Journal of Hazardous Materials 284 (2015) 103–107
Contents lists available at ScienceDirect
Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
Nucleophilic substitution as a mechanism of atrazine sequestration in soil Junhe Lu a,∗ , Juan Shao a , Deyang Kong b a b
Department of Environmental Science and Engineering, Nanjing Agricultural University, Nanjing 210095, China Nanjing Institute of Environmental Science, Ministry of Environmental Protection of PRC, Nanjing 210042, China
h i g h l i g h t s • Atrazine tends to form nonextractable bound residue in soil. • Nucleophilic substitution is a pathway leading to atrazine sequestration in soil. • Sulfur containing amino acids are likely to play an important role as nucleophiles during this process.
a r t i c l e
i n f o
Article history: Received 18 June 2014 Received in revised form 19 October 2014 Accepted 3 November 2014 Available online 7 November 2014 Keywords: Atrazine Sequestration Bound residue Nucleophic substitution
a b s t r a c t Formation of nonextractable residue was widely observed as a sink of atrazine (ATZ) in soil. However, the mechanisms by which ATZ binds to soil organic matter remain unclear. In this study, we demonstrated that neucleophilic substitution could serve an important pathway causing ATZ sequestration. The carbon bonded to the chlorine in ATZ molecule is partially positively charged due to the strong electronegativity of chlorine and is susceptible to the attack of nucleophiles such as aniline. Since aromatic amines are relatively rare in natural soils, amino acids/peptides were hypothesized to act as the main nucleophiles in real environment. However, substantially ATZ transformation was only observed in the presence of those species containing thiol functionality. Thus, we speculated that it was the thiol group in amino acids/peptides acting as the nucleophile. Nitrogen in amino acids was in fact not an active nucleophile toward ATZ. In addition to the sulfur-containing amino acids, other thiol compounds, and sulfide were also proved to be reactive to ATZ. Thus, the sequestration potential of ATZ probably correlates to the availability of thiol compounds in soil. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Atrazine (1-chloro-3-ethylamino-5-isopropylamino-2,4,6triazine, ATZ) is the most commonly applied herbicide to control broadleaf weeds and grasses. It was estimated that over 50 million pounds of active ATZ ingredients are applied annually in United States to corn, soybeans, sorghum, and other crops [1]. Once applied to the soil, ATZ has great chance to enter surface and ground waters through runoff or infiltration [2,3], because it is water soluble and relative conservative in the environment with low volatility, weak sediment partitioning, and relatively slow degradation rates (i.e., a half-life of months to years) [2]. As a result, ATZ is the most commonly detected herbicide in ground and surface waters [1,4]. The U.S. EPA has set the drinking water
∗ Corresponding author. Tel.: +86 25 84395164; fax: +86 25 84395210. E-mail address: [email protected] (J. Lu). http://dx.doi.org/10.1016/j.jhazmat.2014.11.001 0304-3894/© 2014 Elsevier B.V. All rights reserved.
maximum contaminant level (MCL) at 3 ppb, but ATZ concentrations often rise far above this level, especially after herbicide application and during runoff [5,6]. ATZ is a contaminant of environmental concern because its presence has been associated with causing imbalances in hormone levels in animals, possibly disrupting reproductive, and developmental processes [7–9]. Short term ATZ exposure above the drinking water MCL can potentially cause heart, lung, and kidney congestion, low blood pressure, muscle spasms, weight loss, and damage to the adrenal glands. Long term exposure to ATZ above the drinking water MCL has been linked to weight loss, cardiovascular damage, retinal, muscle degeneration, and cancer [10]. The high solubility and mobility of ATZ in combination with its relative resistance to degradation and significant health impacts makes ATZ contamination a serious threat to the environmental and human health [11], particularly in rural communities where ATZ is frequently used in agricultural activities but effective water treatment measures are not in place.
