Environ. Sci. Technol. 2010, 44, 4981–4987
Effect of Chitosan on the Enantioselective Bioavailability of the Herbicide Dichlorprop to Chlorella pyrenoidosa YUEZHONG WEN,† YULI YUAN,† HUI CHEN,† DONGMEI XU,‡ KUNDE LIN,§ A N D W E I P I N G L I U * ,†,§ MOE Key Laboratory of Environmental Remediation & Ecosystem Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310029, China, College of Biology and Environmental Engineering, Zhejiang Shuren University, Hangzhou 310015, China, and Research Center of Green Chirality, College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032, China
Received February 14, 2010. Revised manuscript received May 16, 2010. Accepted June 3, 2010.
Chitosan, a natural polysaccharide, is widely regarded as a biocompatible and potential carrier for controlled-release drug formulations. However, the potential effect of chitosan on the environmental behavior of contaminants is poorly understood, especially on chiral chemicals. In this study, the changes in bioavailability of the chiral herbicide dichlorprop to Chlorella pyrenoidosa by chitosan were investigated. The dissipation of (S)enantiomer in Chlorella pyrenoidosa culture media without chitosan was faster than that of the herbicidally active (R)enantiomer, whereas it was inversed to (R)-enantiomer being faster than (S)-enantiomer when chitosan was added into the media. In the absence of chitosan, the toxicity of (R)enantiomer to Chlorella pyrenoidosa was more potent than that of the (S)-enantiomer. On the contrary, in the presence of chitosan, (R)-enantiomer was less toxic than (S)-enantiomer. These observations clearly suggest that chitosan changed the enantioselective bioavailability of dichlorprop. Fluorescence spectroscopic analysis showed that the interaction between chitosan and dichlorprop enantiomers depended greatly on the steric structure of dichlorprop, which offers a possible explanation as to why the addition of chitosan changed the enantioselective dissipation of dichlorprop by Chlorella pyrenoidosa. This work suggests that the enantioselective behaviors of chiral compounds in the environment might be shifted when interactions with other chiral receptors coexist.
Introduction The extensive use of pesticides in agriculture is becoming a serious environmental concern because of the potential runoff and leaching of these compounds through the soil * Corresponding author tel: +86-571-8832-0666; fax: +86-5718832-0884; e-mail:
[email protected]; current address: MOE Key Laboratory of Environmental Remediation & Ecosystem Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310029, China. † Zhejiang University. ‡ Zhejiang Shuren University. § Zhejiang University of Technology. 10.1021/es100507p
2010 American Chemical Society
Published on Web 06/10/2010
resulting in contamination of the surface and groundwater. To reduce the leaching potential and prevent groundwater contamination and maintain the efficacy of pesticides, various controlled release formulations (CRFs) of pesticides have been developed during the past few decades. Natural polysaccharides have especially been receiving increasing attention due to their biocompatibility and useful biological reactivity (1, 2). Up to 25% of the members of several classes of pesticides are chiral (3), and this ratio is increasing as compounds with more complex structures are introduced into use (4). Enantioselective biological effects associated with chiral pesticides have received considerable attention (5-7). It is found that environmental factors can affect the chiral preference of pesticides. Lewis et al. (5) have investigated the effect of organic nutrients on enantioselectivity of methyl dichlorprop. They found that enrichment with organic nutrients caused soils that preferentially transformed the (+) enantiomer of dichlorprop to shift to strongly preferentially transforming the inactive (-) enantiomer in samples from Brazil and North America. However, in soils from Norway that already preferentially transformed the (-) enantiomer, the addition of nutrients caused the microorganisms preferring the (+) enantiomers to increase in activity but most samples (74%) still preferred the (-) enantiomer. In addition, other studies (8-10) have shown that the soil pH and redox conditions are important factors affecting the chiral preference of pesticides and such effects were thought to be the result of the activity of different microorganisms and enzyme systems involved in the degradation process. However, little information is available about the influence of the interaction between chiral pesticides and polysaccharides on their bioavailability and