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Isomer-Specific Degradation of Branched and Linear 4-Nonylphenol Isomers in an Oxic Soil Jun Shan,†,‡ Bingqi Jiang,†,‡ Bin Yu,†,‡ Chengliang Li,§ Yuanyuan Sun,†,|| Hongyan Guo,†,‡ Jichun Wu,†,|| Erwin Klumpp,§ Andreas Sch€affer,^ and Rong Ji*,†,‡ †
State Key Laboratory of Pollution Control and Resource Reuse, Nanjing University, 163 Xianlin Avenue, 210046 Nanjing, China School of the Environment, Nanjing University, 163 Xianlin Avenue, 210046 Nanjing, China § Agrosphere Institute, IBG-3, Research Centre J€ulich, D-52426 J€ulich, Germany School of Earth Science and Engineering, Hydrosciences Department, Nanjing University, 210093 Nanjing, China ^ Biology 5, Environmental Biology and Chemodynamics, RWTH Aachen University, D-52056 Aachen, Germany
)
‡
bS Supporting Information ABSTRACT: Using 14C- and 13C-ring-labeling, degradation of five p-nonylphenol (4-NP) isomers including four branched (4-NP38, 4-NP65, 4-NP111, and 4-NP112) and one linear (4-NP1) isomers in a rice paddy soil was studied under oxic conditions. Degradation followed an availability-adjusted first-order kinetics with the decreasing order of half-life 4-NP111 (10.3 days) > 4-NP112 (8.4 days) > 4-NP65 (5.8 days) > 4-NP38 (2.1 days) > 4-NP1 (1.4 days), which is in agreement with the order of their reported estrogenicities. One metabolite of 4-NP111 with less polarity than the parent compound occurred rapidly and remained stable in the soil. At the end of incubation (58 days), bound residues of 4-NP111 amounted to 54% of the initially applied radioactivity and resided almost exclusively in the humin fraction of soil organic matter, in which chemically humin-bound residues increased over incubation. Our results indicate an increase of specific estrogenicity of the remaining 4-NPs in soil as a result of the isomer-specific degradation and therefore underline the importance of understanding the individual fate (including degradation, metabolism, and bound-residue formation) of isomers for risk assessment of 4-NPs in soil. 4-NP1 should not be used as a representative of 4-NPs for studies on their environmental behavior.
’ INTRODUCTION Nonylphenols (NPs) occur ubiquitously as endocrine-disrupting micropollutants in the environment.13 The major source of NPs in the environment is the degradation of the widely used nonionic surfactant nonylphenol polyethoxylates.3,4 NPs are more toxic than their parent compounds.5 Recently, p-nonylphenols (4-NPs) have been found to interfere with the secretion of cytokines in human placenta at environmental concentration levels (from pmol L1 to nmol L1).6 In treated sludge of wastewater treatment plants (i.e., biosolids), the concentration of 4-NPs varied from a few mg kg1 up to several thousand mg kg1.2,7 Increasing land application of biosolids as fertilizer can release large amounts of NPs directly into soil.2 Much attention has been paid to degradation of NPs in both oxic and anoxic environments, and most of the studies were carried out using technical NP (tNP) mixtures or the isomer 4-NP1 (e.g., refs 814). tNP is a mixture of ortho- and parasubstituted NP isomers, and the latter (4-NPs) are the predominant components comprising 8694% of tNP mixtures.15 The alkyl chains of NP isomers in tNP are all branched, and the r 2011 American Chemical Society
isomer 4-NP1 with a linear nonyl chain actually does not exist in the mixtures.16 4-NP isomers with different α-C substitutions and branching patterns of the nonyl chain may have different degradation rates as indicated by degradation experiments with pure cultures of the bacteria Sphingomonads (Sphingomonas and Sphingobium).16,17 Isomers with less bulkiness at the α-C were degraded more efficiently via a type II ipso substitution,17 whereas the linear 4-NP1 was not metabolized as a single-carbon source by these bacteria.18,19 Isomer-specific degradation of organic compounds depends on the microbial community.20 While several studies showed that the isomeric composition of 4-NPs in natural environments was significantly different from that of tNP mixture, other studies did not observe marked differences in some environmental matrixes.17 Though isomer-specific degradation of 4-NPs has Received: January 19, 2011 Accepted: August 8, 2011 Revised: August 5, 2011 Published: August 08, 2011 8283
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Table 1. Parameters of the Availability-Adjusted First-Order Model (eq 1) for Degradation of the 4-Nonylphenol Isomers in the Rice Paddy Soil and Predicted Amounts of Nondegradable NP in the Soila
a
Values followed by different letters in columns are significantly different (P < 0.05).
