Structure–Biodegradability Relationship of Nonylphenol Isomers in Two Soils with Long-Term Reclaimed Water Irrigation

Abstract

Nonylphenol (NP), as one of the typical endocrine disrupter chemicals (EDCs), has a high detection concentration and frequency in reclaimed water. This research focused on the degradation of NP isomers in two typical reclaimed water irrigation fields in Daxing, China, and Florida, USA. The results showed that the half-lives of NP isomer degradation in the soil of China and Florida were 2.03–8.66 d and 5.16–11.83 d, respectively. The degradation of NP isomers was structure-specific. Isomers of NP5, NP2, NP11, and NP3 had the highest degradation rates in the two soils; NP12, NP7, and NP6 were the isomers with medium degradation rates; and NP4, NP1, NP10, NP9, and NP8 had the slowest degradation rates. Steric hindrance and mean information index for the magnitude of distance (I_DWbar) were found to be the better indexes for measuring the degradation of NP isomers compared with the length of the side chain, the type of the substitute, and the molecular connectivity. This study offers insights into the characteristics of NP isomers and two reliable indicators for measuring the degradation of NP isomers, which could provide data support for the environmental fate and the health risk assessment of NP in the future.

1. Introduction

The development and utilization of reclaimed water resources has become an important way to alleviate agricultural water shortage all over the world [1]. It is estimated that in China, the amount of reclaimed water resources will reach 70 billion m³, and it is predicted that the amount of reclaimed water irrigation would reach 16.5 billion m³ by 2030 [2,3]. Reclaimed water irrigation can provide necessary nutrients for crops [4], while the organic substance, especially the emerging environmental endocrine disruptors (EDCs), in water may induce human health risks through food chain. EDCs have strong estrogen activity, which has carcinogenic, teratogenic, and mutagenic effects and could cause risks to humans by food chain accumulation [5,6].

Nonylphenol (NP) is one of typical EDCs in reclaimed water with a high detection concentration and frequency. NP is the degradation production of nonylphenol polyoxyethylene ether (NPEO_S), which is widely used as a non-ionic surfactant. The concentration of NP in sewage or reclaimed water usually varies between ND and 8 μg L⁻¹ [7,8,9,10]; the corresponding value in groundwater is generally lower than 3.85 μg L⁻¹ [8,11,12,13,14], which has mainly been caused by the landfill leachates, sewage irrigation, and septic tank leachates [15]. The concentration of NP in fertilization farmlands and sewage irrigation fields generally ranges from 0.01 to 27,882 μg kg ⁻¹ [16,17,18]. NP is a complicated mixture of varieties of isomers with a highly branched alkyl chain, and approximately 86–94% consists of 4-NP [19]. The structures of 66 4-NP isomers have been identified, 21 major components of which are α-quaternary isomers, and one of the six significant minors are α-tertiary ones [20,21]. The estrogenic effect of NP varies greatly for the isomers with different molecular structures [22]. NP has been ranked as a priority pollutant in the European Union directive 2013/39/EU [23]. There is no specific regulation on the limit of NP in reclaimed water. However, some recommended values have been provided in some research. Hao et al. [24] suggested that the standard of NP in reclaimed water should be 2 μg L⁻¹ when the proportion of supplying reclaimed water for urban lakes is smaller than 50%.

Research on NP biodegradation has often been referred to during wastewater treatment, while the biodegradation of its isomers in soils has been relatively seldom [25,26,27,28]. Lu and Gan [26] studied the degradation of NP isomers in sediments and found that the half-lives of NP isomers ranged from 0.9 to 13.2 d in aerobic condition and from 15.6 to 20.1 d in a relatively anoxic condition. The ranking of half-lives of NP isomers was NP111 > NP112 > NP38 > NP65 in sludge, and 4.01–9.48% of the ¹⁴C-NP111 was mineralized after 36 days of incubation [19]. The biodegradability of NP was related to the structures of the isomers. Ikunaga et al. [29] found the isomers with α-dimethyl and α-ethyl-α-methyl substituents had higher degradation rates (0.356 h⁻¹ and 0.554 h⁻¹, respectively), while the degradation rate of isomers with α-methyl-α-propyl isomer was much lower (0.036 h⁻¹). Das and Xia [30] studied the degradation of NP isomers in biosolid composting. The half-lives of isomers with α-methyl-α-propyl substituents and isomers with α-dimethyl substituents were 12.6 ± 0.3 d and 0.8 ± 0.1 d, respectively. Gabriel et al. [31] found the isomers with small substituents in α position, and four to six carbon atoms in the alkyl chain had a higher degradation rate. However, there remains a knowledge gap on the relationship between the half-lives and the structures.