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J. Lu et al. / Journal of Hazardous Materials 284 (2015) 103–107
A thorough understanding of ATZ transformation in environment is critical for scientifically assessing the environmental risks associated with its application and for devising effective management and remediation means to mitigate its negative environmental and human health impacts. Extensive studies have been done in this topic and useful information obtained. ATZ undergoes both biotic and abiotic transformation in soil through two primary types of pathways: degradation and sequestration. While information regarding ATZ degradation is abundantly available [12–16], that relating to its sequestration is scarce. Evidences from experiments using 14 C-atrazine indicted that ATZ sequestration indeed occurred in soil resulting in the formation of ATZ bound residue with soil organic matter (SOM). The extent of such bound residue formation however varied greatly from case to case, which seemed to depend on factors that had yet to be understood. Barriuso and Koskinen found that nonextractable residues were formed immediately after atrazine application to unsterilized agricultural soil, and increased with the herbicide residence time in soil [17]. Jablonowski et al. tested a lysimeter soil containing long-term aged 14 C-ATZ for over twenty years and found 8.8% of the original 14 C activity remained in the top soil, most of which was bound to soil organic matter and could not be extracted [18]. Lesan and Bhandari concluded that longer atrazine-soil contact times resulted in enhanced nonextractable residue formation, and over an 84-day contact period, 35–50% of the pre-loaded 14 C-ATZ was found ending with residues in the organic components of soil [19]. All the above studies show distinctly that sequestration of ATZ and/or their metabolites occurs in soil, but mechanistic understanding about ATZ sequestration is still limited. In this research we demonstrated that neucleophilic substitution was a potential pathway leading to ATZ sequestration in environment, and certain sulfur containing amino acids/peptides probably played an important role as nucleophiles during this process. 2. Experimental 2.1. Chemicals and material ATZ was purchased from Sigma–Aldrich (St. Louis, MO). Phenol, gallic acid, catechol, resorcinol, quinol, salicylic acid, veratry alcohol, 4-methoxylphenol, aniline, glycine, cysteine, glutathione, methionine, mercaptoethanol, and Na2 S were all purchased from Aladdin (Shanghai, China). HPLC grade methanol and acetone were also from Sigma–Aldrich. Other chemical were reagent grade or better. All solutions were freshly prepared in deionized water.
be reactive to ATZ in previous experiments. The soil of yellow brown earth was collected from Xiamafang Park in Nanjing, China. The soil was dried, grinded, and sieved through 20-mesh screen before use. The pH of the soil was 5.73. Each reaction sample contained 5.0 g dry soil and sterilized before mixing with appropriate amount of ATZ dissolved in acetone to achieve a concentration of 10 mg/kg. After acetone was evaporated, 2.0 mL solution of 50 mM humic constituent or amino acid was spiked. The molar ratio of ATZ/humic constituent was approximately 1/440. After the soil samples was incubated in 20 ◦ C in dark for 6 day, the residue ATZ was extracted with 10 mL acetone twice facilitated with sonication. The extracts were combined and concentrated to 1 mL using a gentle stream of nitrogen gas. ATZ in the sample was quantified as described above. Control samples spiked with only ATZ were prepared and treated using identical procedure. Three replicates were performed for each reaction condition. 2.3. Products characterization To characterize the reaction products, selected samples with substantial ATZ removal were analyzed using an Agilent G6410B Triple Quad Mass spectrometer with an electron spray ionization source. The ionization source was operated in positive mode (ESI+). Nitrogen was used as desolvation gas and maintained at flow rate of 10 L/min. The desolvation temperature was set at 350 ◦ C. The mass analyzer was first run at scanning mode from m/z 100 to 1000. Secondary MS of the suspected reaction products were obtained subsequently. Fragmentor and collision energy were experimentally optimized. 3. Results and discussion 3.1. Removal of ATZ in the presence of humic constituents The complexity of SOM renders difficulty in the elucidation of xenobiotic–SOM interactions. A variety of substituted phenolic and anilinic compounds, so called humic constituents that mimic the structure of SOM were incubated with ATZ in an attempt to identify the specific functionalities that form covalent bonds with ATZ. It was revealed in Fig. 1 that ATZ was removed to different extent in the presence of different phenolic compounds. No reduction was seen in samples incubated with phenol or salicylic acid after 5 months. Appreciable attenuation was found in samples with gallic acid, veratry alcohol, catechol, resorcinol, quinol, and 4-methoxylphenol. Nonetheless, the removal in the presence of aniline was most pronounced and precipitants were observed. The removal increased consistently over time and exceeded 30% after
2.2. Reaction setup Reactions between ATZ and SOM were first explored in solutions in a series of glass vials as batch reactors. Each reactor contained 50 mL solution of 0.05 mM ATZ and one of the humic constituents (phenolic/anilinic compounds or amino acids listed in the previous paragraph) at concentrations from 0 to 50 mM. The pH of the solution was buffered at 5.8 using 1.0 mM phosphate. The reactors were kept in dark in an incubator maintained at 20 ◦ C. After predetermined incubation time, 1.0 mL sample was taken from each of the vials and analyzed using a Hitachi L-2000HPLC equipped with a photo-diode array (PDA) detector. A C18 reverse phase column (Hitachi LaChrom, 5 M × 250 mm × 4.6 mm) was used for separation. An isocratic elution consisting of 70% methanol and 30% water at a flow-rate of 1.0 mL/min was used as mobile phase. Controls with only ATZ were also prepared. Three replicate experiments were performed for each reaction condition. Reactions of ATZ was further explored in soil samples spiked with humic constituents and amino acids that were determined to
Fig. 1. Removal of ATZ in the presence of humic constituents. (Conditions: [ATZ]0 = 0.05 mM, initial concentration of the humic constituent was 0.50 mM, pH 5.8).
J. Lu et al. / Journal of Hazardous Materials 284 (2015) 103–107
Fig. 2. MS of ATZ and the adduct with nucleophiles.