been suggested in biosolids2 and in wastewater treatment plants,21 it has not been proven in the soil environment yet. Because 4-NP1 is distinctly different from the branched 4-NP isomers in sorption to soil and estrogenicity,17,22,23 it is necessary to evaluate whether 4-NP1 can represent tNP mixture in terms of their environmental fate. To our knowledge, no comparison study on the degradation of 4-NP1 and tNP isomers has been performed in any environmental matrix. Formation of bound (nonextractable by organic solvents) residues, via physical enclosure in or chemical binding to soil organic matter, is one typical fate of organic xenobiotics in soil and is regarded as an important detoxification process.2426 4-NPs formed bound residues in soil, sediment, and earthworm.2730 It has been shown that in pure culture of Sphingomonas sp., residues of 4-NP111 were able to covalently bind to humic acids after being metabolized.31 However, little is known about the characteristics of the bound residues of 4-NPs in soil, and no study has yet been performed to evaluate the relative importance of the different binding mechanisms in the bound-residue formation of 4-NPs in soil. Here, we synthesized four branched 4-NP isomers, some of which were ring-14C or ring-13C labeled, and studied the degradation of these isomers and the linear isomer 4-NP1 in a rice paddy soil under oxic conditions with the following objectives: (1) to elucidate isomer-specific degradation of 4-NP isomers in soil and (2) to characterize the bound residues of 4-NPs in soil.
’ MATERIALS AND METHODS NP Isomers and Other Chemicals. Four nonlabeled and branched 4-NP isomers (4-NP111, 4-NP112, 4-NP38, and 4-NP65, see Table 1 for their chemical structures) containing a quaternary α-carbon at the alkyl chain, and two isotope-labeled isomers,
i.e., ring-14C-labeled 4-NP111 (14C-4-NP111) and ring-13C-labeled 4-NP38 (13C-4-NP38), were synthesized via FriedelCrafts alkylation (for detailed information about the syntheses, see the Supporting Information). 4-NP1 was purchased from Alfa Aesar (Shanghai, China) with >98% purity. N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) was purchased from SigmaAldrich (Shanghai). Other chemicals were chromatographic or analytical grade. A stock solution of a mixture of five 4-NP isomers (14C-4NP111, 4-NP112, 13C-4-NP38, 4-NP65, and 4-NP1) was prepared in methanol at a concentration of 16.6 μmol mL1 with a molar ratio of the five isomers in the mixture at about 1:1:1:1:1 (each isomer at about 3.3 mmol L1). 13C-4-NP38 was used for signal separation of 4-NP38 from 4-NP111 in gas chromatography mass spectrometry (GC-MS) chromatograms by using selected ion monitoring (SIM, see the Supporting Information). 14C-4NP111 was used for localization and quantitative determination of 4-NP111 and its residues in soil. Soil. A gleyic hydragric Anthrosol soil, derived from a silt loam deposit, was collected from the Changshu Experimental Station of the Chinese Academy of Sciences in Jiangsu Province, China, and brought to the laboratory in a nylon bag. The rice paddy soil contained 2.5% total organic carbon, 0.16% nitrogen, 46.7% clay, 37.9% silt, and 15.4% sand and had a pH (0.01 M CaCl2) of 6.31. The soil was air dried, sieved through 2 mm and stored at room temperature shortly before use. Degradation Experiments. About 18 μL of the stock solution of the 4-NP isomer mixture was added with a microsyringe to 0.2 g of soil. The soil was mixed and transferred into a 100 mL serum flask containing 4.8 g of soil. The whole soil was then thoroughly mixed and kept overnight to evaporate the methanol solvent according to Zhang et al.32 The homogeneity of the 4-NP distribution within the soil was proved by determining the radioactivity of soil subsamples (0.020.05 g) from the flask 8284
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Environmental Science & Technology (recovery = 96 ( 3.5%, n = 3). After solvent evaporation, 1.2 mL distilled water was added to adjust the soil moisture to 70% of the maximal water-holding capacity. The flask was then closed with a polytetrafluoroethylene-wrapped rubber stopper. The 14 CO2 released from the soil was absorbed by 1.0 mL of NaOH (1 M) contained in one 6 mL vial, which was suspended from the bottom of the stopper. In total, 54 flasks were prepared. The spiked soils in the flasks had a total 4-NP concentration of 59.6 μmol kg1 soil (dry weight; about 12 μmol kg1 for each isomer) and a specific radioactivity of 3.7 MBq kg1 soil (dry weight). The flasks were incubated at 20 ( 1 C in the dark. The flasks were opened for 0.5 min each day for exchange of headspace with fresh