ecotoxicity. It is worth noting that the interactions between polysaccharides and some low molecular chiral chemicals are enantioselective, because polysaccharides contain chiral glucose units (11). Therefore, it is necessary to reveal how polysaccharides affect the bioavailability and ecotoxicity of chiral chemicals. Dichlorprop is an acid herbicide with a moderate water solubility of 620 mg/L. It is widely used on wheat, corn, sorghum, and olive trees to control broad-leaf weeds. Dichlorprop is weakly adsorbed by soil particles (12) and can pose a potential risk to surface and groundwater contamination, particularly if heavy rain events occur shortly after the herbicide application. Therefore, the design of formulated forms of dichlorprop with reduced mobility is of particular interest. Chitosan, a natural polysaccharide, is widely regarded as a biocompatible and potential carrier for controlled drug release formulations (13-16). However, drug transport of chitosan is dependent on its positive charges, which may pose a problem at physiologic pH. To overcome this difficulty, nanoparticle chitosan systems were developed, partly because the nanoparticles could protect a labile drug load from chemical and enzymatic hydrolysis. Chlorella pyrenoidosa is a green unicellular alga and has been widely used to evaluate the impacts of pesticides and their bioavailability in aquatic systems, since they are sensitive to organic contaminants at environmentally relevant concentrations (17, 18). To evaluate the possible effect of chitosan on the enantioselective environmental behaviors of chiral contaminants, we investigated the influence of chitosan on the enantioselective dissipation and toxicity of the chiral herbicide dichlorprop in the freshwater alga Chlorella pyrenoidosa. VOL. 44, NO. 13, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Materials and Methods Chemicals. (R)-Dichlorprop, (S)-dichlorprop, and (Rac)dichlorprop with 99% purity were synthesized according to Camps’ method (19). Chitosan nanoparticles and fluorescein isothiocyate (FITC)-labeled chitosan were prepared according to the literature procedures (20-22); the final concentration of FITC in the reaction medium was controlled to give a label to D-glucosamine residue ratio of 1:50. Details on the preparation of chitosan nanoparticles and FITC-labeled chitosan are given in Supporting Information Text S1 and Text S2. Chitosan was supplied by the Zhejiang Yuhuan Ocean Biochemical Company (the degree of deacetylation was 90%). Other reagents were commercially available and used without further purification. Stock solution of 300 mg/L dichlorprop and 100 mg/L chitosan (either as dissolved chitosan or as chitosan nanoparticles) were also prepared in sodium phosphate buffer (pH 6.0). Algal Culture. The freshwater microalgae Chlorella pyrenoidosa were used as a test organism. The initial stock organisms were obtained from the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). The algae were cultivated in algal growth media (sterilized HB-IV), which is composed of the following chemical ingredients (mg/L): (NH4)2SO4, 200; Ca(H2PO4)2 · H2O, 30; MgSO4 · 7H2O, 80; NaHCO3, 100; KCl, 25; FeCl3, 1.5; and 0.5 mL/L soil extract, at 24 ( 1.0 °C in an incubator under continuous illumination of 4500-5000 lx. In each incubator, the cultures were shaken five times per day to ensure optimal growth. The algae were periodically inoculated into the fresh media to keep the cells in the logarithmic growth phase and to prepare them for subsequent bioassays. Dichlorprop Dissipation Experiment. To evaluate the dissipation of dichlorprop by Chlorella pyrenoidosa, a proper amount of algae (to reach a final density of 8.0 × 105 cells/ mL) was inoculated into flasks containing 50 mL of test solution. All the test solutions were prepared with the HB-IV media. For the treatments without chitosan, the test solutions contained 3 mg/L of (S)-, (R)-, or (Rac)-dichlorprop. To determine the effect of chitosan, a set of test solutions contained 3 mg/L dichlorprop and 1.95 mg/L chitosan (either as dissolved chitosan or as chitosan nanoparticles), which were incubated at room temperature for 24 h prior to inoculation. Dichlorprop-free solutions with 1.95 mg/L chitosan were also included to serve as control. All treatments were conducted in triplicate. At times of 0, 2, 7, 12, 24, 48, and 72 h, an aliquot of 1.0 mL algal solution was withdrawn from the flasks and centrifuged at 6000 rpm for 15 min. The supernatant was collected and immediately subjected to HPLC analysis to determine dichlorprop residue. Algal Growth Inhibition Assay. The algal growth inhibition