This work focused on photo-assisted crude peroxidase mediated transformations of chlorinated phenols (CPs) from spiked and industrial wastewaters and the identification of reaction products formed. Garden radish Raphanus sativus was the source of crude peroxidase. No chlorine bearing compounds were detected by gas chromatography-high resolution mass spectrometry analysis. Under identical test conditions, the concentrations of 4-chlorophenol and 2,4-dichlorophenol were demoted to zero from 514 mg/L, 652 mg/L and that of 2,4,6-trichlorophenol and pentachlorophenol were reduced to 18 mg/L and 37 mg/L from 790 mg/L and 1066 mg/L, respectively (high-pressure liquid chromatography analysis). Chloride ion release profiles also showed a progressively increasing trend. A neat chemical oxygen demand removal to the extent of 63–79% was achieved in the case of spiked wastewater sample and to the extent of 77% for industrial wastewaters. A hypothesis reaction scheme was also suggested to comprehend the mechanism of degradation reactions.

NOMENCLATURE

  • ARP

    arthromyces ramosus peroxidase

  • CP

    chlorophenol

  • 4-CP

    4-chlorophenol

  • 2,4-DCP

    2,4-dichlorophenol

  • 2,4,6-TCP

    2,4,6-trichlorophenol

  • PCP

    pentachlorophenol

  • GC-HRMS

    gas chromatography-high resolution mass spectrometry

  • COD

    chemical oxygen demand

  • (OH.)

    hydroxyl radical

  • RS

    Raphanus sativus

  • mg/L

    parts per million

  • HPLC

    high-pressure liquid chromatography

  • HRP

    horseradish peroxidase enzyme

  • SBP

    soybean peroxidase

  • UV

    ultraviolet

INTRODUCTION

Chlorophenols (CPs) are used as antiseptics, wood preservatives, herbicides, insecticides and fungicides (Xin-hua et al. 2005). CPs may be present in the aquatic environment in free or complex form, adsorbed on suspended inert solids or benthic sediments, or carried in biological tissues. They are also detected in surface and groundwaters (Howard 1989). Formed as by-products of pulp and paper, dyestuff, pharmaceutical and agrochemical industries (Ahlborg & Thunberg 1980) and incomplete incineration, CPs are highly toxic and poorly biodegradable (Mathialagan & Viraraghavan 2005).

The recalcitrant nature of CPs tends to aggravate the problem of wastewater remediation (Sharma et al. 2013). Several national and international conventions and regulations are already enforced to prevent the release of these pollutants into the environment (Lei et al. 2010). The conventional methods available for CP can be extremely costly when the ultimate objective is the total remediation of an effluent to fulfill the environmental legislations. Therefore the necessity is to explore detoxification processes entailing lower treatment costs by way of lesser consumption of chemicals and formation of fewer unwanted products of reaction which in the long run are imperative to sustainable development. Removal of aromatic compounds from wastewater by using peroxidase (Nicell et al. 1992; Buchanan & Han 2000; Sakurai et al. 2003) and hydrogen peroxide (Wright & Nicell 1999) has been extensively investigated. Nicelle et al. (1992) reported that when the horseradish peroxidase enzyme (HRP) catalyzed oxidation of aromatic compounds by H2O2, the reaction products were found to undergo a non-enzymatic polymerization to form water insoluble aggregates. Buchanan & Han (2000) investigated the ability of arthromyces ramosus peroxidase (ARP) to catalyze phenol removal from aqueous solution in the presence/absence of polyethylene glycol. The stoichiometry of phenol removal to peroxide consumption was observed to increase from 1.0 to 1.2 as initial phenol, peroxide and enzyme concentrations were increased. Wright & Nicell (1999) investigated the application of soybean peroxidase (SBP) to catalyze the polymerization and precipitation of aqueous phenols by hydrogen peroxide. They reportedly concluded that SBP can act on a broad range of compounds while retaining its catalytic ability over wide ranges of temperature and optimal activity at pH 6.4.