air. Water loss from the flask due to evaporation during incubation was compensated by adding the same amounts of deionized water to the soil. At incubation times of 0, 5, 10, 15, 20, 27, 34, 43, and 58 days, three flasks were sacrificed for analysis of radioactivity in the NaOH trap, concentrations of the 4-NP isomers, and formation of metabolites and bound residues of 14 C-4-NP111 in the soil (see below). Flasks with sterilized soils were set as controls. Soil sterilization was achieved by autoclaving the soil at 120 C for 1 h three times in three consecutive days. All experiments were performed in triplicate. Extraction and Analysis of Soil. Soil samples after incubation were freeze dried and extracted with methanol (20 mL) three times and ethyl acetate (10 mL) once by repeated ultrasonic suspension (0.09 kW, 20 kHz), shaking (220 rpm, 1 h), and centrifugation (8000g, 25 min). The supernatants were combined, and aliquots were taken for quantification of radioactivity by liquid scintillation counting (LSC, see the Supporting Information). The residual supernatants were rotary evaporated at 40 C to approximate dryness and redissolved in 1 mL of anhydrous ethyl acetate. Aliquots were analyzed by thin layer chromatography (TLC) followed by autoradiography for determination of free 14C-4-NP111 and its metabolite and by GC-MS for concentration determination of the five 4-NP isomers (see the Supporting Information). Radioactive determination showed that the freeze-drying and extraction processes had a recovery of 93.1 ( 1.5% (n = 3) for 4-NP isomers. Preliminary experiments showed that after these consecutive extractions with methanol and ethyl acetate the extraction procedure was sufficient and exhaustive. The soil humic substances containing the residual parts of 4-NPs after exhaustive organic solvent extraction, i.e., bound residues,33 were fractionated into fulvic acids, humic acids, and humin according to Shan et al.34 The residual soils with humin were freeze dried, and the humin was silylated according to Butenschoen et al.35 Such silylation procedure involves substitution of active hydrogens of functional groups (such as OH, NH2, dNH, SH, COOH) present in humin by the silyl moiety, which leads to disintegration of humic aggregates that were normally held together by the hydrogen bonds and other noncovalent interactions.36 According to Haider et al.,36 the radioactivity in the supernatant released from the humin by the silylation procedure was attributed to 14 C-4-NP111 residues, which were bound to humin of soil organic matter via physicochemical interactions, whereas the remaining radioactivity in the pellet (i.e., insoluble humin fraction) represented the residues, which were chemically bound to humin via covalent bondings. Data Analysis. Degradation of many organic pollutants in the environment follows pseudo-first-order-kinetics.37 When organic pollutants (e.g., NPs) are released into the soil, their bioavailability will decrease over time due to aging processes, such as
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adsorption and irreversible binding to soil matrix.38 Thus, we applied availability-adjusted first-order kinetics 37 to model the degradation kinetics of 4-NP isomers in soil αt
Ct ¼ C0 ekð1 e
Þ=α
ð1Þ
where C0 and Ct (μmol kg1) are the concentrations of 4-NP isomers at times 0 and t (day), k is the degradation rate constant (day1), and α is the positive constant called the unavailability coefficient (day1), which is the kinetic constant of first-order kinetics of aging processes that 4-NP isomers became unavailable in the soil (for more detail, see the Supporting Information). The half-life (t1/2) of 4-NP isomers can be derived from eq 1 and is expressed as 1 0:693α t1=2 ¼ ln 1 ð2Þ α k When t is infinite, eq 1 gives the amounts of 4-NP isomers remaining in the soil, i.e. Ct f ∞ ¼ C0 ek=α
ð3Þ
Therefore, the percentage of nondegradable 4-NP isomers (Punavailable) in the soil can be predicted Punavailable ¼ ek=α 100%
ð4Þ
In addition to eq 1, the first-order kinetics with one constant parameter (eq 5) might also be appropriate to describe the 4-NP degradation process with the unavailable part in soil Ct ¼ ðC0 C∞ Þekt þ C∞
ð5Þ
where k is the degradation rate constant (day1) and C∞ is the unavailable amount of 4-NP isomers (μmol kg1). The percentage of nondegradable 4-NP isomers in the soil may be calculated as C∞/C0 100%. Fitting of the data to the model was carried out using iterative nonlinear regression by Sigma Plot 11.0. Significance analyses were performed using the student’s t test, and the statistical probability P < 0.05 was considered significant.