5 month. Thus, amine group seemed play a critical role in facilitating ATZ transformation. We hypothesize that the removal of ATZ was due to the formation of adducts with aniline via nucleophilic substitution pathway, because amine is a common nucleophile and the carbon atom with chlorine attached in the triazine ring is partially positively charged induced by the electronegative chlorine and thus, susceptible to nucleophilic attack [20]. Oxygen attracts electrons more tightly making hydroxyl group a weaker nucleophilic than amine in aqueous phase, thus, less ATZ was removed in the presence of phenolic compounds [21]. Nucleophilic substitution mechanism was verified by MS analysis of the products. The data are given in Fig. 2. ATZ has a MW of 215, corresponding to the peak of m/z 216 in MS with ESI+ due to the formation of protonated cation. It is noted that this peak is accompanied by a (M + 2) isotope peak with abundance of approximately 1/3 of M peak, which is characteristic to Cl. In comparison with the control, m/z 216 shrank substantially and m/z 302 appeared as the most abundant peak in the reaction sample. The compound corresponding to this peak does not have the characteristic (M + 2) isotope, and its MW exactly fits to nucleophilic substitution pathway in which the Cl attached to the triazine ring was substituted by the aniline with the elimination of an HCl molecule (273 = 215 + 93 − 35). Secondary MS of this compound further rationalizes this statement (Supporting data, Fig. S1). 3.2. Removal of ATZ in the presence of nitrogen and sulfur nucleophiles Aniline moiety is relatively scarce in natural soil. Soil is indeed rich of amine (or amino) functionalities in forms of peptides and amino acids from biomass decomposition. Therefore, amino acids and peptides are presumed to play an important role in ATZ sequestration. Thiol compounds are another group of compounds in soil that could potentially serve as reactive nucleophiles. Generally, sulfur is more nucleophilic than nitrogen due to its larger size, which makes it readily polarizable and its lone pairs electrons readily accessible [21]. Sulfur nucleophiles are mainly certain
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(Conditions: [ATZ]0 = 0.05 mM, [nuclophiles]0 = 20 mM, pH 5.8, ESI+).
Fig. 3. Removal of ATZ in the presence of different nucleophiles. (Conditions: [ATZ]0 = 0.5 mM, [nuclophiles]0 = 20 mM, pH 5.8.0, ESI+).
sulfur-containing amino acids and peptides such as cysteine and glutathione. To verify these hypotheses, ATZ was incubated with glycine, cysteine, and glutanthione. Glycine contains amino group while the other two contain both amino and thiol groups. Data illustrated in Fig. 3 reveal that more ATZ was transformed in the presence of cysteine or glutanthione than equal molar content of aniline as expected. But almost no reduction of ATZ was seen in the presence of glycine with other conditions identical (data not shown). According to this, we postulate that the nitrogen in ␣amino acid was not ready to donate its lone pare electrons due to the electron withdrawn carbolic group nearby. The role of N in amino acids/peptides is probably limited in ATZ sequestration in soils. Hence, the removal of ATZ in cysteine and glutanthione was more likely due to the thiol group. MS analysis revealed that the MW of reaction product with cysteine was 301 (Fig. 2), consisting with nucleophilic substitution pathway with the elimination of an HCl (301 = 215 + 121 − 35). However, it cannot be differentiated whether the Cl was substituted by amine-N or thiol-S solely by MW. MS/MS analysis of the reaction product provided more insights in
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of thiols among the C–S compounds unknown, we still believe it is mostly these species that cause the formation of ATZ bound residue. This is because the residue concentration of ATZ in soil is usually in g/kg level [30–32] which is approximately three orders of magnitude less than that of total organosulfur. Thus, the availability of the sulfur nucleophiles is unlikely a limitation in the long run. 4. Conclusions
Fig. 4. Transformation of ATZ in soil fortified with various nucleophiles. (Conditions: [ATZ]0 = 10 mg/kg, [nucleophile]0 = 50 mM, pH 5.73, 6 days incubation).
this regard. A characteristic fragment m/z 214 which corresponding to a thiol substituted ATZ was generated upon collision of the molecular ion (m/z 302). The spectrum and proposed fragmentation pathway are given in Supporting data (Fig. S2). Sulfur-containing amino acids in sediments and terrestrial systems are derived from the decomposition of the biomass of animals, plants, microorganisms, and algae. Further degradation of these amino acids generates hydrogen sulfide, methanethiol, and other volatile organosulfur compounds [22]. Data illustrated in Fig. 3 demonstrated that these species were also potential nucleophiles to ATZ. The removal of ATZ in the presence of varying concentrations of sulfide appeared similar trend with glutathione. Due to the low solubility of methanethiol, we used mercaptoethanol to mimic the volatile thiol compounds. Although this compound seemed not as effective as sulfide, the enhanced removal of ATZ was still evident (Fig. 3). Sulfide and volatile organosulfurs are responsible for the offensive odor commonly found in eutrophic freshwater and terrestrial systems [23]. We speculate that transformation of ATZ bound residue would be facilitated in such conditions.