In this study, two typical reclaimed water irrigation fields in Daxing, Beijing, China, and in Lake City, Florida, USA, were chosen to carry out the experiment. Herein, we aimed to explore the relationship between the degradation and the structures, as well as screen the potential indicators for measuring the degradation of NP isomers.

2. Materials and Methods

2.1. Chemicals and Reagents

4-NP isomer mixture standard (0.25 g, 100% purity, CAS: 25154-52-3) was purchased from Dr. Ehrenstorfer GmbH. 4-NP isomer standard was dissolved in the methanol as a stock solution with a concentration of 1000 mg⋅L⁻¹. 4-n-Nonylphenol (98% purity, CAS: 104-40-5) served as a surrogate substance with a concentration of 1000 mg⋅L⁻¹, purchased from Sigma-Aldrich (St. Louis, MI, USA); P-N-nonylphenol, served as the internal standard (Ring-¹³C6, 1.2 mL, 100% purity, CAS: 211947-56-7) at 100 μg⋅mL⁻¹ in nonane; dichloromethane (1.325 g·cm³, CAS: 75-09-2, HPLC grade) was purchased from Honeywell. Sodium azide (NaN₃ 99.7%) was purchased from Sigma-Aldrich, and reagent water (18.3 MΩ. cm resistivity) was prepared. Sodium chloride (NaCl) and anhydrous sodium sulfate (Na₂SO₄) were purchased from Sigma-Aldrich and baked in a muffle oven at 400 °C for 24 h.

2.2. Sample Collection

Two types of soils were used to conduct the degradation experiment. One was from a reclaimed water irrigation field for more than 40 years in Daxing, Beijing, China (39°36′ N, 116°21′ E), which covers an area of 500 m². The Daxing irrigation field is located in a warm region with an annual mean temperature of 14.0 °C and an average precipitation of 554.5 mm, where the rainy season is from June to September. Another soil was collected from the field in Lake City, FL, USA (29°39′0.48″ N, 82°21′55.06″ W), with an area of 2300 m² [32], which has been irrigated with reclaimed water for more than 60 years. It has a humid subtropical climate with a mean temperature of 20–25 °C and a precipitation of 100 cm annually. The five-point sampling method was used in both fields (0–20 cm). A total of 5 topsoil samples in each field were taken using a stainless-steel spade and mixed thoroughly. Afterwards, the soil samples were chosen by quartation and stored in brown glass bottles at 4 °C.

The properties of China soil were detected by the Micro Structure Analytical Laboratory, Beijing. The properties of China soil and Florida soil are shown in Table S2. The data of soil properties of Florida were obtained from the website of the U.S. Department of Agriculture.

2.3. Experimental Design

The soil was 0.25 mm sieved to remove large particles and then weighed in a series of 10 g aliquots into 250 mL brown jars. The jars were placed in the dark for 21 days for the microbes to be acclimatized. A total of 30 μL of the stock solution (1000 mg⋅L⁻¹) of the 4-NP isomer mixture and 3 μL 4-n-nonylphenol (1000 mg⋅L⁻¹) as surrogate substance were dissolved into 1 mL methyl alcohol, and then we dissolved the methyl alcohol into 5 mL deionized (DI) water. The DI water was transferred into above-mentioned the 250 mL brown jars containing 10 g soil. The jars were sealed and shaken for 30 min to ensure homogeneity. The background NP concentration in China soil was detected, which was below the method detection limit (MDL), while the background concentration of NP in Florida soil was not detected. The final actual concentrations of NP and surrogate substance in Florida soil were 3 mg⋅kg⁻¹ and 0.3 mg⋅kg⁻¹, respectively, after the stock solution was added.

