Environ. Sci. Technol. 2001, 35, 713-716
Hydrolysis of an Organophosphate Ester by Manganese Dioxide DARREN S. BALDWIN Murray-Darling Freshwater Research Centre and the CRC for Freshwater Ecology, P.O. Box 291, Albury, New South Wales 2640, Australia JAMES K. BEATTIE* AND LYNETTE M. COLEMAN School of Chemistry, University of Sydney, Sydney, New South Wales 2006, Australia DAVID R. JONES EWL Sciences, P.O. Box 39443, Winnellie, Northern Territory 0821, Australia
Amorphous manganese dioxide facilitates the hydrolysis of p-nitrophenyl phosphate to p-nitrophenol and orthophosphate despite insignificant adsorption of p-nitrophenyl phosphate or p-nitrophenol to the manganese dioxide. At pH 8, the orthophosphate product is released into solution; at pH 4 and pH 6, some remains adsorbed. The rate of hydrolysis is an order of magnitude more rapid than the same reaction facilitated by iron oxides. Because manganese dioxides are ubiquitous components of soils and sediments, this suggests the possibility of significant abiotic pathways for the formation of bioavailable orthophosphate from phosphate ester precursors.
Introduction The bioavailability of phosphorus is an important factor in controlling aquatic plant growth and eutrophication. The adsorption of phosphate by mineral phases has been extensively studied. Strong binding occurs with, for example, iron and aluminum hydrous oxides that are positively charged within the range of pH values typically found for natural waters. Manganese oxides, however, are negatively charged around pH 7 and have much lower affinities for anions such as phosphate. Nevertheless, phosphate does bind to manganese oxides at neutral pH (1). In seawater, the adsorption of phosphate by amorphous manganese dioxide is comparable to that by goethite on a mass basis despite the lower affinity of phosphate for the manganese surface. The higher surface area and site density of the amorphous manganese oxide leads to significant adsorption. We have recently shown that mineral phases also facilitate the hydrolysis of organophosphate esters, indicating another role in their influence on phosphorus availability (2). Three manganese dioxide samples were examined in a survey of the effects of metal oxides on the hydrolysis of p-nitrophenyl phosphate (NPP) (3). Two of the oxides, an amorphous manganese dioxide and akhtenskite (-MnO2) (4), effected the hydrolysis of NPP without exhibiting marked adsorption of the NPP in the initial stages of the reaction. The third form of manganese dioxide, pyrolusite, adsorbed NPP before hydrolysis occurred. Since the amorphous manganese dioxide * Corresponding author e-mail:
[email protected]; phone: +61 2 9351 3797; fax: +61 2 9351 3329. 10.1021/es001309l CCC: $20.00 Published on Web 01/12/2001
2001 American Chemical Society
exhibited a very fast rate of hydrolysis without initial adsorption of significant NPP, this oxide has been investigated further to determine if the mechanism for the facilitated hydrolysis reaction with manganese dioxide differs from that of oxides that adsorb much of the NPP prior to hydrolysis occurring. The mechanism of the hydrolysis reaction was studied by first characterizing the surface of the oxide and then determining the pH dependence of the reaction. The effects on the rate of hydrolysis of the concentrations of NPP and oxide and of added phosphate were examined. Manganese dioxide is a strong oxidizing agent, so the possibility of reductive dissolution of the oxide by oxidation of the product, p-nitrophenol, during the hydrolysis reaction was also explored (5).