It has been shown that ATZ tends to be sequestrated in natural soil environment [18,19], but the underlying mechanisms by which ATZ binds to SOM remain largely unexplored. This is the first research that demonstrates nucleophilic substitution could be an important mechanism and sulfur-containing amino acids/peptides play a critical role as the nucleophile. We conceive this process would diminish xenobiotic transport, bioavailability, and toxicity [33]. Understanding such mechanisms and governing factors is of great importance to accurately predict the environmental behavior of ATZ, making scientific environmental risk assessment possible. On the other hand, mechanistic understanding of ATZ sequestion may catalyze novel ideas to mitigate ATZ contamination by taking advantage of such processes in management and/or remediation programs. Acknowledgements This research was funded by Natural Science Foundation of China (51178224) and the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institute. The content of the paper does not necessarily represent the views of the funding agencies. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhazmat. 2014.11.001.
3.3. Removal of ATZ in soil The above data demonstrated that nucleophilic substitution could in principle serve as a reaction pathway leading to the formation of ATZ bound residue. Considering soil is a complex matrix and the reactivity of the nucleophiles can be affected by a number of factors, the transformation ATZ in soil fortified with various nucleophiles was investigated and the results are shown in Fig. 4. In compare with the control with no external nucleophiles addition, the removal of ATZ in soils amended with aniline, glutathione, and mercaptoethanol was significantly enhanced, suggesting these nucleophiles played a key role in ATZ transformation. However, cysteine and sulfide which facilitated ATZ transformation in water samples did not show any effects in soil. Such results were not unexpected because cysteine tends to couple to each other to form cystine via formation of S S bound [24,25] and the sulfide is very insoluble in soil at normal conditions due to complexation with metals. It is noted that slight removal of ATZ was still observed in the control soil samples, although the incubation time was only 6 days. This suggests the presence of natural nucleophiles in the soil. We believe these nucleophiles are mostly sulfur compounds. It was estimated that more than 90% sulfur in soil is organosulfur and associated with soil organic matter [26–28]. Organosulfur contents of 194–853 mg/kg were reported in the rhizosphere soil of North America Great Plains. The contents in grassland and farmland soils are 135–411 mg/kg. More than 2/3 of the total organosulfur is in form of carbon–sulfur (C–S) compounds and the others are sulfuric acid esters (ester–SO4 ) [29]. In spite of the typical fraction
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J. Lu et al. / Journal of Hazardous Materials 284 (2015) 103–107 [12] J.K. Struthers, K. Jayachandran, T.B. Moorman, Biodegradation of atrazine by agrobacterium radiobacter J14a and use of this strain in bioremediation of contaminated soil, Appl. Environ. Microbiol. 64 (1998) 3368–3375. [13] C. Accinelli, G. Dinelli, A. Vicari, P. Catizone, Atrazine and metolachlor degradation in subsoils, Biol. Fertil. Soils 33 (2001) 495–500. [14] H.D. Skipper, C.M. Gilmour, W.R. Furtick, Microbial versus chemical degradation of atrazine in soils1, Soil Sci. Soc. Am. J. 31 (1967) 653–656. [15] R.T. Mandelbaum, L.P. Wackett, D.L. Allan, Mineralization of the s-triazine ring of atrazine by stable bacterial mixed cultures, Appl. Environ. Microbiol. 59 (1993) 1695–1701. [16] W.W. Mulbry, Purification and characterization of an inducible s-triazine hydrolase from rhodococcus corallinus NRRL B-15444R, Appl. Environ. Microbiol. 60 (1994) 613–618. [17] E. Barriuso, W.C. Koskinen, Incorporating nonextractable atrazine residues into soil size fractions as a function of time, Soil Sci. Soc. Am. J. 60 (1996) 150–157. [18] N.D. Jablonowski, S. Koeppchen, D. Hofmann, A. Schaeffer, P. Burauel, Spatial distribution and characterization of long-term aged 14C-labeled atrazine residues in soil, J. Agric. Food Chem. 56 (2008) 9548–9554. [19] H.M. Lesan, A. Bhandari, Contact-time-dependent atrazine residue formation in surface soils, Water Res. 38 (2004) 4435–4445. [20] E.J. Acosta, M.B. Steffensen, S.E. Tichy, E.E. Simanek, Removal of atrazine from water using covalent sequestration, J. Agric. Food Chem. 52 (2004) 545–549. [21] P.R. Wells, Linear free energy relationships, Chem. Rev. 63 (1963) 171–219. [22] X. Lu, C. Fan, W. He, J. Deng, H. Yin, Sulfur-containing amino acid methionine as the precursor of volatile organic sulfur compounds in algea-induced black bloom, J. Environ. Sci. (China) (25 2013) 33–43. [23] F. Einsiedl, B. Mayer, T. Schaefer, Evidence for incorporation of H2S in groundwater fulvic acids from stable isotope ratios and sulfur K-edge X-ray absorption near edge structure spectroscopy, Environ. Sci. Technol. 42 (2008) 2439–2444.