Despite the great deal of research (Ghioureliotis & Nicell 1999; Akhtar & Husain 2006; Ashraf & Husain 2009) demonstrating the effectiveness of peroxidase assisted remediation of model and industrial wastewaters, the implementation of this process on a large scale is deterred by the high cost of enzyme purification. Hence an additional way to facilitate enzymatic treatment by using crude enzymes is investigated.

The motivation for this work stemmed from the fact that plant materials possess inexpensive enzymatic agents for CP removal thus alleviating the need for enzyme isolation. The effectiveness of utilizing the same is investigated for selected CP congeners namely, 4-chlorophenol (4-CP), 2,4-dichlorophenol (2,4-DCP), 2,4,6-trichlorophenol (2,4,6-TCP) and pentachlorophenol (PCP). More specifically this research is focused on: (a) remnant concentrations of parent pollutant; (b) mineralization aspects by chemical oxygen demand (COD) and chloride release study (per Standard Methods); and (c) gas chromatography-high resolution mass spectrometry (GC-HRMS) analysis of reaction products during the applied treatments. A hypothesis reaction scheme is also suggested for the transition compounds and intermediates that may have been formed during the course of the reaction.

EXPERIMENTAL

Materials

The 4-CP was purchased from Kemphasol (Mumbai, India); 2,4-DCP was obtained from Suvidha (Mumbai, India); 2,4,6-TCP and PCP were obtained from Sigma-Aldrich (USA). Methanol (Qualigens, Mumbai, India), acetone (Molychem, Mumbai, India) and hexane (ACS Chemicals, Ahmedabad, India) were used as solvents. Hydrogen peroxide (30%) was obtained from Molychem (Mumbai, India). HCl, NaOH, H2SO4 and Na2HPO4 (Qualigen, Mumbai, India) were utilized for pH adjustment and phosphate buffer preparation. The concentration of CP congeners was 4.0 mM and that of H2O2 was 9.79 mM. Distilled water was used in cleaning and experimentation. The industrial effluent was collected from a local industrial area (Ankleshwar, Gujarat, India), acidified and kept at 4°C until use. It was characterized (Table 1) and subjected to identical conditions of degradation.

Table 1

Physicochemical characterization of effluent

Parameters Values 
Color Dark yellow to brown 
pH 5.8 
COD 145,000 
Cl 71 mg/L 
CP concentration (4-CP, 2,4-DCP, 2,4,6-TCP and PCP) 1719.97 mg/L 
4-CP, 60.17 mg/L; 
2,4-DCP, 660.7 mg/L; 
2,4,6-TCP, 892 mg/L; 
PCP, 107.1 mg/L 
Parameters Values 
Color Dark yellow to brown 
pH 5.8 
COD 145,000 
Cl 71 mg/L 
CP concentration (4-CP, 2,4-DCP, 2,4,6-TCP and PCP) 1719.97 mg/L 
4-CP, 60.17 mg/L; 
2,4-DCP, 660.7 mg/L; 
2,4,6-TCP, 892 mg/L; 
PCP, 107.1 mg/L 

Experimental procedure

The crude peroxidase enzyme was extracted in phosphate buffer (pH 9.5) from fresh garden radish (Raphanus sativus) by mechanical grating and pressing. The batch reactor for all the experiments was open to the atmosphere. The CP–crude peroxidase–H2O2 mixture in the ratio 1:2:2, was magnetically stirred for 30 minutes and then subjected to UV irradiation (254 nm, Philips, New Delhi, India) for 60 min. All 10 mL water samples were drawn at an interval of 10 min each and the pH was noted (pH meter, CL-46, Toshniwal, Ahmedabad, India). Samples with and without H2O2 served as controls for all experiments. Identical test conditions were maintained for spiked and industrial wastewaters.

