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Page 1 of 19 Environmental Science: Processes & Impacts
Photodegradation of α-cypermethrin in soil in the presence of trace

metals (Cu2+, Cd2+, Fe2+ and Zn2+)
Authors: Nazia Rafiquea* and Saadia R. Tariqa
Affiliations: aDepartment of Chemistry, Lahore College for Women University,
Lahore, Pakistan
E-mail: [email protected]
[email protected]
*Corresponding Author:
Nazia Rafique
Phone No. +992-42-9208201-9

Fax: +992-42-2873869
E-mail: [email protected]
1
Environmental Science: Processes & Impacts Page 2 of 19
1 Photodegradation of α-cypermethrin in soil in the presence of trace metals (Cu2+,

2 Cd2+, Fe2+ and Zn2+)
3 Nazia Rafiquea* and Saadia R. Tariqa
a
4 Department of Chemistry, Lahore College for Women University, Lahore, Pakistan.
5 *Corresponding Author: Tel: +92-42-9208201-9, Fax: +92-42-2873869
6 E-mail: [email protected]
7 Abstract
8 The influences of trace metals (Cu2+, Zn2+, Cd2+ and Fe2+) on the photodegradation of α-cypermethrin
9 (α-CYM) in agricultural soil were studied. The soil samples were spiked with α-cypermethrin
10 with/without the presence of metal ions, irradiated under UV irradiation chamber for a regular period of
11 time and analyzed by using HPLC. The dark control sterile and unsterile soil samples spiked with α-
12 cypermethrin and selected trace metals were incubated for the same interval of time at 25oC. The
13 results obtained indicated that photodegradation of α-cypermethrin followed first-order and biphasic
14 kinetics. The photodegradation half-lives (t½) of α-cypermethrin were found to be increased from 2.3
15 hours to 7.9, 5.4 and 3.2 hours in the presence of Cu2+ Zn2+ and Cd2+ respectively. Fe2+ increased the
16 photodegradation kinetics from -0.299 h1- to -1.849 h1- and varied the t1/2 from 2.32 to 0.37 h1- in the
17 soil. Microbes also affected the degradation of α-cypermethrin in metal contaminated soil. The
18 degradation rate was inhibited in unsterile soil and the order of inhibition was found to be: Zn2+< Cd2+<
19 Cu2+. The degradation/ persistence of α-cypermethrin were affected linearly with the increasing soil
20 metal concentrations. Cd2+ and Fe2+ accelerated the abiotic dissipation by increasing the reaction rate
21 from -0.024 h1- to -0.032 h1- and -0.029 h1- respectively.
22
23 Key words: photodegradation; trace metals; α-cypermethrin; agricultural soil; HPLC
24 1. Introduction
25 The use of pesticides to increase the crop production is a common practice in the world. These
26 practices however, generate residues that may be noxious to the environment. The accumulation and
27 degradation of these pesticides and their dispersion in the environment depends on the characteristics
28 and overall functions of the ecosystem1 . α-Cypermethrin (α-CYM) is widely used to control the
29 Helicoverpa spp., the major pests of cotton. It is highly hydrophobic as reflected by its low water
30 solubility and high octanol–water partition coefficient (Table 1). Low solubility and high lipoaffinity
31 make it a highly toxic agent to fish and aquatic invertebrates even at very low levels (<0.5 µg L1-, LD50
32 values)2. Moreover, it is metabolized and eliminated significantly more slowly by fish than by
33 mammals or birds that explains its higher toxicity to fish than other organisms3. Generally, the lethality
34 of pyrethroids to fish increases with the increasing octanol/water partition coefficients4. US
35 Environmental protection agency (EPA) has also classified it as a possible human carcinogen.
36 A large proportion of cotton grown is irrigated by drainage water, thus the risk of environmental
37 damage may also be significant5, 6. Moreover, pesticides when applied to soil as insecticides are not
38 selective and may also kill beneficial soil microorganisms7, 8. α-Cypermethrin is moderately persistent
39 in the soil environment with field half-lives ranging from 4 to 12 weeks9, 10. Due to its high
40 hydrophobic property, it causes strong sorption to soil particles, which may cause buildup of bound
41 residues11- 13.
42 Organic wastes and sludge are commonly applied to the agricultural soils as a source of organic
43 material and to improve the soil properties14. However, some studies have shown that the addition of
44 organic manure, and N and P fertilizers can affect the pesticide degradation in the soils15-18. Moreover,
45 the use of these materials can lead to the problems associated with their heavy metal contents,
46 especially their successive applications may result in heavy metal accumulation in the soil.
2
47 Pyrethroid can undergo photolysis in the soil with half-lives ranging from 5 to 170 days9. Enhanced
48 concentrations of heavy metals and their strong binding with soil organic matter and clay minerals may
49 lead to their persistence in the soil. This results in a slow dispersion of synthetic pyrethroids and their
50 potential for long-term effects on beneficial soil microorganisms and aquatic species1, 19. Liu et al.
51 (2007) have reported that the presence of Cu2+ (10 mg kg1−) in the soil may inhibit the degradation of
52 cypermethrin (increases t1/2 from 8.1 to 10.9 d) that may be explained as the reduction in activity of
53 bacterial biomass due to Cu2+ 20. Some of the metals like iron are known to enhance the degradation of
54 pesticides and reduce their half-lives21, 22.The dissipation/persistence of pesticides in presence of trace
55 metals was due to their effect on growth rate of the pesticide degrading bacterial populations 23, 24. For
56 example, the carbendazim degrading Variovorax and the diuron degrading Rhodococcus strains were
57 extremely sensitive to Cd2+ as it decreased their degrading activity even at low concentrations. Cu2+
58 ions strongly inhibited the degradation process of ethylenethiourea (ETU) which is an important
59 degradation product of ethylenebisdithiocarbamate fungicides while 2,4D-degradation by Variovorax
60 was highly accelerated by Cu2+ ions. Zn2+, Cu2+ and Mn2+ (20-50 mg L1-) accelerated the carbendazim
61 and diuron degradation23, 24. Therefore the goal of the present study was to determine the influence of
62 Cu2+, Cd2+, Fe2+and Zn2+ ions on the dissipation/persistence of α-cypermethrin in the soil. The study is
63 important because the trace metal levels in agricultural soil can enhance the catalytic photodegradation
64 of pesticides. So major hazards related to excessive and repeated use of pesticides in the agricultural
65 soils may be abated in this way.
66 2. Materials and Methods