The experimental treatments and control treatments (CK) were set up, and each treatment had three replicates. The experimental treatments were the China soil and Florida soil with non-sterilization. The control treatments were the ones with sterilization. Soil sterilization was achieved by heating at 121 °C for 4 h each day continuously for 7 days, and then sodium azide was added. All the samples were in an aerobic environment with the brown jars open. A total of 2 mL DI water was added into each jar every two days to keep the maximum water holding capacity of the soil. On 1, 3, 7, 14, 21, 28, 42, and 56 d of the experiment, the concentrations of NP remained in the soil after the degradation was detected by gas chromatography–mass spectrometry (GC-MS).

2.4. Sample Treatment and Analysis

Soil samples of 10 g were dried by freezing and extracted by 20 mL methanol three times and 10 mL ethyl acetate once by repeated ultrasonic suspension (0.09 kW, 20 kHz), shaking at 220 rpm for 1 h, and centrifugation at 8000× g for 25 min. The supernatants were combined and rotary evaporated at 40 °C to approximate dryness, and then dissolved in 1 mL of anhydrous ethyl acetate. P-N-Nonylphenol (Ring-13C6, 99%) as the internal standard was added into each sample.

Gas chromatography–mass spectrometry (GC-MS) (Agilent 6890N/5975, Santa Clara, CA, USA) was used for the determination of NP isomers. GC-MS detection was performed using capillary column DB-5MS (60 m × 0.25 μm × 0.25 mm, Agilent Co., Santa Clara, CA, USA). The details of detection can refer to the former research [33]. The MDLs of NP isomers were 0.86–2.65 μg kg⁻¹, and the recovery ranged between 70% and 110%, which met the standards of the USEPA.

2.5. Data Analysis

2.5.1. Half-Life

Half-life (t_1/2) refers to the time at which the quantity reduces to half of its initial value. The following was the first-order kinetic formula, and the half-life can be calculated by the following equation:

Cs = C_i × (1/2) ^T/(t_1/2⁾

(1)

where t_1/2 refers to the half-life; Cs denotes the concentration of pollutants in soils after degradation (mg⋅L⁻¹); Ci denotes the initial concentration of NP in soils (mg⋅kg⁻¹); and T denotes the whole degradation time (d).

2.5.2. Calculation of Molecular Descriptors

The calculation of topological steric index was according to the research of Cao and Liu [34]. Molecular connectivity χ indices and information indices, which were the common topological indices, were calculated by the equations Formula (2)–(4). Values of molecular descriptors such as steric index and the mean information index for the magnitude of distance (I_DWbar) of each isomer were cited from the study of Lu and Gan [26], which were calculated by Molconn-Z (version 4.12S, eduSoft, La Jolla, CA, USA). The definitions can be found in the work of Todeschini et al. [35]. Molecular descriptors of steric index and I_DWbar were checked to ensure that there was a variation in the values.

The first topological parameter reported in the literature is the Wiener index. The rapid development of computer promotes the research and application of the molecular topological index method [36,37,38]. Randic [39] proposed the molecular branching index χ (it is later called the simple molecular connectivity index). The calculation method was as follows:

$$1_{\chi }{}^v={\sum }_{i=1}^n{({\delta }_i^v\times {\delta }_j^v)}^{-0.5}$$

(2)

$$2_{\chi }{}^v={\sum }_{i=1}^n{({\delta }_i^v\times {\delta }_j^v\times {\delta }_k^v)}^{-0.5}$$

(3)

where χ is the branching index; n is the number of bonds; and δ_i, δ_j, and δ_k represent the point valence of two adjacent atoms. The point valence is the numerical representation of the vertex atom, which contains the structural information of the atom. In the branching index, the midpoint value is the number of non-hydrogen atoms connected by vertex atoms, that is, the number of connecting edges of vertex atoms in the hidden hydrogen graph and the branching degree of atoms.