Analysis

The residual concentrations were measured under the following experimental conditions by high-pressure liquid chromatography (HPLC) (Jasco, UK) analysis: column: Hypersil ODS/Kromasil C18 SGE 250 × 4.6 (5 μm); mobile phase: acetonitrile: water (40:60); pH: 2.6, adjustment using phosphoric acid; flow: 1.0 mL/min; temperature: 30 °C; range: bipolar, 1250 mV sample per second; detector: JASCO HPLC; detection: UV 210 nm. The GC-HRMS (Agilent 7890-Jeol AccuTOF GCV; USA) conditions were: mass range: 10–2000 amu; mass resolution: 6000; EI/CI source time of flight analyzer). In order to avoid interference in HPLC analysis, the blank run of oxidants was used for comparison with initial experimental results. The COD and chloride ion were analyzed per Standard Methods (Clesceri et al. 1998). As per Standard Methods, most of the organic matters are destroyed when boiled with a mixture of potassium dichromate and sulphuric acid producing carbon dioxide and water. A sample is refluxed with a known amount of potassium dichromate in sulphuric acid medium and the excess of dichromate is titrated against ferrous ammonium sulphate and ferroin as indicator. The amount of dichromate consumed is proportional to the oxygen required to oxidize the oxidizable organic matter. For Cl determination, the sample is titrated with silver nitrate solution. The end point is marked by formation of brick red silver chlorate formation. Results reported are averages of triplicate sample analysis. The results of reproducibility study were noted to be within 5% error.

RESULTS and DISCUSSION

Crude peroxidase in CP congener degradation

Peroxidases are predominantly heme proteins which utilize hydrogen peroxide (H2O2) or organic hydroperoxides as cosubstrate to oxidize a variety of organic and inorganic substrates (Demarche et al. 2012) (Table 2). The selected CP congeners are dechlorinated/mineralized by UV and crude peroxidase enzyme from Raphanus sativus at room temperature. The pH of the reaction medium dictates the extent of CP degradation and the nature of the reaction products formed (Sharma et al. 2013). The fluctuations in pH being minimal, the natural pH (of about 6–7) of the system under conditions of test is not altered. It is reported that in the presence of H2O2, peroxidase enzymes catalyze the oxidation of various chlorinated phenols, anilines, and other aromatics to free radicals, which may combine to form insoluble polymers (Dunford & Stillman 1976). It is known that H2O2 upon illumination with approximately, λ < 370 nm, undergoes homolytic splitting into (OH.) radicals, resulting in the mineralization of a great variety of pollutants (Dainton & Rowbottom 1953; Legrini et al. 1993).