’ RESULTS AND DISCUSSION Isomer-Specific Degradation of 4-NP Isomers in Soil. The five (four branched and one linear) 4-NP isomers degraded at different rates in the active rice paddy soil during 58 days of incubation under oxic conditions (Figure 1). The degradation kinetics of the isomers was fitted to the availability-adjusted firstorder model (eq 1), the first-order model with one constant parameter (eq 5), and the simple first-order model. The goodness-of-fit of the three models is summarized in Table S2 in the Supporting Information. The values of the reduced chi square (χ2reduced) for these models are far from the optimal value of 1. Among the three models, the availability-adjusted first-order model (eq 1) had the best goodness-of-fit and is most rational for describing the gradually increasing unavailability of 4-NP isomers in soil over incubation time. Also, the residual plots of this model show a random distribution of the residues around the zero line (see Figure S1 in the Supporting Information). Therefore, we prefer to apply the availability-adjusted model to fit the degradation data of the five 4-NP isomers in order to compare their persistence in the soil. The values of parameters k, α, and t1/2 as well as the goodness-of-fit of the model (eq 1) for the 8285
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Figure 1. Degradation kinetics of five 4-NP isomers in an active rice paddy soil at 20 C under oxic conditions. Points are experimental data, and lines are their fitting curves according to the availability-adjusted first-order kinetics (eq 1). All values are means with standard deviations of three individual experiments.
individual 4-NP isomers are summarized in Table 1. The k ranged from 0.09 to 0.58 day1 with the following increasing order: 4-NP1 (0.58 day1) > 4-NP38 (0.38 day1) > 4-NP65 (0.13 day1) > 4-NP112 (0.10 day1) > 4-NP111 (0.09 day1), showing that the linear isomer 4-NP1 degraded most rapidly in the soil (t1/2 = 1.4 days), while the branched 4-NP111, the main component of tNP mixtures,15 was degraded considerably more slowly (t1/2 = 10.3 days). α varied from 0.04 to 0.20 day1, and 4-NP1 had the highest unavailability factor, which is in good agreement with its highest sorption affinity in soils.22 The unavailable amounts of the five isomers in the soil predicted by eq 4 ranged from 2.9 to 24.4% of the initial spiking with the following decreasing order: 4-NP111 (24.4%) > 4-NP112 (7.9%) > 4-NP1 (5.6%) > 4-NP65 (3.3%) > 4-NP38 (2.9%) (Table 1). 4-NP111 and 4-NP112, which were more recalcitrant, also had higher unavailable percentage in the soil. Dissipation of 4-NP112 in two German agricultural soils was described by biexponential kinetics with t1/2 of 4.24.3 days,32 which is in agreement with that in the rice paddy soil (Table 1). By using defined 4-NP isomers, we clearly showed that the degradation of 4-NP isomers in soil was isomer specific. Rapid disappearance and mineralization of 4-NP1 has been observed in soils (t1/2 < 2 days) and sediment (t1/2 < 4 days) under oxic conditions,8,9,11,13,39,40 which is in agreement with the degradation rate of 4-NP1 in the soil (t1/2 = 1.4 days, Table 1). Because 4-NP1 does not exist in technical 4-NP mixtures,16 considering the different degradation behaviors of 4-NP isomers in the soil (Table 1), rapid degradation of 4-NP1 may not represent the real 4-NP persistence in these oxic environments. The observed higher recalcitrance of the branched 4-NP isomers than 4-NP1 can be attributed to the alkyl chain structure at the benzene ring. The branched isomers have a quaternary α-C on the alkyl chain (Table 1), and this structure is regarded resistant to ω- and β-oxidation.16 The length of the side chain at α-C seems to be the most important factor for their degradation. The three isomers with an ethyl side chain at α-C (4-NP111, 4-NP112, 4-NP65) showed a longer t1/2 than 4-NP38 with two methyl side chains at α-C (Table 1). The branch number of the
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Figure 2. Relative amounts of radioactivity recovered as CO2 (right vertical axis), extractable, and bound residues (left vertical axis) of 14C-4NP111 during incubation in active (closed symbols) and sterilized (open symbols) rice paddy soils. The sum of radioactivity in CO2, extractable, and bound residues gave the total recovery. Values are means with standard deviations of three replicates.