In order to characterize heteroatoms, Kier and Hall [40] made a series of corrections on the basis of Randic point valence and revised it to the following calculation:

$${\delta }_i^v=\frac{Z_i^v-h_i}{Z-Z_i^v-1}$$

(4)

where δ_i^v is the point valence of i atom, Z is the total number of electrons of the atom, Z_i^v is the number of valence electrons of the atom, and h_i is the number of hydrogen atoms directly connected to the i atom. The point valence distinguishes different types of bonds—single bond, double bond, and triple bond—and can also be used to calculate the point valence of some heteroatoms, such as S, P and O. The point price calculated by Equation (4) is called the point price δ_v.

3. Results and Discussion

3.1. Degradation Kinetics of Nonylphenol Isomers

Figure 1 shows the degradation of 12 NP isomers in China non-sterilized (CN) soil and Florida non-sterilized (FN) soil (take as an example the total ion chromatogram of NP isomers during the degradation on the first and fifth days in CN soil, as shown in Figure S1. Obviously, the NP isomer hardly degraded in sterilized control groups, but in the non-sterilized experimental group, all the NP isomers initially underwent a rapid degradation phase, then a slow stage, while different isomers degraded at different degrees. The degradation rates of NP2 and NP5 were stable on the eighth day in CN soil, with the degradation of 85.62% and 81.90%, respectively. Conversely, NP1, NP3, NP4, NP6, NP7, NP11, and NP12 in CN soil were stable until 14 d. This indicated that NP2 and NP5 had a relatively high degradation rate. However, NP8, NP9, and NP10 were in a falling trend and did not reach a stable phase during the whole experiment. After 14 days, the degradation of all isomers in CN soil was above 80%. In FN soil, the degradation of NP isomers was different from that in CN soil. Except for NP5 and NP11, the other 10 isomers were not stable until 27 d. The degradation of NP5 and NP11 tended to be stable on the 8th and 14th days, with removal efficiencies of 84.31% and 80.44%, respectively. In general, the average removal efficiency of the isomers in CN soil and FN soil were 86.80% and 81.22%, respectively.

The degradation of NP was mainly affected by two factors: (1) the structures of the isomers, and (2) the species of microorganisms [41]. Gabriel et al. [31] studied the degradation of α-quaternary NP isomers via a bacterial strain that was isolated from activated sludge. Up to 99.7% of NP 128 and NP 119 were removed, while only 30.5% NP193b dissipated after incubation at 9 d. Likewise, Toyama et al. [42] found that the degradation rates of eight isomers were different in the soil improved by biosolids, which indicated that the degradation of NP isomers in soil was specific as well.

It can be seen that the degradation of NP isomers in CN and CF soils conformed to the first-order kinetics with the linear correlation coefficients (R²) greater than 0.90 (

Table 1. NP degradation kinetic equations of 12 isomers in CN soil.

Isomers	First-Order Kinetic Equation	R2	K (d−1)	Half-Life (d)
NP1	y = −0.108x	0.98	0.108	6.42
NP2	y = −0.228x	0.98	0.228	3.04
NP3	y = −0.140x	0.99	0.140	4.95
NP4	y = −0.110x	0.95	0.110	6.30
NP5	y = −0.341x	1.00	0.341	2.03
NP6	y = −0.120x	0.97	0.120	5.78
NP7	y = −0.122x	0.99	0.122	5.68
NP8	y = −0.080x	0.98	0.080	8.66
NP9	y = −0.085x	0.98	0.085	8.15
NP10	y = −0.090x	0.98	0.090	7.70
NP11	y = −0.165x	0.98	0.165	4.20
NP12	y = −0.135x	0.97	0.135	5.13
tNP	y = −0.131x	0.97	0.131	5.29

Note: y indicates lnc/c₀; x indicates time (t); tNP: indicates the total NP.

and

Table 2. NP degradation kinetic equations of 12 isomers in FN soil.

