure 5c), compared with fulvic acid alone, there was a 78% increase in the absorbance of the composite at 376 nm. At 20 mg/L of fulvic acid and 50 mg/L of nZnO (Figure 5d), the UV absorbance of the composite was significantly higher than those of individual compounds. These findings demonstrate that humic acid chemically reacts with nZnO. Similarly, in another study, humic acid was shown to mitigate the toxicity of Cu2+ and Mn2+ to bacterial cells through interaction mechanisms [64].
As shown in Figure 5e, at 1000 mg/L of glucose and 10 mg/L of nZnO, the UV absorbance of the glucose and nZnO composite was significantly higher than those of the individual compounds (174%). A further increase was observed at 2000 mg/L of glucose (Figure 5f). At 50 mg/L of nZnO (Figure 5g,h), the absorbance of the composite was lower than the sum of the absorbances of the individual compounds. Previous studies have shown that glucose can chelate Zn2+, shifting the UV absorption peak of the composite product. This indicates that glucose reacts with nZnO in a concentration-dependent manner.
As shown in Figure 5i,j, when aspartic acid concentrations of 5–200 mg/L and 10 mg/L of nZnO were used, the characteristic peak of the nZnO–aspartic acid composite at 376 nm was lower than the sum of the absorbances of the individual compounds. At 50 mg/L of nZnO (Figure 5k), the UV absorbance of the composite at 376 nm originated mainly from a single nZnO molecule. For 5 mg/L aspartic acid and 10 mg/L nZnO (Figure 5l), the absorption of the nZnO–aspartic acid composite was generally the same as that of aspartic acid. At higher aspartic acid/nZnO content ratios, the UV absorbance of the nZnO–aspartic acid composite decreased compared with those of the individual pollutants. This indicated a strong reaction between aspartic acid and nZnO.

3.4.2. Crystal Structure Analysis

Figure 6 shows the XRD spectra of the individual nZnO and nZnO–PAC composites. The characteristic peaks of nZnO appeared at 31.74°, 34.34°, 36.17°, 47.39°, 56.51°, 62.82°, 67.84°, and 69.04°, with no impurity peaks, which is consistent with the standard spectrum. The XRD spectrum of the nZnO–PAC composite exhibited characteristic peaks of nZnO at 31.96°, 34.67°, and 36.39°. The characteristic peaks of AlOCl appeared at 11.47°, 23.08°, and 60.36°, indicating the presence of PAC. New Zn(OH)2 characteristic peaks appeared at 39.14° and 46.40°, which can be attributed to hydrolysis via Zn2+ release from nZnO, as well as the reaction between PAC and nZnO. Under acidic conditions, the products of PAC hydrolysis, such as Al(OH)2+, Al(OH)2+, Al2(OH)24+, and Al3(OH)45+, may react with positively charged humic acid, suggesting that a similar binding mechanism may exist between PAC and Zn2+ [65]. Meanwhile, nCuO reacts with PAC and chitosan, resulting in changes in its crystal structure [66]. Therefore, the XRD spectra of nZnO exhibited changes in the chemical components and crystal structure with the addition of PAC, which may be associated with the antagonistic effect of the composite on the inhibition of phosphorus removal. Some studies have reported the removal of coexisting metal-based engineered nanoparticles (TiO2, Ag, and CuO) in natural water using PAC [66]. This may have been caused by a reduction in electrostatic repulsion between PAC and the nanometals, which promoted their precipitation, leading to the production of Zn(ClO4)2 [67]. Similarly, nTiO2 was reduced by 84.8% when 25 mg/L of polyaluminum ferric chloride was added [68]. Coagulants containing positively charged ions, such as aluminum and iron, can enhance ionization within the reaction system, thereby improving coagulation efficiency, including adsorption, charge neutralization, and interparticle bridging [69].