Table 2

Review of CP congener degradation by various biological sources

Sr no. CP congener Experimental conditions Biological source Remarks Authors 
2,4,6-TCP pH, 3.0–7.0; H2O2, 10−3M for all the experiments. The oxidative dehalogenation of TCP carried out at 25 °C in small flasks (4 mL) with CPO (5 × 103 UL−1) as catalyst. Additionally, kinetic measurements carried out at pH 5.0 varying H2O2 concentration at two fixed TCP concentrations (10−5 and 10−4 M) Chloroperoxidase (CPO) Kinetic data were adjusted to the Hill equation and the kinetic parameters were obtained: n = 1.7 ± 0.2, vmax = (8.8 ± 0.3) × 10−5 M min−1, the pseudo-Michaelis constant Ks* = (8.6 ± 0.5) × 10−5 M, kcat = 677 ± 84 min−1 and the catalytic efficiency = (8.9 ± 0.6) × 106 M−1 min−1. The sigmoidal curve was related to the cooperative binding of the substrate to the enzyme, so that the binding of the first substrate molecule may help the binding of the second one Diaz-Diaz et al. (2010)  
4-CP Free and immobilized SBP and UV (produced by a KrCl excilamp) were used to treat 4-CP solutions, 50–500 mg L−1 SBP and KrCl excilamp (free/immobilized enzyme) Excilamp facilitates complete 4-CP elimination between 5 and 90 min. The enzyme removed 80% of the 4-CP concentrations, up to 250 mg L−1. At 500 mg L−1 the immobilized system shows higher removal efficiency due to increased enzyme stability in the presence of higher formation of by-products Gomez et al. (2009)  
4-CP pH 7.0, temperature 30 °C, and a reaction time of 90 min. the enzyme was dissolved in phosphate buffer 0.1 mol dm–3 and reagents 4-CP and H2O2 were dissolved in distilled water. H2O2 solution used was 100 mmol dm–3 and 4-CP solution was 20 mmol dm–3 SBP and H2O2 In the absence of PEG, good elimination of >90%, almost 90% for an enzyme concentration of 7.5 × 10–3 mg cm–3 and a stoichiometric ratio of 1:1 for both substrates, when concentrations of 1.5 mmol dm–3 or less were used is achieved Bodalo et al. (2006)  
4-CP Experiments conducted in a jacketed batch reactor (30 °C) of 50 mL total volume. Substrates, SBP and H2O2 were aqueous solutions Immobilized peroxidase The kinetic study of the immobilized SBP and H2O2 system used the expanded version of the Dunford mechanism, which extends the peroxidase cycle to the reaction products thus permitting a more general kinetic equation Gomez Carrasco et al. (2011)  
2,6-DMCP 5 mM solution of TCP in 100 mM potassium phosphate buffer at pH 7; protein concentrations, 500 and 260 Lm for DHP and HRP, respectively. Reactions were initiated by addition of a calculated amount of H2O2 HRP and dehalo-peroxidase Based on quantum mechanical/molecular mechanical (QM/MM) modeling and experimentally determined chlorine kinetic isotope effects, it was concluded that two sequential one electron oxidations led to a cationic intermediate that strongly resembles a Meisenheimer intermediate-a commonly formed reactive complex Szatkowski et al. (2011)  
2,4,6-TCP 
4-CP 
2,4-DCP Expression in yeast of lignin peroxidase (LiP) with improved 2,4-DCP degradability by DNA shuffling. LiP from P, chrysosporium was shown to mineralize/oxidize other recalcitrant compounds Lignin peroxidase (LiP) The resulting variants showed approximately 1.6-fold improved 2,4-DCP degradation activity and stability against H2O2 compared with the parent strain. The kinetic properties of the variants toward 2,4-DCP and H2O2 were also increased compared with the wild-type for all three mutants studied Ryu et al. (2008)  
Sr no. CP congener Experimental conditions Biological source Remarks Authors 
2,4,6-TCP pH, 3.0–7.0; H2O2, 10−3M for all the experiments. The oxidative dehalogenation of TCP carried out at 25 °C in small flasks (4 mL) with CPO (5 × 103 UL−1) as catalyst. Additionally, kinetic measurements carried out at pH 5.0 varying H2O2 concentration at two fixed TCP concentrations (10−5 and 10−4 M) Chloroperoxidase (CPO) Kinetic data were adjusted to the Hill equation and the kinetic parameters were obtained: n = 1.7 ± 0.2, vmax = (8.8 ± 0.3) × 10−5 M min−1, the pseudo-Michaelis constant Ks* = (8.6 ± 0.5) × 10−5 M, kcat = 677 ± 84 min−1 and the catalytic efficiency = (8.9 ± 0.6) × 106 M−1 min−1. The sigmoidal curve was related to the cooperative binding of the substrate to the enzyme, so that the binding of the first substrate molecule may help the binding of the second one Diaz-Diaz et al. (2010)  
4-CP Free and immobilized SBP and UV (produced by a KrCl excilamp) were used to treat 4-CP solutions, 50–500 mg L−1 SBP and KrCl excilamp (free/immobilized enzyme) Excilamp facilitates complete 4-CP elimination between 5 and 90 min. The enzyme removed 80% of the 4-CP concentrations, up to 250 mg L−1. At 500 mg L−1 the immobilized system shows higher removal efficiency due to increased enzyme stability in the presence of higher formation of by-products Gomez et al. (2009)  
4-CP pH 7.0, temperature 30 °C, and a reaction time of 90 min. the enzyme was dissolved in phosphate buffer 0.1 mol dm–3 and reagents 4-CP and H2O2 were dissolved in distilled water. H2O2 solution used was 100 mmol dm–3 and 4-CP solution was 20 mmol dm–3 SBP and H2O2 In the absence of PEG, good elimination of >90%, almost 90% for an enzyme concentration of 7.5 × 10–3 mg cm–3 and a stoichiometric ratio of 1:1 for both substrates, when concentrations of 1.5 mmol dm–3 or less were used is achieved Bodalo et al. (2006)  
4-CP Experiments conducted in a jacketed batch reactor (30 °C) of 50 mL total volume. Substrates, SBP and H2O2 were aqueous solutions Immobilized peroxidase The kinetic study of the immobilized SBP and H2O2 system used the expanded version of the Dunford mechanism, which extends the peroxidase cycle to the reaction products thus permitting a more general kinetic equation Gomez Carrasco et al. (2011)  
2,6-DMCP 5 mM solution of TCP in 100 mM potassium phosphate buffer at pH 7; protein concentrations, 500 and 260 Lm for DHP and HRP, respectively. Reactions were initiated by addition of a calculated amount of H2O2 HRP and dehalo-peroxidase Based on quantum mechanical/molecular mechanical (QM/MM) modeling and experimentally determined chlorine kinetic isotope effects, it was concluded that two sequential one electron oxidations led to a cationic intermediate that strongly resembles a Meisenheimer intermediate-a commonly formed reactive complex Szatkowski et al. (2011)  
2,4,6-TCP 
4-CP 
2,4-DCP Expression in yeast of lignin peroxidase (LiP) with improved 2,4-DCP degradability by DNA shuffling. LiP from P, chrysosporium was shown to mineralize/oxidize other recalcitrant compounds Lignin peroxidase (LiP) The resulting variants showed approximately 1.6-fold improved 2,4-DCP degradation activity and stability against H2O2 compared with the parent strain. The kinetic properties of the variants toward 2,4-DCP and H2O2 were also increased compared with the wild-type for all three mutants studied Ryu et al. (2008)  