assay was carried out using Chlorella pyrenoidosa according to the updated Organisation for Economic Cooperation and Development (OECD) Guideline 201 for the freshwater algal growth inhibition assay. The toxicity of dichlorprop and chitosan was evaluated independently in preliminary experiments that generated the concentration-response curves for Chlorella pyrenoidosa. The optimal molar ratio (dichlorprop/chitosan) was also determined to be 1:1 (3 mg/L dichlorprop and 1.95 mg/L chitosan) in the preliminary experiment. To do the inhibition test, the algae were precultured in HB-IV media at 24 ( 1.0 °C to maintain the exponential growth phase. A certain amount of algae suspension was inoculated into the test solutions (pH 6.0) in flasks (all flasks were sterilized prior to use) to reach a final density of 8.0 × 105 cells/mL in the final 50.0 mL solution. Test solutions were the same as used in the dissipation experiment described before. After inoculation, all flasks were incubated at 24 ( 1.0 °C under continuous illumination of 4500-5000 lx. The flasks were shaken five times per day and repositioned daily 4982
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within the cultural chamber to minimize any possible spatial differences in illumination and temperature. At times of 0, 24, 48, 72, 96, 120, and 144 h, an aliquot of suspension from each treatment was collected to analyze the density of algal cell. According to linear equations between direct cell counts and optical density at 680 nm (OD680), the inhibition of algal growth relative to the control was determined by measuring the OD680 using a Shimadzu UV-2401PC UV-visible spectrometer (Tokyo, Japan). Each treatment was conducted in quadruplicate. The growth inhibition rate was calculated as Inhibition rate ) (control absorbance sample absorbance)/control absorbance
(1)
Chemical Analysis. Dichlorprop residue in the supernatant was determined on a reversed-phase HPLC system coupled with an SPD-M10A photodiode array detector. The detection wavelength was 234 nM. A Kromstar C18 column (4.6 mm ×250 mm) was employed for the separation. The isocratic mobile phase consisted of 80% methanol and 20% water at a flow rate of 0.8 mL/min (each 500.0 mL mobile phase also contained 0.1 mL of 85% concentrated phosphoric acid). The injection volume was fixed at 10 µL. Fluorescence spectra were recorded using a Hitachi F-2500PC fluorescence spectrometer (Tokyo, Japan). The scanning parameters were as follows: excitation and emission slit width of 15 nm, scanning speed of 1500 nm/min, Xe flash lamp light source, excitation wavelength of 470 nm, and scanning range of 495-550 nm. All the experiments were conducted in triplicate. The effects of chitosan (30 µM) on dichlorprop at different concentrations (0-2.25 mM) were investigated. Three hundred microliters of 500 µM FITClabeled nanoparticles or FITC-labeled chitosan molecules were added to dichlorprop solution and the volume was fixed to 5 mL with 0.1 M HAc (pH ) 2.8). The concentrations of dichlorprop were 0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.2, 1.6, and 2.25 mM. The samples were put in a constant temperature slot, and the temperature was controlled at 20 or 25 °C. The mixtures were incubated for 24 h. The scanning parameters were as follows: excitation and emission slit width of 15 nm, scanning speed of 1500 nm/min, Xe flash lamp light source, excitation wavelength of 470 nm, and scanning range of 495-550 nm. The chitosan nanoparticles were examined by transmission electron microscopy (TEM) using a JEM-1200EX (MinatoKu, Japan). The surface charge characteristics and particle sizes of chitosan nanoparticles were measured by an electrophoretic light scattering spectrophotometer (NANO-ZS90, Malvern Instruments Ltd., Malvern, England). The X-ray photoelectron spectroscopy (XPS) study was performed on the electron spectrometer Thermo ESCALAB 250 (Thermo Electron Corporation, MA); the samples were excited with X-rays over a specific 400-µm area using monochromatic Al KR radiation (1486.6 eV) at 150 W. Chitosan (as received neutral form) and nanoparticle chitosan samples were vacuum-dried at room temperature for 72 h prior to analysis. All binding energies were referenced to the C1s neutral carbon peak at 284.8 eV, and surface elemental stoichiometries were determined from sensitivity-factor corrected peak ratios. Statistical Analysis. The data were analyzed using the Origin 6.0 program (OriginLab, Northampton, MA) according to the methods provided by the manufacturer of the test kit. The differences were considered statistically significant when p was less than 0.05 or 0.01.
