REMEDIATION OF CHLORPYRIFOS CONTAMINATED SOILS BY LABORATORY SYNTHESIZED ZERO-VALENT NANO IRON (ZVNI) PARTICLES: EFFECT OF pH AND ALUMINIUM SALTS

Degradation of the insecticide chlorpyrifos in contaminated soils was investigated using laboratory synthesized zero-valent nano iron (ZVNI) particles. The synthesized ZVNI particles were determined as nano scale by scanning electron microscopic (SEM) and transmission electron microscopic (TEM) analysis. The zero valent state (Fe0) of iron was confirmed by EDAX analysis and the morphology of the ZVNI particles was studied by XRD. Batch experiments were conducted by treating the chlorpyrifos contaminated soil with ZVNI, our results indicate that 90% of chlorpyrifos was degraded after 10 days of incubation. Only 32% degradation was observed with micro zero valent iron(mZVI) and no considerable degradation was attained without ZVNI. The degradation of chlorpyrifos has followed the first-order kinetics with a rate constant and a half-life of 0.245 day-1 and 2.82 days respectively. Degradation was monitored at two different pH values i.e. pH 10 and pH 4. Chlorpyrifos degradation rate constant was increased as the pH decreases from 10 to 4. The corresponding rate constant and half-lives were 0.43day-1 and 1.57days for pH 4, 0.18day-1 and 3.65days for pH 10. In addition, an attempt was made by augmenting Al2(SO4)3 with ZVNI and it was found that the degradation rate of chlorpyrifos was greatly enhanced and the rate constant was rapidly increased from 0.245day-1 to 0.60day-1. Hydrolysis and stepwise dechlorination pathway of chlorpyrifos with ZVNI was the dominant reaction.


Introduction
Chlorpyrifos [O,O-diethyl O-3,5,6-trichloropyridin-2-yl phosphorothioate], an organophosphorous insecticide, is widely used worldwide in agriculture and residential pest control [1].It is effective against a broad spectrum of insect pests on a variety of crops like grain, cotton, fruit, nut, and vegetable crops [2].It has low water solubility, 2 mgL −1 , but it is highly soluble in many organic solvents [3].Chlorpyrifos has high soil sorption coefficient (  = 13.4 to 1862 mL g −1 ) depending on the soil type with a half-life of 10 to 120 days in different soils [4,5].Like other organophosporous pesticides, its insecticidal action is due to the inhibition of the enzyme acetyl cholinesterase, resulting in the accumulation of the neurotransmitter, acetylcholine, at nerve endings [6,7], this results in the excessive transmission of nerve impulses, which causes a potential risk to the humans and other organisms.Chlorpyrifos industry has developed fastly in the past years worldwide, especially in some developineg countries, such as China and India.Many disputes over chlorpyrifos were also happened in the aspect of application security for its high neurotoxicity.Recently, there are several reports indicating the presence of chlorpyrifos in cold drinks [8,9], bottled drinking water and blood of farmers in Punjab, India.erefore, more attention should be paid to the degradation of chlorpyrifos and their residues in the soil, water, and waste sites contaminated by chlorpyrifos.Conventionally, bioremediation is a good option for the degradation of chlorpyrifos, but it has several operational constraints, like slow rate of biodegradation, requiring long time, and incomplete degradation of chlorpyrifos gives more harmful degradation products [10].Recently, nanotechnology represents a new generation of environmental technology that provides highly effective solutions to environmental cleanup and remediation by using zero-valent nano iron (ZVNI) (particle size 1-100 nm) as a chemical reductant [11].
e high effectiveness of ZVNI is due to its high surface area and surface reactivity, which is about 10-10,000 times higher than microscale zero-valent iron (mZVI) [12].Pesticides which are persistent in aerobic environments are more readily degraded under reducing conditions [13], thus generating a reducing environment in contaminated soils.As a powerful reducing agent, zero-valent iron for reductive dechlorination of toxic halogenated pesticides attracted a great deal of attention since the 1980s [14].
Remediation with ZVNI can promote rapid abiotic degradation via reductive dehalogenation.When halogenated pesticide pollutants are treated with ZVNI, oxidation of ZVNI to Fe (II) provides electrons for dechlorination [15].Under aerobic conditions, oxygen is the usual electron acceptor while, under anaerobic conditions, e − release from the reaction of ZVNI with water can be coupled to the reaction of chlorinated compound according to the equations [16]: ZVNI has been successfully used for remediation of halogenated pesticides and herbicides, chlorinated solvents, and heavy metal contaminants [17][18][19].Based on relevant literature cited on pesticide treatment options, ZVNI-based remediation may be a feasible and attractive option.Iron is inexpensive, nontoxic and environmentally compatible.Researchers showed that ZVNI has been successfully used to transform halogenated pesticides, chlorinated solvents, and polychlorinated biphenyls [20].
e objective of the study described in this paper was to quantify the effectiveness of our laboratory synthesized zero-valent nano iron (ZVNI) to remediate chlorpyrifos contaminated soils and investigate the effect of pH and aluminium salts on the rate of degradation of chlorpyrifos.A pH stat was used to determine the change of pH during the degradation by ZVNI.Finally, the mechanism of the transformation of chlorpyrifos on treatment with ZVNI was analyzed according to the experimental results.were procured in their highest grades available from Sigma-Aldrich (St. Louis, MO, USA).Ethanol, toluene, and all other solvents used were of analytical grade.Pure water was deionized and double distilled with a Milli-Q-water puri�cation system (Millipore).