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[24] H. Alwael, D. Connolly, L. Barron, B. Paull, Development of a rapid and sensitive method for determination of cysteine/cystine ratio in chemically defined media, J. Chromatogr. A 1217 (2010) 3863–3870. [25] K. Kargosha, S.H. Ahmadi, M. Zeeb, S.R. Moeinossadat, Vapour phase Fourier transform infrared spectrometric determination of l-cysteine and l-cystine, Talanta 74 (2008) 753–759. [26] J. Eriksen, Soil sulfur cycling in temperate agricultural systems, Adv. Agron. 102 (2009) 55–89. [27] D. Solomon, J. Lehmann, I. Lobe, C.E. Martinez, S. Tveitnes, C.C. Du Preez, W. Amelung, Sulphur speciation and biogeochemical cycling in long-term arable cropping of subtropical soils: evidence from wet-chemical reduction and SK-edge XANES spectroscopy, Eur. J. Soil Sci. 56 (2005) 621–634. [28] F.J. Zhao, J. Lehmann, D. Solomon, M.A. Fox, S.P. McGrath, Sulphur speciation and turnover in soils: evidence from sulphur K-edge XANES spectroscopy and isotope dilution studies, Soil Bio. Biochem. 38 (2006) 1000–1007. [29] J. Wang, D. Solomon, J. Lehmann, X. Zhang, W. Amelung, Soil organic sulfur forms and dynamics in the great plains of North America as influenced by long-term cultivation and, Geoderma 133 (2006) 160–172. [30] P. Skladal, A.P. Deng, V. Kolar, Resonant mirror-based optical immunosensor: application for the measurement of atrazine in soil, Anal. Chim. Acta 399 (1999) 29–36. [31] G. Min, S. Wang, H. Zhu, G. Fang, Y. Zhang, Multi-walled carbon nanotubes as solid-phase extraction adsorbents for determination of atrazine and its principal metabolites in water and soil samples by gas chromatography–mass spectrometry, Sci. Total Environ. 396 (2008) 79–85. [32] I. Baranowska, H. Barchanska, R.A. Abuknesha, R.G. Price, A. Stalmach, ELISA and HPLC methods for atrazine and simazine determination in trophic chains samples, Ecotoxicol. Environ. Saf. 70 (2008) 341–348. [33] J. Dec, J.M. Bollag, Phenoloxidase-mediated interactions of phenols and anilines with humic materials, J. Environ. Qual. 29 (2000) 665–676.
Contents lists available at ScienceDirect
Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
Nucleophilic substitution as a mechanism of atrazine sequestration in soil Junhe Lu a,∗ , Juan Shao a , Deyang Kong b a b
Department of Environmental Science and Engineering, Nanjing Agricultural University, Nanjing 210095, China Nanjing Institute of Environmental Science, Ministry of Environmental Protection of PRC, Nanjing 210042, China
h i g h l i g h t s • Atrazine tends to form nonextractable bound residue in soil. • Nucleophilic substitution is a pathway leading to atrazine sequestration in soil. • Sulfur containing amino acids are likely to play an important role as nucleophiles during this process.
a r t i c l e
i n f o
Article history: Received 18 June 2014 Received in revised form 19 October 2014 Accepted 3 November 2014 Available online 7 November 2014 Keywords: Atrazine Sequestration Bound residue Nucleophic substitution
a b s t r a c t Formation of nonextractable residue was widely observed as a sink of atrazine (ATZ) in soil. However, the mechanisms by which ATZ binds to soil organic matter remain unclear. In this study, we demonstrated that neucleophilic substitution could serve an important pathway causing ATZ sequestration. The carbon bonded to the chlorine in ATZ molecule is partially positively charged due to the strong electronegativity of chlorine and is susceptible to the attack of nucleophiles such as aniline. Since aromatic amines are relatively rare in natural soils, amino acids/peptides were hypothesized to act as the main nucleophiles in real environment. However, substantially ATZ transformation was only observed in the presence of those species containing thiol functionality. Thus, we speculated that it was the thiol group in amino acids/peptides acting as the nucleophile. Nitrogen in amino acids was in fact not an active nucleophile toward ATZ. In addition to the sulfur-containing amino acids, other thiol compounds, and sulfide were also proved to be reactive to ATZ. Thus, the sequestration potential of ATZ probably correlates to the availability of thiol compounds in soil. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Atrazine (1-chloro-3-ethylamino-5-isopropylamino-2,4,6triazine, ATZ) is the most commonly applied herbicide to control broadleaf weeds and grasses. It was estimated that over 50 million pounds of active ATZ ingredients are applied annually in United States to corn, soybeans, sorghum, and other crops [1]. Once applied to the soil, ATZ has great chance to enter surface and ground waters through runoff or infiltration [2,3], because it is water soluble and relative conservative in the environment with low volatility, weak sediment partitioning, and relatively slow degradation rates (i.e., a half-life of months to years) [2]. As a result, ATZ is the most commonly detected herbicide in ground and surface waters [1,4]. The U.S. EPA has set the drinking water
∗ Corresponding author. Tel.: +86 25 84395164; fax: +86 25 84395210. E-mail address: [email protected] (J. Lu). http://dx.doi.org/10.1016/j.jhazmat.2014.11.001 0304-3894/© 2014 Elsevier B.V. All rights reserved.