Results and Discussion Physicochemical Properties of Chitosan and Nanoparticle Chitosan. A previous study has shown that proper characterization of nanoparticle properties is essential to understanding their behavior (23). Subtle changes in nanoparticle
TABLE 1. Surface Functionality Properties of Chitosan and Nanoparticle Chitosan from the X-ray Photoelectron Spectroscopy Analysis element
chitosan
chitosan nanoparticle
C (%) O (%) N (%)
60.3 32.7 7.0
56.8 36.4 6.8
preparation, storage, or use during experiments can greatly affect results. A TEM image for the nanoparticle chitosan in Figure S1 shows numerous tiny particles in the form of agglomerates. The diameters of the individual (primary) particles were R, indicating that the spontaneous reaction of (S)-dichlorprop with chitosan would occur more easily.
The main attractive forces between small organic molecules and biological macromolecules are hydrophobic interactions, hydrogen bonds, Van Der Waals forces, and electrostatic forces (31-33). Ross (32) reported that hydrophobic interactions can make positive contributions toward enthalpy of binding (∆H) and entropy of binding (∆S). On the other hand, hydrogen bonds and Van Der Waals forces will contribute negatively to ∆H and ∆S while electrostatic forces may result in ∆H < 0 and ∆S > 0. As indicated by ∆H > 0 and ∆S > 0 in Table 2, it can be speculated that the hydrophobic interactions might play a leading role in the interaction between chitosan and dichlorprop. Qaqish and Amiji (21) have studied the interactions between chitosan and mucin using FITC-labeled chitosan. Their research also showed that electrostatic interactions were not the primary mode of chitosan-mucin association. For instance, at a pH value of 4.0, more than 99% of the D-gluosamine residues and 96% of the sialic acid residues would be ionized. However, the fluorescence polarization data showed that the highest increase in polarization occurs at more acidic pH (2.9). At a pH of 2.9, even though almost 100% of the D-glucosamine would be ionized, only 20% of the sialic acid residues are ionized. Chitosan consists of one amino group and two free hydroxyl groups on each glucose ring. The protonation of the amine groups is necessary for the attraction of acid groups, thus the electrostatic attraction between chitosan and dichlorprop should play a critical role. However, Qaqish and Amiji’s research results suggest that in addition to electrostatic interactions, there could be other attractive forces involved in chitosan-small organic molecule binding such as hydrogen bonding and/or hydrophobic interaction. This opinion is consistent with our result reported above. Because the chiral interactions are governed by hydrophobic interactions (34), to focus on chiral interaction between chitosan and dichlorprop, our experiments to characterize the interaction of chitosan with dichlorprop were conducted at pH 2.8 so as to exclude the effect of ionization of D-glucosamine of chitosan (almost 100% ionization in pH 2.8). Because the electrostatic interactions and hydrophobic interactions are all present in the range of pH 2.8-6.0, therefore, the chiral interaction mechanism proposed at pH 2.8 should be valid at the pH of the toxicity and dissipation experiments (pH 6.0). To interpret the experimental results regarding to chiral interaction between chitosan and dichlorprop, we employed molecular mechanics and dynamics simulations of the interaction. The molecular mechanics model Version 2 (MM2) from Chem3D Ultra 8.0 (Cambridge Soft) was used to compute the steric energy of chitosan-dichlorprop complexes. Chitosan structure is formed by single helices because the energy of formation of a single helix is not higher than 12.5 kJ/mol (35). It has been reported that the phenyl groups of organic molecules and the glucose units of chitosan are ordered in the gels in a face-to-face arrangement (11). Thus, the chitosan dimer shown in Figure S3 was used as a model of chitosan. Considering the strongly electrostatic binding of the -NH2 groups of chitosan, we hypothesized that the carboxyl of dichlorprop was bound to the NH2 groups of chitosan, the MM2 computed the steric energy for an optimized structure of an (R)-dichlorprop-chitosan complex and an (S)-dichlorprop-chitosan complex (Figure 4). The results show that the steric energy of an (R)-dichlorprop-chitosan complex is 0.9493 kJ · mol-1, greater than the steric energy of an (S)-dichlorprop-chitosan complex (-0.0311 kJ · mol-1). From Figure 4, a face-to-face orientation between the phenyl group of dichlorprop and the glucose units of chitosan can be seen. The primary OH group at C6 is also sterically easily bound to the aromatic residues in dichlorprop. The difference is the location of the position of the -CH3 group of dichlorprop. In an (R)-dichlorVOL. 