alkyl chain also seems to be a factor affecting isomer degradation. The isomers with an alkyl side chain branched at two positions (such as 4-NP111 at α-C and γ-C and 4-NP112 at α-C and δ-C) exhibited a longer t1/2 than isomer 4-NP65 with only one branch at α-C (Table 1). The observation on the bacterial degradation of 4-NP isomers by Sphingomonas, that isomers with quaternary α-C and a more branched alkyl chain degraded more rapidly and 4-NP1 did not degrade,18,19 is in contrast to those on the degradation of isomers in the rice paddy soil. However, Sphingobium xenophagum Bayram degraded 4-NP isomers with quaternary α-C, except for 4-NP65, with rates on the same order as those in the paddy soil.17 The degradation rates of the 4-NP isomers in the paddy soil were in good agreement with the order of the estrogenicity of the isomers,17,23 i.e., the half-live of the isomers decreased with decreasing estrogenicity. All Sphingomonads bacteria (Sphingomonas and Sphingobium) capable of metabolizing branched 4-NP isomers were isolated from active sludge of wastewater treatment plants.17 The difference between the isomer-specific degradation by Sphingomonads in pure culture and our findings in the soil (Table 1, Figure 1) indicates that microbial communities responsible for degradation of 4-NPs in engineered media may be different from those in environmental media, suggesting that degradation of 4-NPs involves many mechanisms. Pathways other than the ipso substitution used by Sphingomonads41,42 might play a more important role in the 4-NP degradation in the oxic rice paddy soil. Mineralization and Bound-Residue Formation of 4-NP111. Degradation of the 4-NP isomers in the rice paddy soil was accompanied by mineralization and formation of bound residues. Figure 2 shows the course of mineralization and formation of organic solvent-extractable and bound residues of 14C-4-NP111 over an incubation of 58 days in the active and sterilized soils. Figure 2 also shows good recoveries of radioactivity (96107%) 8286
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Figure 3. Radioactivity recovered from extractable 14C-4-NP111 and its metabolite (left vertical axis), and relative amounts of the metabolite to the extract (right vertical axis) during incubation of 14C-4-NP111 in the soils under oxic conditions. No metabolite was found in the sterilized soil. Values are means with standard deviations of three replicates.
of the experiments, suggesting that the volatility of 14C-4-NP111 was negligible in the soil, which is in agreement with the low amount of volatiles formed when incubating 14C-4-NP111 in an agricultural sandy loam soil (1.7% for 135 days incubation).27 The mineralization of 14C-4-NP111 in the rice paddy soil was low (about 5% of the initially applied 14C within 58 days) and did not have a lag phase (Figure 2), indicating that microorganisms in the soil did not need an apparent adaptation time for mineralizing 4-NP111. In the sterilized soil less than 0.5% of 14C-4-NP111 was mineralized (Figure 2), indicating that the mineralization of 14C4-NP111 in the active soil was attributable to microbial activity. During incubation of 58 days in the soil, the extractable radioactivity decreased to 38.3% of the initial radioactivity whereas the bound radioactivity increased rapidly to 31.1% within the beginning 5 days and to 54.4% at the end of the incubation (Figure 2). Comparing the degradation of 4-NP isomers in the active and sterilized soils, it can be concluded that formation of the bound residues was apparently related to the microbial activity. Formation of bound residues is regarded as a consequence of aging processes of organic pollutants in soil, for which the main mechanisms are sorption and diffusion;38 however, our results highlight the role of microbial activity in aging processes. The soil microbes would increase formation of bound residues of organic pollutants by incorporating the pollutants or their metabolites into soil organic matter. The extractable radioactivity was analyzed by TLC followed by autoradiography (see the Supporting Information). One metabolite of 14C-4-NP111 was found in the active soil, but no metabolite was detected in the sterilized soil. The metabolite had a higher Rf value (0.75) on TLC than that of the parent 14C-4-NP111 (0.38), indicating that the metabolite was less polar than 4-NP111. Formation of the metabolite was rapid. After 5 days of incubation, the amounts of the metabolite in the extract accounted already for 24.2% of the initial radioactivity and appeared to be stable during incubation (Figure 3), whereas the relative amounts of the metabolite in the extractable residues increased and the
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Figure 4. Relative distribution of bound residues of 14C-4-NP111 within fractions of humic substances during incubation for 58 days in the active soil under oxic conditions.