67 2.1 Test Materials and Reference Standards
68 Reference standard of α-cypermethrin (99% purity) was obtained from Sigma-Aldrich, Ltd. (USA).
69 The physical properties of α-cypermethrin as provided by “OECD25 guidelines for the
70 photodegradation of pesticides on soil surface”are listed in Table 1. HPLC grade methanol, acetonitrile,
71 ferrous sulphate, zinc chloride, cadmium chloride, copper sulphate (CuSO4•5H2O) and anhydrous
72 Na2SO4 (analytical Grade) were purchased from Merck (Darmstadt, Germany). Highly pure double
73 distilled water for use during experiment was prepared with a Milli-Q system from Millipore-Waters
74 Co. (Bedford, MA). Na2SO4 was baked at 500°C for 4 h before the beginning of experiment and then
75 stored in an airtight glass bottle until use.
76 2.2 Soil collection and characterization

77 Soil (0-20 cm top soil) used in the study was collected from botanical garden of Lahore College for
78 Women University, Lahore. Prior to use, the soil was passed through 2 mm sieve, and maintained at a
79 75% water holding capacity (WHC) in accordance with the method described elsewhere26. It was then
80 stored in the dark at 20°C until analysis. Soil texture was determined by using the hydrometer27. The
81 physical and chemical properties of the soil sample were measured by using the methods of Saltanpour
82 and Schwap (1977)28 and summarized in Table 2. Soil was cleaned from pesticides by stirring it with
83 acetone for 24 h (three times) and after decanting the acetone, it was dried first at room temperature
84 and then in oven at 105 oC. Soil sample were sterilized by autoclaving for 2 h in a capped 100-mL
85 Erlenmeyer flask at 121°C29.
86 2.3 Photochemical experimental set up

87 Irradiation of the soil samples was performed in a self-designed photoreactor, equipped with a 6-W UV
88 tube (Atlas, Linsengericht, Germany), surrounded with a thermopore jacket and water bath that
89 circulated water through the floor of the photolysis chamber for temperature control. An electric fan (3
90 volt) fitted inside the radiation chamber allowed constant purging of the sample headspace. The spiked
91 soil samples contained in Pyrex petri plates were continuously irradiated with the UV tube placed 23
92 cm above. A reference plate containing unspiked soil sample was also irradiated for the same time
93 interval. Soil moisture values were recorded first after every hour and subsequently after every 6 h. If
94 necessary at each sampling, the weight of each soil tray was manually adjusted with distilled water to
95 ensure that the soil was being maintained at its initial weight and moisture content.
96
3
97 2.4 Control sterilized and unsterilized soil dark samples