Experimental Section Materials. Amorphous manganese(IV) dioxide was made by slowly adding a KMnO4 solution (400 mL; 0.8 M) heated to 65 °C to a MnSO4 solution (300 mL; 1.6 M) heated to 90 °C and then heating for a further 20 min at 90 °C. The resulting brown suspension was washed with about 2 L of hot distilled water until the filtrate was no longer pink in color to remove excess KMnO4. The product was resuspended in cold distilled water and filtered again to remove excess electrolytes. This process was repeated until the filtrate had a conductivity of 0.3 mS cm-1. After this washing, the oxide was placed in an evaporating dish and oven-dried for 1 h at 100 °C. Characterization Methods. The specific surface area of the prepared manganese dioxide was determined using a Quantasorb apparatus. Adsorption isotherm data were analyzed by the BET surface area method. The samples were degassed overnight at 150 °C. Nitrogen was used as the carrier gas, and a nitrogen-helium mix was used for the adsorptiondesorption cycles. The powder X-ray diffraction pattern of the oxide samples was recorded with a Siemens Kristoloflex X-ray diffractometer. The sample was prepared by grinding in an agate mortar and pestle. The pattern was recorded over 35° at a collection rate of 1°/min. The pH of the point of zero charge, pHpzc, of the manganese dioxide was determined by performing acid-base titrations of the oxide suspended in three different electrolyte concentrations of 0.001, 0.01, or 0.1 M KCl. The oxide loading was 1 g/L. The titration curves were plotted on the same graph, and the point of intersection of the three curves was taken to be the pHpzc. The number of phosphate adsorption sites was determined by suspending the oxide in 0.01 M KCl prepared with doubly distilled water (0.1 g; 100 mL) and adjusting the pH of the suspension to pH 6.00 with dilute HCl or NaOH. A solution of Na2HPO4 solution was then added to give final concentrations of 1.6, 16, or 1.6 × 102 µM phosphate. The suspensions were stirred for 6 h and filtered through 0.4-µm Nucleopore polycarbonate membrane filters. The phosphate remaining in solution was determined colorimetrically using the Murphy and Riley molybdenum blue method (6). Each experiment was duplicated and corrected for the blank. Hydrolysis Experiments. The catalyzed hydrolysis of the model phosphate ester, p-nitrophenyl phosphate (NPP), was examined because of the ease with which the concentrations of both the nitrophenyl phosphate and the hydrolysis product, p-nitrophenol (NP), can be determined. In acidic conditions, the absorbance peaks for NP and NPP are superimposed. However, under basic conditions the NP is deprotonated to form the nitrophenolate ion, which is bright VOL. 35, NO. 4, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Time dependence of NPP and NP concentrations at pH 6 at an initial concentration of 62 µM NPP in a suspension of 1 g/L of amorphous MnO2. yellow and has an absorbance maximum at 400 nm, quite distinct from NPP, which has an absorbance maximum at 310 nm. The molar extinction coefficients at 310 and 400 nm, respectively, are as follows: NP, 1.17 × 103 and 1.83 × 104 M-1 cm-1; NPP, 1.00 × 104 and 63 M-1 cm-1. The hydrolysis reaction was initiated by adding a known concentration of NPP to a suspension of 0.25 g of manganese dioxide in 250 mL of 0.01 M KCl prepared with Milli-Q water with the pH adjusted by the addition of HCl or NaOH. The solid oxide was kept in suspension by magnetic stirring, and the temperature was maintained at 25.0 ( 0.1 °C in a water bath. The pH was measured before the addition of the NPP and subsequently through the reaction time. Aliquots of the suspension were removed 5 min after initiation and at subsequent regular intervals. The concentrations of NPP and NP were measured colorimetrically after the 5-mL aliquot of the suspension was syringe-filtered through a 25 mm diameter Advantec glass fiber GA55 filter placed over a 0.4 µm Nucleopore polycarbonate filter. The fate of phosphate released by the hydrolysis was investigated by experiments at pH 4.0 and pH 8.0 with an initial nitrophenyl phosphate concentration of 35 µM. The hydrolysis reactions were monitored as described above by measuring the production of nitrophenol. Additional 5-mL aliquots were removed for analysis of phosphate with an autoanalyzer using the colorimetric method developed by Murphy and Riley (6).