extractable free 14C-4-NP111 decreased (Figure 3), indicating the progressive transformation of 14C-4-NP111 into the metabolite by soil microbial organisms. Fractionation of Bound Residues of 4-NP111. The bound residues of 14C-4-NP111 in the soil were fractionated according to their alkaline solubility into fulvic acids, humic acids, and humin. The relative distribution of the bound residues in these fractions is shown in Figure 4. Most of the bound radioactivity was located in the humin fraction, already amounting to >84% after incubation for 5 days. The humin-bound residues increased during incubation and accounted for 96% of the total bound residues at the end of incubation. The predominance of the humin-bound residues indicates that 14C-4-NP111 and its transformation products favor interacting with the humin fraction of soil organic matter, a mechanism which needs further investigation. The preferential binding of 4-NP111 residues to humin fraction in the soil was in contrast to the observation in the pure culture of the bacterium Sphingomonas sp. TTNP3 in the presence of humic acids, where 4-NP111 residues were relatively homogenously distributed within the humic molecules of various sizes.31 This supports the above conclusion that degradation of 4-NPs in the rice paddy soil took place by different pathways other than the ipso substitution by the Sphingomonas and indicates again the complexity of mechanisms for 4-NP degradation in the environment. Bound residues of pollutants in humin fraction may be formed through physicochemical enclosure in or chemical binding to humic matter26,36 or by strong sorption to black carbon, a possible sequestration mechanism for hydrophobic organic pollutants in soil.43 Pollutants which are covalently bound to soil organic matter are considered as an integral portion of soil organic matter and have little or no risks to the environment.24 The silylation procedure was used to distinguish the chemically bound residues from that via physicochemical enclosure.36,44 Silylation of the humin-bound 14C-4-NP111 residues showed that both amounts of chemically bound residues and total huminbound residues increased during incubation up to 20% and 52% of the initially applied 14C-4-NP111, respectively (Figure 5). In contrast, in the sterilized soil, the amounts of these residues were 8287
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derivation. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Phone: (86) 25-8968 0581. E-mail:
[email protected].
’ ACKNOWLEDGMENT This study was supported by the National Science Foundation of China (grant nos. 20777033, 20977043, and 41030746). ’ REFERENCES
Figure 5. Formation courses of total and chemically humin-bound residues of 14C-4-NP111 during incubation in the active (closed symbols) and sterilized (open symbols) rice paddy soils under oxic conditions. Values are means with standard deviations of three replicates.
low (5.5% and 13.7% of initial, respectively) at the end of incubation (Figure 5). Chemically bound residues of 14C-4NP111 in the humin fraction resulted from covalent binding of 14 C-4-NP111 or its metabolites, such as hydroquinone or shortchain organic acids, to soil organic matter through ester or ether bonds.16,29,31 Oxidative coupling of phenolic compounds to soil organic matter, which may be mediated by enzymes (such as laccase) or abiotic catalyst (such as manganese dioxide),25,45 could also contribute to the binding of 14C-4-NP111 residues to the humin. The increase in residues chemically bound to humin in the active soil during incubation (Figure 5) suggested that 14C4-NP111 was transformed continuously into stable residues in soil by binding to the soil matrix. Environmental Implications. Land application of biosolids releases large amounts of 4-NPs into the soil environment. The present study showed that 4-NP isomers with higher estrogenicity are more persistent in the rice paddy soil and less available for microbial degradation. This differential degradation and preservation of 4-NPs will result in an increase of specific estrogenicity of the remaining 4-NPs in the soil. This is the first direct evidence for the isomer-specific fate of 4-NPs in the environment and suggests that risk assessment of 4-NPs in the soil environment, probably also in the aquatic environment, should consider the specific fate of different isomers. Using the linear 4-NP1 as a model compound for studies on the environmental behavior of 4-NPs, such as fate and ecotoxicity, is not relevant. The observations about rapid degradation of 4-NPs in the environment based on studies with 4-NP1 can lead to an underestimation of their environmental persistence. Since considerable amounts of 4-NPs metabolites may be released into soil, their contribution to the ecological risk of 4-NPs in the soil should be evaluated, whereas the strong binding of NPs and their metabolites to humin during the humification process may reduce the risk in oxic soil.
’ ASSOCIATED CONTENT
bS
Supporting Information. Details of 4-NP isomer syntheses, GC-MS analysis, radioactivity determination, and model
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