Preparations of ZVNI Particles.
Unlike other conventional methods, ethanol was used as a stabilizer during the preparation of ZVNI particles.Preparation of ZVNI was achieved by the reduction of 30% ethanolic solution of 1 M ferrous sulfate with equal volume of 1.6 M sodium borohydride (6 mL min −1 ), addition of ethanol provides a protection layer for each iron nano particle.Here, the control of dropping rate is very important because quick addition may cause aggregation of ZVNI precipitates; in contrast very slow addition may cause the oxidation of nanoparticles which are formed sequentially.Ferrous iron was reduced and zerovalent iron particles were precipitated according to the equation [21]: When all the nanoparticles were precipitated on the bottom of the beaker, the supernatant was removed, and then the solid iron nano particles were washed with ethanol.Aer thoroughly drying the nanoscale iron, air was allowed to slowly bleed over the iron for a period of approximately 8 h to passivate the iron.e resulting black clusters of iron were ground and �ne ZVNI powder was stored under nitrogen environment for later use.

Nanoparticle Characterization.
Transmission electron microscope (TEM) image was taken by using Philips TECH-NAI FE 12 microscope.Morphological studies of the synthesized ZVNI particles were carried out by using scanning electron microscope (SEM) �tted with an Energy-Dispersive X-ray Analysis (EDAX) System (model CARL-ZEISS EVO MA 15).e sample was observed at 30,000 x magni�cation with an accelerating voltage of 20 kV.X-ray diffraction (XRD) analysis of ZVNI was performed using a Seifert 3003 TT X-ray diffractometer, the analysis was performed at 40 kV and 40 mA with Cu-K radiation at a wavelength of 1.542 A ∘ .Brunauer-Emmett-Teller (BET-)-speci�c surface area of ZVNI was determined by N 2 gas adsorption using Micromeritics ASAP-2000, USA.Microscale zero-valent iron particles (mZVI) were prepared by mechanically grinding the commercial �ne iron powder to diminish the particle size by electrical mortar grinder with a 130-W motor.