44, NO. 13, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Acknowledgments We thank the anonymous reviewers for their constructive suggestions to improve the clarity and quality of this manuscript. This study was supported by the National Natural Science Foundation of China (20977078, 20907046, and 20837002), the National Basic Research Program of China (2009CB421603), the Research Projects of Zhejiang Education Department (Y200803107), and the Program for Changjiang Scholars and Innovative Research Team in Chinese Universities (IRT 0653). FIGURE 4. 3D molecular structures of chitosan-dichlorprop complexs generated by CS Chem3D Ultra 8.0 with the MM2 molecular mechanics force-field method. prop-chitosan complex, the -CH3 group is located between the phenyl and chitosan, increasing the steric resistance. However, in an (S)-dichlorprop-chitosan complex, the -CH3 group was far from the phenyl and chitosan. Thus, (S)dichlorprop was easy bound to chitosan. Thus, it indicated that chitosan can change the distribution ratios of dichlorprop between the liquid that contained chitosan and the algae and this change was driven by the binding constants. In Figure 2a chitosan was not present and greater binding of (S)-dichlorprop to algae would lower the amount of (S)-dichlorprop in the supernatant relative to (R)-dichlorprop. In Figure 2b chitosan was present and the 3-fold greater chitosan binding constant of the (S)-dichlorprop increased the remains of (S)-dichlorprop relative to (R)-dichlorprop. Environmental Implications. These results have important implications for controlled pesticide and drug release techniques, public health, and environmental protection. The chiral preference of the herbicide dichlorprop by chitosan, for example, raises new questions with respect to the application of the controlled drug release formulations using chitosan as a carrier, which can change the enantioselective behaviors of chiral chemicals. This is a fortuitous situation for dichlorprop, since the (R)-enantiomer is known to be the active herbicides, while the (S)-enantiomer is simply “ballast”. The lower toxicity and lower dissipation rate of the herbicidally active (R)-enantiomer in the environment are favorable for the application of chitosan. However, it is unlikely that chitosan always has a positive effect on the biological activity of all chiral chemicals. If the effects are adverse, the higher toxicity and higher dissipation rate of the active enantiomer in the environment in the presence of chitosan would not be favorable for the application of chitosan because it would pose a risk to public health and the environment. Although this study considered only one small molecule (dichlorprop) and one large molecule (chitosan), the concept is relevant for other chiral compounds and the findings may have broad implications. For instance, such interactions may diminish or enhance the chiral selectivity in the efficacy of a chiral drug or pesticide when chiral macromolecules are used a carrier. On the other hand, in the environment, chitosan has been proposed for a number of agricultural applications (36, 37). Thus, there are large residual amounts of chitosan and chitin in environment. When chiral pesticides enter the aquatic environment after their application as herbicides, they may interact with chiral receptors, leading to a possible shift in bioavailability. As polysaccharide such as chitosan may be ubiquitous, interactions of chiral contaminants with these macromolecules may affect the direction and degree of chiral selectivity in the behavior of chiral contaminants. Thus, the effects of polysaccharide such as chitosan and chitin on the safety of chiral chemicals need to be explored. 4986
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Supporting Information Available Texts S1-S2, and Figures S1-S3 as described in this paper. This material is available free of charge via the Internet at http://pubs.acs.org.
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