maximum contaminant level (MCL) at 3 ppb, but ATZ concentrations often rise far above this level, especially after herbicide application and during runoff [5,6]. ATZ is a contaminant of environmental concern because its presence has been associated with causing imbalances in hormone levels in animals, possibly disrupting reproductive, and developmental processes [7–9]. Short term ATZ exposure above the drinking water MCL can potentially cause heart, lung, and kidney congestion, low blood pressure, muscle spasms, weight loss, and damage to the adrenal glands. Long term exposure to ATZ above the drinking water MCL has been linked to weight loss, cardiovascular damage, retinal, muscle degeneration, and cancer [10]. The high solubility and mobility of ATZ in combination with its relative resistance to degradation and significant health impacts makes ATZ contamination a serious threat to the environmental and human health [11], particularly in rural communities where ATZ is frequently used in agricultural activities but effective water treatment measures are not in place.
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J. Lu et al. / Journal of Hazardous Materials 284 (2015) 103–107
A thorough understanding of ATZ transformation in environment is critical for scientifically assessing the environmental risks associated with its application and for devising effective management and remediation means to mitigate its negative environmental and human health impacts. Extensive studies have been done in this topic and useful information obtained. ATZ undergoes both biotic and abiotic transformation in soil through two primary types of pathways: degradation and sequestration. While information regarding ATZ degradation is abundantly available [12–16], that relating to its sequestration is scarce. Evidences from experiments using 14 C-atrazine indicted that ATZ sequestration indeed occurred in soil resulting in the formation of ATZ bound residue with soil organic matter (SOM). The extent of such bound residue formation however varied greatly from case to case, which seemed to depend on factors that had yet to be understood. Barriuso and Koskinen found that nonextractable residues were formed immediately after atrazine application to unsterilized agricultural soil, and increased with the herbicide residence time in soil [17]. Jablonowski et al. tested a lysimeter soil containing long-term aged 14 C-ATZ for over twenty years and found 8.8% of the original 14 C activity remained in the top soil, most of which was bound to soil organic matter and could not be extracted [18]. Lesan and Bhandari concluded that longer atrazine-soil contact times resulted in enhanced nonextractable residue formation, and over an 84-day contact period, 35–50% of the pre-loaded 14 C-ATZ was found ending with residues in the organic components of soil [19]. All the above studies show distinctly that sequestration of ATZ and/or their metabolites occurs in soil, but mechanistic understanding about ATZ sequestration is still limited. In this research we demonstrated that neucleophilic substitution was a potential pathway leading to ATZ sequestration in environment, and certain sulfur containing amino acids/peptides probably played an important role as nucleophiles during this process. 2. Experimental 2.1. Chemicals and material ATZ was purchased from Sigma–Aldrich (St. Louis, MO). Phenol, gallic acid, catechol, resorcinol, quinol, salicylic acid, veratry alcohol, 4-methoxylphenol, aniline, glycine, cysteine, glutathione, methionine, mercaptoethanol, and Na2 S were all purchased from Aladdin (Shanghai, China). HPLC grade methanol and acetone were also from Sigma–Aldrich. Other chemical were reagent grade or better. All solutions were freshly prepared in deionized water.