3.4.3. Functional Group Analysis

In the FTIR spectrum of fulvic acid (Figure 7a), the peak at 3381 cm−1 corresponds to the O-H stretching vibration, whereas the peak at 2958 cm−1 indicates the stretching vibration of carbon bonds in aromatic rings. The absorption peak at 1604 cm−1 is ascribed to the stretching of the carbonyl C=O bond, whereas the peak at 1406 cm−1 is associated with the stretching vibration of the benzene ring skeleton of the C-H bond [70,71]. The 1207 cm−1 and 1045 cm−1 peaks are related to the stretching of C-O bonds [70]. In the range of 900–600 cm−1, the peak arises due to the substitution of a benzene ring at 638 cm−1. Comparing the infrared spectrum of fulvic acid with that of the nZnO–fulvic acid composite shows a new absorption peak at 3142 cm−1, which may be associated with the formation of a coordination compound resulting from the reaction between Zn2+ and the hydroxyl group of phenols [71,72]. The typical peak at 3142 cm−1 of the composite can be ascribed to the stretching vibration of the O-H bond, indicating that Zn2+ complexes interact with hydroxyl O-H. Furthermore, nZnO releases Zn2+, adsorbs fulvic acid through electrostatic interactions, and coordinates with the carboxyl (–COOH) and hydroxyl (–OH) groups of aliphatic carbons and phenols [73,74]. In addition, the oxygen-containing functional groups of fulvic acid can specifically bind to Zn2+ released from nZnO [71,72,75].
As shown in Figure 7b the FTIR spectrum of glucose shows four characteristic absorption peaks at 1144, 1103, 1057, and 1020 cm−1. The peak at 1020 cm−1 is assigned to the resonance absorption ascribed to the vibration of the C-H bond. The other three absorption peaks occurred because of the stretching of the C-O bond and the bridge ring structure of glucose. In the FTIR spectrum of the glucose–nZnO composite, the absorption intensity at 2180 cm−1 increased significantly because of the binding reaction of the hydroxyl group from glucose and Zn2+ from nZnO. The peak for the carbonyl group (C=O) at 1659 cm−1 shifted to a lower wavenumber of 1636 cm−1, suggesting that Zn2+ was bound to the carbonyl group, leading to a peak shift. Glucose can form complexes with heavy metal ions (Cd2+, Pb2+, and Zn2+) in wastewater [76,77].
Figure 7c shows the FTIR spectra of aspartic acid and its complex with nZnO. In the infrared spectra of a single aspartic acid molecule, the absorption peak at 3015 cm−1 can be attributed to the N-H stretching vibration of the amino functional group. Peaks assigned to the N-H bending vibration are at 1609 cm−1 and 1512 cm−1 [71,78,79]. An absorption peak at 1134 cm−1 is observed for the C-O-C stretching vibration. There is a characteristic peak for C=O at 1678 cm−1 [71,79,80]. At 1414 cm−1 and 1308 cm−1, the two peaks derived from the carboxylate anion (-COO-) indicate that the carboxyl group in the molecule becomes an anion in the form of an inner salt. The absorption peak of the -COO- shifts slightly from 1308 cm−1 to 1317 cm−1, suggesting that the carboxyl group and Zn2+ undergo a complexation reaction. Carboxyl groups enable ion exchange and covalent chelation with cationic heavy metals [81].

4. Conclusions

In this study, we examined the interactions and regulatory effects of coagulants and DOM on the toxicity of nZnO to phosphorus-accumulating organism during phosphorus removal in sewage treatment. We found that PAC and PFC effectively reduced nZnO toxicity and achieved maximum detoxification efficiencies of 95.5% and 89.3%, respectively, at a concentration of 50 mg/L. Furthermore, UV spectroscopy revealed significant synergistic interactions between PAC and nZnO, with higher absorbance when together than individually, as corroborated by XRD analysis. For the nZnO–fulvic acid composite, UV spectroscopy and FTIR confirmed that fulvic acid interacted with Zn2+ released from nZnO through the carboxyl functional groups, achieving 89.4% detoxification at 2.5 mg/L of nZnO. The UV absorbance of the nZnO–PAC composite increased by 35% compared with the sum of the absorbances of individual PAC and nZnO samples. Glucose exhibited stronger detoxification at high nZnO concentrations, with significant coordination at the carbonyl groups of phenol. Aspartic acid showed 11.2% detoxification at 20 mg/L when exposed to 2.50 mg/L of nZnO but increased toxicity by 20.9% at a high concentration of 100 mg/L, highlighting its concentration-dependent effects. Overall, coagulants and DOM significantly reduced nZnO toxicity, with coagulants forming stable complexes and DOM reducing free Zn2+ via coordination reactions. These mechanisms reduced the inhibition of phosphorus removal by phosphorus-accumulating organism upon exposure to nZnO. Fulvic acid and glucose resulted in consistent detoxification, whereas the effect of aspartic acid varied. These findings deepen the understanding of coagulant/DOM–nZnO interactions and their environmental implications, suggesting the need for further research on their behavior in wastewater treatment systems and their potential regulation. Further studies are required to elucidate the molecular detoxification mechanisms of coagulants and DOM influencing the toxicity of nZnO to PAOs by employing advanced technologies, such as high-throughput sequencing.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/w17111563/s1, Figure S1: Effects of individual PAC (a)and PFC (b) on P removal in sewage; Table S1: Micro elements of the synthetic wastewater (100 mL).