Isomers	First-Order Kinetic Equation	R2	K (d−1)	Half-Life (d)
NP1	y = −0.0642x	0.97	0.0642	10.79
NP2	y = −0.0907x	0.91	0.0904	7.67
NP3	y = −0.0794x	0.94	0.0794	8.73
NP4	y = −0.0628x	0.96	0.0628	11.04
NP5	y = −0.1344x	0.99	0.1344	5.16
NP6	y = −0.0703x	0.9	0.0703	9.86
NP7	y = −0.0719x	0.93	0.0719	9.64
NP8	y = −0.0586x	0.92	0.0586	11.83
NP9	y = −0.0658x	0.97	0.0658	10.53
NP10	y = −0.0644x	0.95	0.0644	10.76
NP11	y = −0.1017x	0.91	0.1017	6.81
NP12	y = −0.0793x	0.90	0.0793	8.74
tNP	y = −0.0605x	0.93	0.0605	11.46

Note: y indicates lnc/c₀; x indicates time (t); tNP: indicates the total NP.

). The degradation rate of total NP in CN soil was higher than that in FN soil. The half-lives of isomers in CN and FN soils were 2.03–8.66 d and 5.16–11.83 d, respectively. The degradation rates of NP isomers in CN soil were relatively higher than that in FN soil. The degradation of NP in soil is complicated and affected by varieties of factors: the isomers’ structures, oxygen availability, and temperatures [29]. The ranking of the degradation rate of the isomers in CN soil was NP5 > NP2 > NP 11 > NP3 > NP12 > NP7 > NP6 > NP4 > NP1 > NP 10 > NP9 > NP8. In CF soil, the order of kinetic degradation rate of each isomer was NP5 > NP11 > NP2 > NP3 > NP12 > NP7 > NP6 > NP9 > NP10 > NP1 > NP4 > NP8. It can be seen that NP5, NP2, NP11 and NP3 were the isomers that degraded rapidly in the two soils. Among them, NP5 had the highest degradation rate and the shortest half-life, which was only 2.03 d; the isomers with a medium degradation rate were NP12, NP7 and NP6; and the isomers with the slowest degradation rate were NP4, NP1, NP10, NP9 and NP8. This research showed that the degradation of the NP isomers in soil was specific. Similarly, Yao [27] found the same results in the study of the effect of rice-straw biochar on degradation of NP. He found that NP5 degraded the fastest and NP8 degraded the slowest among the isomers. Lu and Gan [26] found that the degradation rate of NP8 was the lowest among the 19 isomers that have been identified as well.

Shan et al. [43] evaluated the degradation of five isomers under aerobic conditions in paddy soil, and the half-lives were in the order of NP111 (10.3 d) > NP112 (8.4 d) > NP65 (5.8 d) > NP38 > (2.1 d) > NP1(1.4 d). In the reactor of the simulated wastewater treatment process, the degradation order of four NP isomers was as follows: NP3 (75.4%) > NP 111 (42.9%) > NP 170 (40.7%) > NP 194 (36.2%). Das and Xia [30] explored the degradation of NP isomers in the process of biosolid composting and found that under the optimal conditions, the half-life of total NP was 1.3 ± 0.2 d, while the half-life of isomers with α-methyl-α-propyl structure and α-dimethyl structures were 12.6 ± 0.3 d and 0.8 ± 0.1 d, respectively. Hence, observations from the former and current studies suggested that there may be intrinsic, structurally based properties that affected the degradability of NP isomers. Thus, it is essential to dig out a relationship between the degradability and the structure of the isomers to describe the degradation of NP.