Extraction of Pesticide for GC-MS Analysis.
Chlorpyrifos was extracted from 20 g of soil in 50 mL Te�on centrifuge tubes by adding 30 mL toluene and agitated for a period of 4 h at 150 rpm on horizontal shaker at 25 ∘ C. e tubes were then centrifuged at 4000 × g for 10 min, and then the extract was �ltered through anhydrous sodium sulfate and evaporated by using a rotary evaporator (Rotavapor R-210/R-215-BUCHI) at 40 ∘ C. To the �ltrate, 1 mL of toluene was added and analyzed by GC-MS.A GC-MS (JEOL GCMATE II GC-MS) was used for the detection of intermediate products of chlorpyrifos during the degradation.eGC was equipped with a HP-5MS capillary column (30 m × 0.025 mm i.d) in helium carrier gas (1 mL per min) and with splitless injection system.e column was initially maintained at 90 ∘ C for 5 min and then increased to 290 ∘ C at a rate of 8 ∘ C per min and hold at 290 ∘ C for 5 min.e injector and interface temperature were kept at 280 ∘ C and the source temperature at 250 ∘ C. e injection volume was 1 L.Mass spectra were obtained by the electron impact (EI) at 70 eV using SIM mode.   1. Soil was spiked with chlorpyrifos (dissolved in methanol) to get an initial concentration of 20 mg of chlorpyrifos per Kg of soil.Chlorpyrifos retained in soil was extracted with toluene as above.Aer that the aliquots were removed and transferred to 1.5 mL microcentrifuged tubes for GC-MS analysis.Results showed that the recovery of chlorpyrifos was always in the range between 95-98%.20 g of chlorpyrifos contaminated soil was incubated in anaerobic condition with 0.2 g of ZVNI (1%) in 50 mL Te�on centrifuge tubes at 30 ∘ C and a soil/water content of 0.3 Kg/Kg.e sacri�cial samplings were obtained aer soil had been incubated for 2, 4, 6, 8, and 10 days, the trials were also conducted with microsized zero-valent iron (mZVI).e soil was extracted at preselected times with 30 mL toluene as described above and analyzed by GC-MS for identi�cation of intermediates and HPLC for quantitative analysis.
An attempt was also made to carry out the in�uence of aluminium sulfate [Al 2 (SO 4 ) 3 ] on the destruction of chlorpyrifos by ZVNI.20 g of Chlorpyrifos-contaminated soil (20 mg Kg −1 ) was treated with 0.2 g (1%) of ZVNI and 0.2 g (1%) of [Al 2 (SO 4 ) 3 ], 6 mL 0.3 kg/kg(w/w) of water were added.e sacri�cial samplings were obtained aer soil had been incubated for 2, 4, 6, 8, and 10 days.e soil was extracted at preselected times with 30 mL toluene as described previously and analyzed by GC-MS.

Results and Discussion
3.1.Characterizations of ZVNI Particles.Scanning electron microscope (SEM) images of our laboratory-synthesized ZVNI particles show that the surface of ZVNI was rough and looked like aggregated round-shaped and the particle size distribution was in the range of 10-90 nm (Figure 1(a)).Transmission electron microscope (TEM) image shows that most of the particles size of ZVNI was less than 100 nm (Figure 2(a)).Compared to the previous results [21], the size of ZVNI particles of this study is even small and uniform.Ethanol was �rst introduced into ZVNI synthesis [22]; to prevent NZVI oxidation; however, we found that ethanol not only could prevent oxidation, but also acts as a stabilizer during ZVNI synthesis due to its organic properties.In addition, the surface area of ZVNI was signi�cantly increased.XRD spectra of the synthesized ZVNI even show a little oxide peak, the very clear Fe 0 peak was also appeared (Figure 2(b)).As its support, XRD also indicated crystalline structure of the particles.Surface areas were determined by a Brunauer Emmett Teller (BET) N 2 gas adsorption desorption method at 77 K (Micromeritics ASAP-2000, USA).BET surface area analysis gave the surface area of ZVNI as 26.5 m 2 g −1 .e average particle diameter was determined to be approximately 40 nm.e diameter is in accordance with that reported previously.According to the EDAX analysis of nanoscale Fe 0 particles, the composition was found as 86.72% of iron and 13.28% of oxygen (Figure 1(b)).