Author Contributions

Investigation, methodology, writing original draft preparation, S.Q.; investigation, writing original draft preparation, W.Z.; formal analysis, Y.W.; validation, Y.Z.; investigation, J.L.; writing—review and editing, supervision, project administration, Y.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the National Key Research and Development Program of China (2024YFC3908500).

Data Availability Statement

Data available on request due to restrictions eg privacy or ethical.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. PO43− removal by PAOs under different concentrations of individual nZnO.
Figure 1. PO43− removal by PAOs under different concentrations of individual nZnO.
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Figure 2. Detoxification effects of PAC (a) and PFC (b) on nZnO inhibition of PO43− removal by PAOs.
Figure 2. Detoxification effects of PAC (a) and PFC (b) on nZnO inhibition of PO43− removal by PAOs.
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Figure 3. Detoxification effects of fulvic acid (a), glucose (b), and aspartic acid (c) on nZnO inhibition of PO43− removal by PAOs.
Figure 3. Detoxification effects of fulvic acid (a), glucose (b), and aspartic acid (c) on nZnO inhibition of PO43− removal by PAOs.
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Figure 4. UV–Vis spectra of individual PAC, PFC, and nZnO samples and their composites: (a) 10 mg/L nZnO + 50 mg/L PAC; (b) 10 mg/L nZnO + 500 mg/L PAC; (c) 50 mg/L nZnO + 50 mg/L PAC; (d) 50 mg/L nZnO + 500 mg/L PAC; (e) 10 mg/L nZnO + 50 mg/L PFC; (f) 10 mg/L nZnO + 150 mg/L PFC; (g) 50 mg/L nZnO + 50 mg/L PFC; (h) 50 mg/L nZnO + 150 mg/L PFC.
Figure 4. UV–Vis spectra of individual PAC, PFC, and nZnO samples and their composites: (a) 10 mg/L nZnO + 50 mg/L PAC; (b) 10 mg/L nZnO + 500 mg/L PAC; (c) 50 mg/L nZnO + 50 mg/L PAC; (d) 50 mg/L nZnO + 500 mg/L PAC; (e) 10 mg/L nZnO + 50 mg/L PFC; (f) 10 mg/L nZnO + 150 mg/L PFC; (g) 50 mg/L nZnO + 50 mg/L PFC; (h) 50 mg/L nZnO + 150 mg/L PFC.
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Figure 5. UV–Vis absorbance of individual fulvic acid, glucose, and aspartic acid and their composites: (a) 10 mg/L nZnO + 5 mg/L fulvic acid; (b) 10 mg/L nZnO + 20 mg/L fulvic acid; (c) 50 mg/L nZnO + 5 mg/L fulvic acid; (d) 50 mg/L nZnO + 20 mg/L fulvic acid; (e) 10 mg/L nZnO + 1000 mg/L glucose; (f) 10 mg/L nZnO + 2000 mg/L glucose; (g) 50 mg/L nZnO + 1000 mg/L glucose; (h) 50 mg/L nZnO + 2000 mg/L glucose; (i) 10 mg/L nZnO + 5 mg/L aspartic acid; (j) 10 mg/L nZnO + 200 mg/L aspartic acid; (k) 50 mg/L nZnO + 5 mg/L aspartic acid; (l) 50 mg/L nZnO + 200 mg/L aspartic acid.
Figure 5. UV–Vis absorbance of individual fulvic acid, glucose, and aspartic acid and their composites: (a) 10 mg/L nZnO + 5 mg/L fulvic acid; (b) 10 mg/L nZnO + 20 mg/L fulvic acid; (c) 50 mg/L nZnO + 5 mg/L fulvic acid; (d) 50 mg/L nZnO + 20 mg/L fulvic acid; (e) 10 mg/L nZnO + 1000 mg/L glucose; (f) 10 mg/L nZnO + 2000 mg/L glucose; (g) 50 mg/L nZnO + 1000 mg/L glucose; (h) 50 mg/L nZnO + 2000 mg/L glucose; (i) 10 mg/L nZnO + 5 mg/L aspartic acid; (j) 10 mg/L nZnO + 200 mg/L aspartic acid; (k) 50 mg/L nZnO + 5 mg/L aspartic acid; (l) 50 mg/L nZnO + 200 mg/L aspartic acid.