The experiments are conducted without H2O2 first and then with addition of 10 mL H2O2 as an activator for the crude enzyme. Degradation of all CP congeners to the extent of 50% is confirmed by HPLC analysis. It is observed that the addition of H2O2 results in further degradation to the extent of 50–60%. The increased degradation of CPs is attributed to peroxides’ activity. Under identical test conditions, 4-CP is reduced to the extent of 95.8%. In the case of 2,4-DCP, degradation to the extent of 94.58% is achieved and the time course profile exhibits only marginal fluctuations. In the cases of 2,4,6-TCP and PCP the degradation is noted to be to the extent of 95.73% and 95.53%, respectively.

As per HPLC analysis results (Figure 1), 4-CP concentration was demoted to 20 mg/L under test conditions. This concentration is noted to be constant up to 50 min and no further degradation is observed. In the case of 2,4-DCP degradation, the CP decrease is 25 mg/L at 10 min, 24 mg/L remains almost constant up to 40 min and then finally a marginal decrease to 22 mg/L is observed. In the case of 2,4,6-TCP, the decrease is marginal from 18 to 14 mg/L during the observed time interval. In the case of PCP the decrease is from 36 to 32 mg/L in the observed time span. However the time course profile did not show any further decline in residual concentration. This observation leads the authors to believe that free radical scavenging effect may have posed as a limitation to further CP degradation (Sharma et al. 2013). The following reactions are presumed to occur: 
formula
1
 
formula
2
 
formula
3
Given the small time of in situ radical generation and action, an additional 5.0 mL dose of H2O2 is added in all cases. With an incremental addition of 5.0 mL H2O2, the concentrations of 4-CP, 2,4-DCP are demoted to zero under identical test conditions (HPLC analysis) and that of 2,4,6-TCP and PCP are reduced to 18 and 37 mg/L, respectively (Figure 2). This is a significant aspect of the present work.
Figure 1

Comparison of CP congeners concentration with time in synthetic samples (CP0, 4.0 mM; H2O2, 9.79 mM; UV, 254 nm; pH, 6–7).

Figure 1

Comparison of CP congeners concentration with time in synthetic samples (CP0, 4.0 mM; H2O2, 9.79 mM; UV, 254 nm; pH, 6–7).

Figure 2

CP congeners concentration profile showing the effect of H2O2 in synthetic samples (CP0, 4.0 mM; H2O2, 9.79 mM; UV, 254 nm; pH, 6–7).