Results Characterization of the Oxide. The oxide was characterized by X-ray powder diffraction, BET surface area measurements, and pHpzc measurements. The oxide was found to be mostly amorphous material with a weak X-ray diffraction pattern similar to a hydrated R-manganese dioxide (7); the specific surface area was 135 m2 g-1. The pHpzc was approximately 2.3, which is lower than the pHpzc for R-manganese dioxide, pH 4.6 (8). Several other manganese dioxides have been identified with reported pHpzc values of 1.5 and 3.0 for birnessite (9), 1.5 and 2.0 for cryptomelane (9), and 1.5 (8) and 2.25 (10) for δ-manganese dioxide. The pHpzc for this oxide is therefore consistent with the published range of values for preparations of amorphous manganese dioxide. Products of Reaction. The facilitated hydrolysis of NPP by manganese dioxide (1 g/L, equivalent to 11.5 mM MnO2) was followed to completion at pH 4 and at pH 6 and to about 40% completion at pH 8, with initial NPP concentrations of 55, 62, and 32 µM, respectively. The changes in the concentrations of NPP and NP as a function of time are shown for pH 6 in Figure 1, for pH 4 in Figure 2, and for pH 8 in 714
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FIGURE 2. Time dependence of NPP and NP concentrations at pH 4 at an initial concentration of 55 µM NPP in a suspension of 1 g/L of amorphous MnO2.
FIGURE 3. Time dependence of NPP and NP concentrations at pH 8 at an initial concentration of 32 µM NPP in a suspension of 1 g/L of amorphous MnO2. Figure 3. The sum of the concentrations of NPP and NP measured in solution was used to test if either adsorbs on the manganese dioxide surface. Figures 1 and 3 show that the sum of the concentrations of NP and NPP in solution at pH 6 and pH 8 equals the initial NPP concentration over the entire reaction period, which indicates that neither NP nor NPP adsorb significantly to the surface of the oxide at these pH values. However, it is evident from Figure 2 that at pH 4 there is initially some adsorption of the NPP to the manganese dioxide. The sum of NPP and NP increases with time as all of the NPP is hydrolyzed and released as NP, which does not adsorb significantly to the surface of manganese oxides (11). Manganese dioxides are powerful oxidizing agents that can oxidize substituted phenols (5). The possible dissolution of manganese by reaction (reductive dissolution) with nitrophenol produced in the hydrolysis reaction was therefore investigated. The concentration of dissolved manganese in the filtrate from a sample of manganese dioxide before and after the hydrolysis of NPP was determined by graphite furnace AAS. The small increase in Mn(II) found in solution after the hydrolysis reaction had gone to completion was not greater than the blank Mn(II) levels, which indicates that a reductive dissolution mechanism in the reaction of NP and manganese dioxide does not occur. This is consistent with the literature description of the oxidation of substituted phenols by manganese dioxide, which showed that pnitrophenol was unreactive in the presence of manganese
FIGURE 4. pH dependence and NPP concentration dependence of the initial rates of hydrolysis. dioxide (5). This is also consistent with the observed 1:1 stoichiometry between NPP and NP shown in Figure 1. The fate of the phosphate produced by the hydrolysis reaction was determined by measuring the concentration of phosphate in solution during the hydrolysis reactions at pH 4 and pH 8, with an initial NPP concentration of 35 µM. At pH 4, the concentration of phosphate in solution increased to a maximum of 7 µM, approximately 20% of the total phosphate produced by the hydrolysis of NPP. After the hydrolysis reaction was completed, the final solution concentration of