be reactive to ATZ in previous experiments. The soil of yellow brown earth was collected from Xiamafang Park in Nanjing, China. The soil was dried, grinded, and sieved through 20-mesh screen before use. The pH of the soil was 5.73. Each reaction sample contained 5.0 g dry soil and sterilized before mixing with appropriate amount of ATZ dissolved in acetone to achieve a concentration of 10 mg/kg. After acetone was evaporated, 2.0 mL solution of 50 mM humic constituent or amino acid was spiked. The molar ratio of ATZ/humic constituent was approximately 1/440. After the soil samples was incubated in 20 ◦ C in dark for 6 day, the residue ATZ was extracted with 10 mL acetone twice facilitated with sonication. The extracts were combined and concentrated to 1 mL using a gentle stream of nitrogen gas. ATZ in the sample was quantified as described above. Control samples spiked with only ATZ were prepared and treated using identical procedure. Three replicates were performed for each reaction condition. 2.3. Products characterization To characterize the reaction products, selected samples with substantial ATZ removal were analyzed using an Agilent G6410B Triple Quad Mass spectrometer with an electron spray ionization source. The ionization source was operated in positive mode (ESI+). Nitrogen was used as desolvation gas and maintained at flow rate of 10 L/min. The desolvation temperature was set at 350 ◦ C. The mass analyzer was first run at scanning mode from m/z 100 to 1000. Secondary MS of the suspected reaction products were obtained subsequently. Fragmentor and collision energy were experimentally optimized. 3. Results and discussion 3.1. Removal of ATZ in the presence of humic constituents The complexity of SOM renders difficulty in the elucidation of xenobiotic–SOM interactions. A variety of substituted phenolic and anilinic compounds, so called humic constituents that mimic the structure of SOM were incubated with ATZ in an attempt to identify the specific functionalities that form covalent bonds with ATZ. It was revealed in Fig. 1 that ATZ was removed to different extent in the presence of different phenolic compounds. No reduction was seen in samples incubated with phenol or salicylic acid after 5 months. Appreciable attenuation was found in samples with gallic acid, veratry alcohol, catechol, resorcinol, quinol, and 4-methoxylphenol. Nonetheless, the removal in the presence of aniline was most pronounced and precipitants were observed. The removal increased consistently over time and exceeded 30% after
2.2. Reaction setup Reactions between ATZ and SOM were first explored in solutions in a series of glass vials as batch reactors. Each reactor contained 50 mL solution of 0.05 mM ATZ and one of the humic constituents (phenolic/anilinic compounds or amino acids listed in the previous paragraph) at concentrations from 0 to 50 mM. The pH of the solution was buffered at 5.8 using 1.0 mM phosphate. The reactors were kept in dark in an incubator maintained at 20 ◦ C. After predetermined incubation time, 1.0 mL sample was taken from each of the vials and analyzed using a Hitachi L-2000HPLC equipped with a photo-diode array (PDA) detector. A C18 reverse phase column (Hitachi LaChrom, 5 M × 250 mm × 4.6 mm) was used for separation. An isocratic elution consisting of 70% methanol and 30% water at a flow-rate of 1.0 mL/min was used as mobile phase. Controls with only ATZ were also prepared. Three replicate experiments were performed for each reaction condition. Reactions of ATZ was further explored in soil samples spiked with humic constituents and amino acids that were determined to
Fig. 1. Removal of ATZ in the presence of humic constituents. (Conditions: [ATZ]0 = 0.05 mM, initial concentration of the humic constituent was 0.50 mM, pH 5.8).
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Fig. 2. MS of ATZ and the adduct with nucleophiles.
5 month. Thus, amine group seemed play a critical role in facilitating ATZ transformation. We hypothesize that the removal of ATZ was due to the formation of adducts with aniline via nucleophilic substitution pathway, because amine is a common nucleophile and the carbon atom with chlorine attached in the triazine ring is partially positively charged induced by the electronegative chlorine and thus, susceptible to nucleophilic attack [20]. Oxygen attracts electrons more tightly making hydroxyl group a weaker nucleophilic than amine in aqueous phase, thus, less ATZ was removed in the presence of phenolic compounds [21]. Nucleophilic substitution mechanism was verified by MS analysis of the products. The data are given in Fig. 2. ATZ has a MW of 215, corresponding to the peak of m/z 216 in MS with ESI+ due to the formation of protonated cation. It is noted that this peak is accompanied by a (M + 2) isotope peak with abundance of approximately 1/3 of M peak, which is characteristic to Cl. In comparison with the control, m/z 216 shrank substantially and m/z 302 appeared as the most abundant peak in the reaction sample. The compound corresponding to this peak does not have the characteristic (M + 2) isotope, and its MW exactly fits to nucleophilic substitution pathway in which the Cl attached to the triazine ring was substituted by the aniline with the elimination of an HCl molecule (273 = 215 + 93 − 35). Secondary MS of this compound further rationalizes this statement (Supporting data, Fig. S1). 3.2. Removal of ATZ in the presence of nitrogen and sulfur nucleophiles Aniline moiety is relatively scarce in natural soil. Soil is indeed rich of amine (or amino) functionalities in forms of peptides and amino acids from biomass decomposition. Therefore, amino acids and peptides are presumed to play an important role in ATZ sequestration. Thiol compounds are another group of compounds in soil that could potentially serve as reactive nucleophiles. Generally, sulfur is more nucleophilic than nitrogen due to its larger size, which makes it readily polarizable and its lone pairs electrons readily accessible [21]. Sulfur nucleophiles are mainly certain
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(Conditions: [ATZ]0 = 0.05 mM, [nuclophiles]0 = 20 mM, pH 5.8, ESI+).