Degradation of Chlorpyrifos by ZVNI.
Rapid degradation of chlorpyrifos in all trials was observed, the initial chlorpyrifos concentration of 20 mg L −1 was decreased to 90% by reduction with ZVNI aer 10 days of incubation.e relative persistence of commercial formulation of chlorpyrifos and its degradation rate were measured under anaerobic conditions in the laboratory.During the 10 days of incubation, the disappearance of chlorpyrifos was 90% with ZVNI, at the same time the disappearance of chlorpyrifos with mZVI was only 32% aer 10days of incubation (Figure 3(a)).e disappearance of chlorpyrifos with ZVNI followed the �rstorder kinetics (Figure 4).e calculated rate constant and half-life for the degradation of chlorpyrifos with ZVNI were 0.245 day −1 and 2.82 days respectively.We could be able to determine the feasibility of using 1% ZVNI (w/w) to remediate 20 mg Kg −1 chlorpyrifos contaminated soil.Soil pH was increased due to reduction of H 2 O by ZVNI in soil as per the equation: �ercentage of soil organic matter has not signi�cantly changed during the 10 days incubation.Soil aer treating with ZVNI showed the black compound and was found as magnetite by XRD analysis according to the equation: ZVNI promotes dechlorination and hydroxylation of chlorinated compounds [18], chlorpyrifos degradation products were analyzed at preselected times during treatment.e GC-MS analysis showed that ZVNI is able to degrade Chlorpyrifos during the 10 days of incubation.Degradation efficiency was increased with increasing the concentration of nano iron particles (Figure 3(b)), the degradation of chlorpyrifos was 90% aer 10 days of incubation, and the quantitative analysis was done by comparing the peak area of standard chlorpyrifos.e major degradation product was TCP ( ).To analyze the degradation products of chlorpyrifos, the soil was extracted with toluene and analyzed by GC-MS.e results suggested that hydrolysis of chlorpyrifos were occurred followed by dechlorination using zero-valent nano iron, it was inferred that our laboratorysynthesized ZVNI is functioned well as a reductant to degrade chlorpyrifos structure.

Effect of PH and Aluminium Sulfate on Chlorpyrifos
Degradation.Chlorpyrifos degradation by ZVNI was investigated under two pH conditions.We used a pH-stat apparatus (ELICO, LI-120) to control the pH in ZVNI-Chlorpyrifos matrix.To study the effect of soil pH on chlorpyrifos degradation, three replicates of different pH soils were treated with solution of chlorpyrifos dissolved in methanol to give a concentration of 20 mg Kg −1 .e soils were le on a laminar �ow bench for 3 to 4 h to allow evaporation of the solvent, pH of the soil has been adjusted by adding CaCO 3 (to increase soil pH to 10 ) and (NH 4 ) 2 SO 4 (to decrease soil pH to 4 ).Chlorpyrifos degradation rate constant was increased as the pH decreased from 10 to 4. Corresponding rate constant and half-lives were 0.43 day −1 and 1.57 day for pH 4, and they were 0.18 day −1 and 3.65 days for pH 10 (Figure 5).Slower destruction kinetics with increasing pH was previously observed with metachlolor treatment [18].e reason of this is the formation of secondary reductants Fe(II) or Fe(II)-containing oxides and hydroxides on the surface of ZVNI, which hinders the degradation.Lower pH would remove these passivating layers from ZVNI core and makes it free for further reaction with halogenated pesticides.Moreover, acid hydrolysis facilitates the degradation of chlorpyrifos at lower pH.Hence this process is strongly pH-dependent, since hydrolysis of chlorpyrifos was observed effectively at lower pH.Chlorpyrifos degradation efficiency was enhanced when [Al 2 (SO4) 3 ] was added to the soil.In soil incubation experiment, ZVNI with aluminium sulfate salt had successfully removed the total chlorpyrifos aer 6 days of incubation.e rate constant was rapidly increased from 0.24 day −1 to 0.60 day −1 , this indicates that rapid degradation of chlorpyrifos was noticed when ZVNI was augmented with Al 2 (SO 4 ) 3 because high concentration of aluminium salts can restrict passivation by moving the reaction products away from ZVNI surface [23].is supports that availability of Al(III), Fe(II) and Fe(III) during ZVNI oxidation results in increasing Fe(II) in the chlorpyrifos treatment system and thus favoring its degradation.Small, high-charge exchangeable cations, such as Al(III) or Fe(III), produce Bronsted acidity by promoting a reaction with water to release H + ions.e ranking potential Bronsted acid strength for Al(III) is high [24]; hence, Al can act as a Lewis acid by coordinating the moieties of some organic contaminants, bringing them closer to the surface of ironoxides for reductive transformation.