phosphate was only 3 µM as the phosphate released into solution by the faster hydrolysis reaction was slowly re-adsorbed. At pH 8, however, the concentration of phosphorus in solution was equal to the concentration of NPP hydrolyzed, indicating that no significant amount of the 35 µM phosphate was adsorbed to the surface at this pH. The adsorption of phosphate to manganese dioxide is known to decrease with increasing pH (12). Balistrieri and Choa also showed that about 75% of 4.8 µM phosphate was adsorbed on a 100 mg/L suspension of amorphous manganese dioxide at pH 4, but only about 10% (0.5 µM) was adsorbed at pH 8 (13). Rates of Hydrolysis. The rates of hydrolysis of NPP by manganese dioxide (11.5 mM equivalent) were determined between pH 4 and pH 8 with initial NPP concentrations in the range of 5-65 µM (Figure 4). The initial rates were calculated from a least-squares fit of the concentration of NP with time in the first minutes of the reaction during which time the rate was linear. The initial rates of reaction decreased with increasing pH. Below pH 6, the rate increased with increasing initial NPP concentration until a saturation limit was reached above an initial NPP concentration of about 32 µM. The increase in rate is less than first order with respect to NPP concentration, so that the first-order rate constants decrease with the increase in NPP concentration. At pH 6 and above, the dependence of the initial rates on increasing initial NPP concentration decreases still further until at pH 8 there is little dependence on the NPP concentration. This reflects the decreasing adsorption of phosphate and NPP as the pH increases. The hydrolysis reactions were first order with respect to the rate of NP formation except at pH 6 for an initial NPP concentration of 64 µM, for which biphasic behavior was observed. The biphasic behavior disappears if the experiment is conducted with the same initial NPP concentration but with twice the oxide loading. This is consistent with the saturation limit at pH 6 of 32 µM described above and suggests that the biphasic behavior occurs because the surface sites of the oxide are saturated by the substrate concentration of
FIGURE 5. Dependence of the initial rates of hydrolysis of 30 µM NPP on oxide loading (g/L) at pH 4 (upper line) and pH 8 (lower line).
FIGURE 6. Inhibition by 36 µM phosphate of the hydrolysis of 36 µM NPP. 32 µM at the lower oxide level. In the biphasic reaction, the faster rate is ascribed to the NPP adsorbed to the surface in the first fast step of the reaction. The second, slower reaction is that of the excess NPP that must subsequently be adsorbed onto a surface that is increasingly negatively charged by the presence of the phosphate product adsorbed at pH 6. The existence of this biphasic behavior in the presence of excess NPP implies that NPP binds more strongly to the surface than does the product orthophosphate. Oxide Loading Effect. The dependence of the initial rates of reaction on the concentration of manganese dioxide was determined by measuring the rates of the hydrolysis at pH 4 and pH 8 with an initial NPP concentration of 30 µM and oxide concentrations between 0.4 and 2.2 g/L (Figure 5). The initial rates of hydrolysis are linearly dependent on the oxide concentration with slopes of the log-log plots of 1.10 ( 0.09 at pH 4 and 1.06 ( 0.09 at pH 8, which indicate that the hydrolysis reaction is first order with respect to oxide concentration. Product Inhibition. Inhibition of the reaction by phosphate was examined by measuring rate of reaction after addition of equimolar concentrations (36 µM) of NaHPO4 and NPP to suspensions of manganese dioxide at pH 4, pH 6, and pH 8. Figure 6 shows that there is significant product inhibition at pH 4 and pH 6.