Figure 2

CP congeners concentration profile showing the effect of H2O2 in synthetic samples (CP0, 4.0 mM; H2O2, 9.79 mM; UV, 254 nm; pH, 6–7).

The noteworthy results of enhanced CP degradation by UV-enzyme-H2O2 paved the path for remediation of industrial wastewater samples containing 4-CP, 2,4-DCP, 2,4,6-TCP and PCP (Table 1). CP degradation was noted to be 98.41, 98.94, 99.93 and 98.8% for 4-CP, 2,4-DCP, 2,4,6-TCP and PCP, respectively (Figure 3).

Figure 3

CP congeners concentration profile in industrial wastewater samples (C04-CP, 60.17 mg/L; C02,4-DCP, 660.7 mg/L; C02,4,6-TCP, 892 mg/L; C0PCP, 107.1 mg/L; H2O2, 9.79 mM; UV, 254 nm; pH, 6–7).

Figure 3

CP congeners concentration profile in industrial wastewater samples (C04-CP, 60.17 mg/L; C02,4-DCP, 660.7 mg/L; C02,4,6-TCP, 892 mg/L; C0PCP, 107.1 mg/L; H2O2, 9.79 mM; UV, 254 nm; pH, 6–7).

Mineralization studies

In the present investigation, CP congener degradation is followed by COD and chloride ion analysis. The first step in the degradation process by ultraviolet Raphanus sativus (UV-RS) action is presumed to be the dechlorination of the substituted phenols followed by mineralization. Chloride ions are known to be released during the enzymatic coupling processes of chlorophenols (Dec & Bollag 1994). A neat COD decline trend is observed (Figure 4) in cases of all CP congener degradation by UV-RS action. An appreciable COD decrease to the extent of 60–80% is observed for all the selected CP congeners. In the case of industrial effluent treatment, the COD of non-irradiated samples was higher compared to those of the samples subjected to UV irradiation. The COD removal to the extent of 77% was noted in the UV irradiated industrial effluent samples. The chloride ion release is marked by a steady increase (Figure 5). It is observed that the test conditions also enabled enhanced release of chloride ions upon irradiation as compared with the non-irradiated samples. This leads the authors to believe that degradation/mineralization does take place by UV-RS synergistic action.

Figure 4

COD depletion with time (CP0, 4.0 mM; H2O2, 9.79 mM; UV, 254 nm; pH, 6–7).

Figure 4

COD depletion with time (CP0, 4.0 mM; H2O2, 9.79 mM; UV, 254 nm; pH, 6–7).

Figure 5

Chloride ion concentration with time (CP0, 4.0 mM; H2O2, 9.79 mM; UV, 254 nm; pH, 6–7).

Figure 5

Chloride ion concentration with time (CP0, 4.0 mM; H2O2, 9.79 mM; UV, 254 nm; pH, 6–7).

Identification of reaction products (GC-HRMS)

There is scant literature available about the reaction products of CP congener degradation by enzymatic action. Hence the identification of reaction products assumes considerable significance. The presence of diverse compounds in the industrial wastewater matrix may interfere in the enzyme mediated CP degradation process. Hence, reaction products were identified by GC-HRMS (Tables S1–S5, available online at http://www.iwaponline.com/wst/072/269.pdf). The photo-assisted enzymatic CP degradation leads to the formation of reaction products having no chlorine substitutions. The major compounds formed upon 4-CP degradation include butane, (retention time, 3.1 min) oxiranemethanol and glycidol (retention time, 3.8 min each) and acetaldehyde (retention time, 4.3 min) (Figure S1, available online at http://www.iwaponline.com/wst/072/269.pdf).