Degradation Pathway.
During the initial reaction period of chlorpyrifos, TCP was the most predominant by product.e reductive dechlorination by successive loss of chloride ions was con�rmed as the main pathway of degradation of chlorpyrifos with ZVNI.It is in agreement with several studies of dechlorinated compounds [25].According to the by product analysis, the base peak at m/z 197 was identi�ed as the major degraded product TCP.e main fragment ion spectrum of the peak at m/z 197, m/z 131, m/z 97, m/z 111, m/z 95, m/z 127, and m/z 115 are the major dechlorination products of TCP.e peak at m/z 169 was due to the formation of (DETP) 0,0-diethyl thiophosphate which was formed on the hydrolysis of chlorpyrifos (Table 2).e fragment ion peak at m/z 141 was due to the loss of C 2 H 5 OH from DTEP, and the peak at m/z 113 was due to the formation of phosphorothioic acid.In addition to above, the peak at m/z 314 corresponds to [M-HCl] + and the peaks at 286, 258 correspond to the removal of one and two ethylene molecules from [M-HCl] + .e �rst step in the reductive transformation is the formation of TCP.TCP's continous dechlorination resulted in the formation of maleamic acid and another byproduct DETP was transformed to phosphorothioic acid.Hence, the degradation of chlorpyrifos with ZVNI had occurred through a stepwise dechlorination mechanism.e proposed intermediates and pathway with the sequential reductive dechlorination of chlorpyrifos by ZVNI are shown (Figure 6) according to the byproduct analysis.

Conclusion
e results of the present investigation showed that ZVNI can be successfully used to remediate the soils contaminated with chlorpyrifos under anoxic environment at ambient temperature and pressure.Hydrolysis followed by reductive dechlorination was the major process in degradation of chlorpyrifos by ZVNI.e dechlorination product was maleamic acid.e dechlorinated product is environmentally benign and more biodegradable than its parent compound chlorpyrifos.Lowering the pH from 10 to 4 increases the destruction kinetic rates of chlorpyrifos by ZVNI.From the results, it can be suggested that ZVNI may be applicable for the remediation of concentrated chlorpyrifos contaminated soils.Moreover, aluminium salts can successfully increase the destruction of chlorpyrifos by ZVNI, the degradation of chlorpyrifos mainly seemed to be a stepwise dechlorination process.is is for the �rst time that chlorpyrifos has been degraded under reductive environment using ZVNI.Results from this study can be applied for on-site treatment of soil with high concentrations of chlorpyrifos contamination.

F 4 :
First-order kinetic model for chlorpyrifos degradation with ZVNI.

F 5 :
Time-course degradation of chlorpyrifos at different pH values.

3 F 6 :
Proposed pathway for the degradation of chlorpyrifos by ZVNI particles.

T 2 :
Major fragmentation peaks of chlorpyrifos with / values.