3.2. The Relationship between Degradation and Structures

In this study, half-life was used to measure the degradation rate of NP isomers in soil. The structures of the isomers are shown in Table S1. Figure 2a,b(A) showed the relationship between the half-life of each isomer and the length of the side chain, which is indicated by the number of the side chain in the nonyl side chain. Generally, the half-life decreased with the increase of the nonyl side chain. However, when the numbers of carbon atoms in the nonyl side chain were 4 and 5, the corresponding half-life overlapped. It is not the case that the longer the side chain, the shorter the half-lives of isomers. Therefore, the degradation rate of the isomers cannot be judged by the number of carbon atoms in the nonyl side chain. Figure 2a,b(B) shows the relationship between the half-lives and the type of α-substituent of the isomers. Generally, the larger the volume of the substituent at the α position, the smaller of the half-life, but there were the same problems as in the nonyl chain. Similarly, the same problem existed for the type of β-substituents in our study (Figure 2a,b(C)). Therefore, it is difficult to judge the relationship between the degradation of isomers and their structures from the length of the side chain. The molecular connectivity indices of NP isomers were calculated, as shown in Table S3. It can be seen from Figure 2a,b(D–F) that there was no correlation between the half-lives and the molecular connectivity indices of the isomers. Hao et al. [44] explored the relationship between the degradation of NP isomers and their molecular connectivity indexes ²χ^v and ⁴χ^v_pc; however, regressions of these indices were less satisfactory in her study. Steric hindrance was used to describe the alkyl chain at the ipso position, wherein the alkyl chain was attached to the benzene ring. The steric hindrance of each NP isomer is provided in Hall,. It is shown in Figure 2a,b(G) that the half-lives of the isomers had a good positive linear relationship with their steric hindrance (R² = 0.82 for CN soil; R² = 0.86 for FN soil), and the points of the NP isomer were scattered in the 95% confidence interval. Therefore, the half-life can be obtained by calculating the steric hindrance of the isomer, which is a method to estimate the half-life of the NP isomer. To better identify the molecular descriptors, many other topological techniques are commonly used to characterize the molecular structure. The mean information index for the magnitude of distance (I_DWbar) is one of the topological techniques. This index is a measure of branching degree, especially for isomers, wherein a larger value indicates a higher branching degree. For example, the larger I_DWbar in this study indicated a higher branching degree of nonyl branched chain. The I_DWbar of each NP isomer is provided in Table S4. It was found that there was a good linear relationship between the half-life and I_DWbar value (R² = 0.83 for CN soil; R² = 0.94 for FN soil). The larger the I_DWbar value of the NP isomer, the longer the half-life (Figure 2a,b(H)). Compared with steric hindrance, the data points were relatively less scattered, and the 95% confidence interval was narrow. Therefore, I_DWbar and steric hindrance are better evaluation indexes for NP isomers. This finding would be one of methods to measure the degradation of NP isomers in the future. It provides a foundation for the risk assessment of NP isomers.

4. Conclusions

In this paper, the degradation behavior of NP isomers in two reclaimed water irrigation fields was studied. The degradation of each isomer in soil was specific. The half-life of NP degradation in soil was positively correlated with the steric hindrance and mean information index for the magnitude of distance (I_DWbar). Therefore, the half-life of NP isomers can be obtained by the values of the steric hindrance and I_DWbar, which is an effective method to evaluate the half-life of NP isomers and an important parameter for the environmental risk assessment of isomers and in making the health risk assessment of NP more exact. However, this study was conducted indoors and at room temperature, and the whole experiment process was not affected by the natural environment. Therefore, if the degradation of NP in soil is outdoors, causing it to be affected by some other factors, such as temperature, the biological community, or redox potential, this conclusion in this study may require further study. Moreover, the degradation products of NP in soil were not further identified in this study. In fact, the degradation of NP may produce more toxic secondary metabolites, causing more serious risks to the environment. Therefore, to identify the degradation products of NP in soil by secondary mass spectrometry, other methods require further study. Meanwhile, the research in the future requires a focus on the relationship of degradation of NP isomers and the microbial community in the soil. It is worth mentioning that, considering the requirements of NP extraction on soil particles, only soil fraction less than 0.25 mm was used in this study, excluding an important part of the active abiotic and biotic components. Therefore, this work was limited to the small soil component (<0.25 mm). Hence, in further study, the effect of the soil particle size on NP degradation can be studied as well.