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Figure 6. The XRD spectra of the nZnO and PAC composite.
Figure 6. The XRD spectra of the nZnO and PAC composite.
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Figure 7. The FTIR spectra of the fulvic acid (a), glucose (b), aspartic acid (c), and nZnO composite.
Figure 7. The FTIR spectra of the fulvic acid (a), glucose (b), aspartic acid (c), and nZnO composite.
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Table 1. Phosphorus removal efficiency in different biological phosphorus removal systems.
Table 1. Phosphorus removal efficiency in different biological phosphorus removal systems.
Sewage Treatment ProcessWastewater
Type		Phosphorus Concentration in the Influent (mg/L)Mixed Liquor Suspended Solid (g/L)Sludge Volume Index (mL/g)Phosphorus Removal (%)Reference
Aerobic granular sludgePractical1.6–6.58.0 <30.067[32]
Aerobic granular sludgePractical1.6–6.58.0 <30.094[32]
Anaerobic–anoxic–oxicPractical4.0--85[33]
High concentration powder carrier bio-fluidized bedPractical4.0--88[33]
Enhanced biological phosphorus removalPractical1.8–6.12.8 63.683[34]
Aerobic granular sludgePractical-3.5 <50.030–80[35]
Aerobic granular sludgePractical-8.0 -30–70[36]
Enhanced biological phosphorus removalPractical1.8–5.7--93[37]
Partial denitrification coupled anammoxArtificial2.4--91[38]
Anaerobic–anoxic–oxicPractical2.3–4.33.5-96[39]
Enhanced biological phosphorus removalPractical1.7–3.64.1-78–96[40]
Denitrifying phosphorus removalArtificial1.0 --61[41]
Enhanced biological phosphorus removalArtificial10.0 --63This study
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Qu, S.; Zhao, W.; Wang, Y.; Zhang, Y.; Liu, J.; Yang, Y. Effect of Inorganic Coagulant and Dissolved Organic Matter on the Toxicity of Nano-Zinc Oxide to Phosphorus-Accumulating Organisms in Wastewater. Water 2025, 17, 1563. https://doi.org/10.3390/w17111563

AMA Style

Qu S, Zhao W, Wang Y, Zhang Y, Liu J, Yang Y. Effect of Inorganic Coagulant and Dissolved Organic Matter on the Toxicity of Nano-Zinc Oxide to Phosphorus-Accumulating Organisms in Wastewater. Water. 2025; 17(11):1563. https://doi.org/10.3390/w17111563

Chicago/Turabian Style

Qu, Sen, Wen Zhao, Yushu Wang, Yuan Zhang, Jinyi Liu, and Yongkui Yang. 2025. "Effect of Inorganic Coagulant and Dissolved Organic Matter on the Toxicity of Nano-Zinc Oxide to Phosphorus-Accumulating Organisms in Wastewater" Water 17, no. 11: 1563. https://doi.org/10.3390/w17111563

APA Style

Qu, S., Zhao, W., Wang, Y., Zhang, Y., Liu, J., & Yang, Y. (2025). Effect of Inorganic Coagulant and Dissolved Organic Matter on the Toxicity of Nano-Zinc Oxide to Phosphorus-Accumulating Organisms in Wastewater. Water, 17(11), 1563. https://doi.org/10.3390/w17111563

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