Fig. 3. Removal of ATZ in the presence of different nucleophiles. (Conditions: [ATZ]0 = 0.5 mM, [nuclophiles]0 = 20 mM, pH 5.8.0, ESI+).
sulfur-containing amino acids and peptides such as cysteine and glutathione. To verify these hypotheses, ATZ was incubated with glycine, cysteine, and glutanthione. Glycine contains amino group while the other two contain both amino and thiol groups. Data illustrated in Fig. 3 reveal that more ATZ was transformed in the presence of cysteine or glutanthione than equal molar content of aniline as expected. But almost no reduction of ATZ was seen in the presence of glycine with other conditions identical (data not shown). According to this, we postulate that the nitrogen in ␣amino acid was not ready to donate its lone pare electrons due to the electron withdrawn carbolic group nearby. The role of N in amino acids/peptides is probably limited in ATZ sequestration in soils. Hence, the removal of ATZ in cysteine and glutanthione was more likely due to the thiol group. MS analysis revealed that the MW of reaction product with cysteine was 301 (Fig. 2), consisting with nucleophilic substitution pathway with the elimination of an HCl (301 = 215 + 121 − 35). However, it cannot be differentiated whether the Cl was substituted by amine-N or thiol-S solely by MW. MS/MS analysis of the reaction product provided more insights in
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of thiols among the C–S compounds unknown, we still believe it is mostly these species that cause the formation of ATZ bound residue. This is because the residue concentration of ATZ in soil is usually in g/kg level [30–32] which is approximately three orders of magnitude less than that of total organosulfur. Thus, the availability of the sulfur nucleophiles is unlikely a limitation in the long run. 4. Conclusions
Fig. 4. Transformation of ATZ in soil fortified with various nucleophiles. (Conditions: [ATZ]0 = 10 mg/kg, [nucleophile]0 = 50 mM, pH 5.73, 6 days incubation).
this regard. A characteristic fragment m/z 214 which corresponding to a thiol substituted ATZ was generated upon collision of the molecular ion (m/z 302). The spectrum and proposed fragmentation pathway are given in Supporting data (Fig. S2). Sulfur-containing amino acids in sediments and terrestrial systems are derived from the decomposition of the biomass of animals, plants, microorganisms, and algae. Further degradation of these amino acids generates hydrogen sulfide, methanethiol, and other volatile organosulfur compounds [22]. Data illustrated in Fig. 3 demonstrated that these species were also potential nucleophiles to ATZ. The removal of ATZ in the presence of varying concentrations of sulfide appeared similar trend with glutathione. Due to the low solubility of methanethiol, we used mercaptoethanol to mimic the volatile thiol compounds. Although this compound seemed not as effective as sulfide, the enhanced removal of ATZ was still evident (Fig. 3). Sulfide and volatile organosulfurs are responsible for the offensive odor commonly found in eutrophic freshwater and terrestrial systems [23]. We speculate that transformation of ATZ bound residue would be facilitated in such conditions.
It has been shown that ATZ tends to be sequestrated in natural soil environment [18,19], but the underlying mechanisms by which ATZ binds to SOM remain largely unexplored. This is the first research that demonstrates nucleophilic substitution could be an important mechanism and sulfur-containing amino acids/peptides play a critical role as the nucleophile. We conceive this process would diminish xenobiotic transport, bioavailability, and toxicity [33]. Understanding such mechanisms and governing factors is of great importance to accurately predict the environmental behavior of ATZ, making scientific environmental risk assessment possible. On the other hand, mechanistic understanding of ATZ sequestion may catalyze novel ideas to mitigate ATZ contamination by taking advantage of such processes in management and/or remediation programs. Acknowledgements This research was funded by Natural Science Foundation of China (51178224) and the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institute. The content of the paper does not necessarily represent the views of the funding agencies. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhazmat. 2014.11.001.
3.3. Removal of ATZ in soil The above data demonstrated that nucleophilic substitution could in principle serve as a reaction pathway leading to the formation of ATZ bound residue. Considering soil is a complex matrix and the reactivity of the nucleophiles can be affected by a number of factors, the transformation ATZ in soil fortified with various nucleophiles was investigated and the results are shown in Fig. 4. In compare with the control with no external nucleophiles addition, the removal of ATZ in soils amended with aniline, glutathione, and mercaptoethanol was significantly enhanced, suggesting these nucleophiles played a key role in ATZ transformation. However, cysteine and sulfide which facilitated ATZ transformation in water samples did not show any effects in soil. Such results were not unexpected because cysteine tends to couple to each other to form cystine via formation of S S bound [24,25] and the sulfide is very insoluble in soil at normal conditions due to complexation with metals. It is noted that slight removal of ATZ was still observed in the control soil samples, although the incubation time was only 6 days. This suggests the presence of natural nucleophiles in the soil. We believe these nucleophiles are mostly sulfur compounds. It was estimated that more than 90% sulfur in soil is organosulfur and associated with soil organic matter [26–28]. Organosulfur contents of 194–853 mg/kg were reported in the rhizosphere soil of North America Great Plains. The contents in grassland and farmland soils are 135–411 mg/kg. More than 2/3 of the total organosulfur is in form of carbon–sulfur (C–S) compounds and the others are sulfuric acid esters (ester–SO4 ) [29]. In spite of the typical fraction
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