Discussion The hydrolysis of NPP is clearly facilitated by manganese dioxide. The homogeneous rate is negligible by comparison (14). This is consistent with the first-order dependence on the mass of added oxide (Figure 5). At pH 8, this process can VOL. 35, NO. 4, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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be described as catalysis, for both the phenol and phosphate products are released into solution and not adsorbed. However, at pH 4 most of the phosphate remains adsorbed on to the oxide, and added phosphate inhibits the NPP hydrolysis reaction. Manganese dioxide is unusual among the oxides previously surveyed for catalytic activity because it does not adsorb significant amounts of NPP prior to the release of NP into solution. Examples of related heterogeneous catalysis in which there is no significant adsorption of the substrate to the catalytic surface are known. These include the hydrolysis of phenyl picolinate by goethite and titania (15), the oxidation of As(III) to As(V) by manganese dioxide (16), and the oxidation of 1,4-dihydroxybenzene by manganese dioxide (17). All of these reactions involve the adsorption of the substrate to the surface of the oxide. The fact that there is little net absorption of the primary reactant prior to appearance of the product implies that either the equilibrium binding constant is low or that the rate of the primary adsorption process is slower than that of the subsequent reaction of the adsorbed species (that is, the adsorption process is rate limiting). Previous workers have suggested that anion adsorption onto amorphous manganese dioxide is weak because of the net negative surface charge above pH 2 (18-20). The second acid dissociation of NPP has a pKa2 of 5.18. At pH 4, therefore, the monoanion predominates, while at pH 6 and above the dianion is the dominant species. The probably accounts for the limited adsorption of the NPP to the manganese dioxide surface below pH 6 and no measurable adsorption above this pH. The pH dependence of the rate constants for the catalyzed hydrolysis of NPP thus reflects two factors: differences between the monoanion and dianion of NPP and the pH dependence of the surface charge of the manganese oxide. The data do not allow a distinction between the two effects to be made. What is clear is that the reaction is fastest at pH 4, where some of the NPP monoanion is adsorbed and where the surface charge of the oxide is less negative than at higher pH values. The rate constants at pH values less than 6 decrease significantly with increasing initial NPP concentration. At pH 8, however, the rate constants are almost independent of the NPP concentration. This behavior is consistent with stronger adsorption of the monoanion than of the dianion. At low pH, the surface of the oxide becomes saturated at low NPP concentrations. As the concentration of NPP at the surface increases, the net negative surface charge increases making the adsorption of more NPP more difficult. The inhibition by phosphate displays a similar pattern with inhibition at pH 4 and no effect at pH 8. At the lower pH, phosphate from the hydrolysis reaction remains adsorbed to the surface. In addition, added phosphate also binds and therefore blocks active surface sites. This effect should lead to product inhibition and deviation from first-order kinetics as phosphate is produced at pH 4, but the kinetics data were not precise enough to observe this effect. The pKa2 of orthophosphate is 7.2. At pH 8, all the phosphate produced by the hydrolysis reaction is released into solution, reflecting the weak interaction between the surface and the phosphate dianion, and added phosphate does not inhibit the hydrolysis reaction. The hydrolysis of NPP by amorphous manganese dioxide is an order of magnitude more rapid, per gram of oxide, than the same reaction facilitated by hydrous ferric oxide (2). This difference is probably due to the increased acidity of the surface hydroxyl groups of the manganese(IV) surface, with pHpzc of 2.3, as compared with hydrous iron(III) oxide with pHpzc of 8.2. Hence, the rate of reaction decreases from pH 4 to pH 8 for the manganese system, but for the iron system it reaches a maximum at pH 6.9 (21). The observed differences 716
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are consistent with a role for surface hydroxyl groups acting as nucleophiles in the hydrolysis mechanism. This would be an analogous process to that observed for intramolecular hydrolysis of phosphate esters in discrete metal complexes in homogeneous aqueous solution (for example, ref 22). p-Nitrophenyl phosphate has been used as a substrate to measure what is believed to be the activity of phosphatase enzymes in soils (23, 24). Hence, it is possible to compare “enzymatic” activity measured in soil systems with the abiotic rates measured for manganese dioxide in this work. In one study with phosphomonoesterase at its optimum pH between 6 and 7, approximately 1 µmol of p-nitrophenol was released h-1 (g of soil)-1 at 37 °C. This can be compared with the rate at pH 6 shown in Figure 1 of about 50 µmol of NP/g of amorphous manganese. In another study, the maximum hydrolysis rate calculated from the integrated MichaelisMenten equation was of the order 10-100 µmol h-1 (g of soil)-1 (23), comparable to the abiotic rates observed here. While there are many assumptions made in these comparisons, it seems clear that the abiotic process is potentially competitive with the inferred activity of phosphatase enzymes. Similar conclusions have been reached in a parallel study on the hydrolysis of tripolyphosphate also facilitated by amorphous manganese dioxide (12).
Acknowledgments The financial support of the Australian Research Council Small Grants Scheme is gratefully acknowledged.
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Received for review May 29, 2000. Revised manuscript received November 9, 2000. Accepted November 14, 2000. ES001309L