Upon 2,4-DCP degradation by UV-RS, 1,2,4-triazol-3-amine, 5-(1,3,5-trimethyl-4-pyrazolyl)amino (retention time, 3.1 min) and propanenitrile, 3-(5-diethylamino-1-methyl-3-pentynyloxy)- and 2,6 dichlorophenol (retention time, 4.1 min) are detected (Figure S2, online at http://www.iwaponline.com/wst/072/269.pdf). 2,4,6-DCP degradation by UV-RS leads to the formation of butanal, 3-hydroxy- (retention time, 3.1 min) ethanamine, 2-propoxy- (retention time, 3.1 min); acetic acid, [(aminocarbonyl)amino]oxo- (retention time, 3.8 min) and ethanol, 2-(2-aminoethoxy)- (retention time, 3.8 min); 2,4,6-trichlorophenol (retention time, 6.8 min), dimethylphthalate (retention time, 6.8 min) are observed (Figure S3, online at http://www.iwaponline.com/wst/072/269.pdf). PCP degradation results in the formation of phthalic anhydride (retention time, 5.8 min) and 1,2-benzenedicarboxylic acid (retention time, 5.8 min), phthalic acid, 2-isopropylphenyl methyl ester (retention time, 8.3 min), pentachlorophenol (retention time, 13.8 min) (Figure S4, online at http://www.iwaponline.com/wst/072/269.pdf). Industrial wastewater samples upon reaction with UV-RS show the formation of dimethyl phthalate (retention time, 8.2 min) as the degraded product (Figure S5, online at http://www.iwaponline.com/wst/072/269.pdf).

In this investigation appreciably high removal of CP congeners is confirmed by GC-HRMS analysis. The dechlorination process is presumed to be the main pathway for CP congener degradation/mineralization which tends to proceed by (OH.) attack on Cl substitutions. It is presumed that the highly substituted CP congeners, PCP and 2,4,6-TCP may have degraded to 4-CP or 2,4-DCP with stepwise elimination of chlorine atoms. A hypothesis reaction scheme is proposed for the transition compounds and intermediates which may have been formed during the course of CP degradation from industrial wastewaters by UV-RS action (Figure 6 (a)(c)).

Figure 6

Proposed hypothesis reaction scheme for the transition compounds and intermediates which may have been formed during the course of CP degradation by UV-RS action from industrial wastewaters.

Figure 6

Proposed hypothesis reaction scheme for the transition compounds and intermediates which may have been formed during the course of CP degradation by UV-RS action from industrial wastewaters.

The presence of 2,4-DCP, 2,6-DCP and 2,4,6-TCP in 2,4-DCP and 2,4,6-TCP degradation, respectively, may be attributed to scavenging action of (OH.) radicals. The presence of –NH2 in simple straight chain aliphatic compounds and in cyclic intermediates may be due to the proteinaceous nature of the enzyme. It is hypothesized that chloride ions after being removed from the aromatic ring are bound in hydrochloric acid, and also on small-molecular-weight organic acids. The presence of dimeric reaction products is also reported by Laurenti et al. (2002). It is presumed that the (OH.) radical attacks the aromatic carbon with its unpaired electron, forming a C–O bond. This leads to the formation of dimeric intermediates, cyclic structures and simple straight chain aliphatic compounds. Observations from the present investigation further support the hypothesis that dimeric intermediates tend to be formed in the matrix as a result of the peroxidase activity. Thus crude peroxidase activity efficiently facilitated degradation of CP congeners in spiked as well as industrial wastewater matrix.

CONCLUSIONS

A combined photochemical-enzymatic treatment integrating the main advantages from both methods, with an initial photo-degradation process followed by an enzymatic treatment, thus appears to be a promising alternative for CP removal. The photo-assisted enzymatic approach reported here presents an edge over other methods in demoting CP concentration. Since crude peroxidase is employed in this investigation, the cost and isolation of the enzyme are no longer debatable issues. However, issues related to polymerization, suicide action, optimization and determination of the fate of spent enzyme may need to be addressed for effective application of this strategy for other recalcitrant compounds’ degradation. The present investigation thus establishes the feasibility of the use of crude peroxidase enzyme as an inexpensive source of peroxidase in CP congener degradation/mineralization.

ACKNOWLEDGEMENTS

The authors are thankful to M/s Gujarat Insecticides Limited, Ankleshwar, Gujarat for HPLC analysis and Sophisticated Analytical Instrument Facility (SAIF), IIT Mumbai for GC-HRMS analysis.

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