98 In the laboratory, control soil samples were subdivided into two groups to investigate the dissipation
99 rates under sterilized and unsterilized dark conditions. The unsterilized samples were used as bioactive
100 controls and were not given any acetone wash. Each portion (10 g, dry weight) of the sample used for
101 sterilization was autoclaved three times (at 24 h apart) for 30 min each in a capped 100-mL Erlenmeyer
102 flask at 121°C. Double de-ionized water was added to the germ-free (autoclaved) and original (un-
103 autoclaved) soils to obtain the water content of 75% by WHC. These moistened sub-samples were
104 spiked with pesticide and then incubated at 25°C in the dark for 0, 24, 48, 96, 144,192, 384 and 762 h
105 respectively.
106 2.5 Standard solution preparations and spiking procedure

107 The spiking solutions (0.5µg g1-) of α-cypermethrin were prepared by appropriate dilution of
108 stock solution (5µg g1-) with acetonitrile. For metal assisted degradation tests, stock solutions of
109 CuSO4.5H2O, FeSO4.7H2O, CdCl2 and ZnCl2 were prepared at concentrations of 1000 mg L1− in water.
110 These stock solutions were then diluted to 100 mg/L for use as a source of external Cu2+, Fe2+, Cd2+ and
111 Zn2+ ions. Soil samples were spiked with α-cypermethrin at the maximum field concentration of 0.5 mg
112 kg1−. The final concentrations of Cu2+ in the soil were set at 15.9 (control treatment), 25.9, 35.9 and
113 45.9 mg kg1-, for Zn2+ final concentrations were 26.9 (control treatment), 36.9, 46.9 and 56.9 mg kg1-,
114 for Cd2+ 0.7 (control treatment), 10.7, 17.7 and 27.7 mg kg1-and for Fe2+ these final concentrations
115 were 863 (control), 873, 883 and 893 mg kg1- (triplicate samples) of each concentration were measured.
116 After soil treatments, the samples were incubated at 25°C in the dark at a moisture content of 75%. The
117 residual contents in the sterilized and unsterilized samples were monitored at regular intervals as
118 described above.
119 Soil slurries were prepared by mixing 10.0 g of soil (dry weight) with 7.5 mL of water in petri plates.
120 The soil was evenly spread across the plate to a depth of 2 mm and then spiked with appropriate
121 concentration of pesticide. Subsequently, these soil samples were spiked separately with Cu2+, Cd2+,
122 Fe2+ and Zn2+. To this effect, different volumes of diluted metal solutions were dispensed evenly across
123 the soil surface via micro-syringe while maintaining the similar moisture level for all the samples. Soil
124 samples were manually shaken to homogenize them. The petri plates were then placed inside the
125 photoreactor and irradiated for 0, 4, 24, 48, 96, 144,192, 384 and 762 h respectively. Control
126 experiments with no addition of trace metals were carried out simultaneously. After irradiation, the
127 triplicate samples and control were removed from the photoreactor and processed further.
128 2.6 Pesticide Extraction and analysis

129 USE method which is an extension of EPA method 3550C was used for extraction of α-cypermethrin
130 from the soil30. Briefly, the irradiated soil samples were placed in 50 mL Erlenmeyer flasks and
131 extracted with 10 mL of ethyl acetate. These samples were first manually agitated and then exposed
132 thrice to USE in a 100H (80/160 W) ultrasonic bath (Sonorex, Germany) for 15 min. After each
133 extraction, extracts were collected by pouring the extractant through a funnel plugged with a small
134 piece of cotton wool overlaid by a portion of anhydrous sodium sulfate which had been previously
135 washed with the same solvent. In order to achieve the adequate concentration factor, 10 g aliquot of the
136 sample was submitted for extraction and the final extract (ca. 30 mL) was evaporated to dryness using
137 rotary evaporator and gentle steam of nitrogen without need of any clean-up procedure and
138 reconstituted in 1 mL acetonitrile. The extraction method showed good efficiency and reproducibility
139 with mean recoveries of 73–92% with standard deviations lower than 2.4% for the whole procedure.
140 α-Cypermethrin was analyzed by using the method of Metwally et al., 199731 and Martnez et al..
141 199632. HPLC system consisting of Agilent model 1100 pump, equipped with DAD detector, an
142 autosampler (model G1313A) and C8 chromatographic column (Bondsil, 15x0.46 cm, 5 urn particle
143 size, Analytichem International) was used for analysis. Mobile phase (acetonitrile/water 75/25) at flow
144 rate of 1 mL/ min was used. The areas of eluted peaks detected at 225 nm were recorded by using a
145 multi- wavelength UV detector Model G 1315B. The retention time of α-cypermethrin under the above
146 conditions was 8.3 min. Calibration was performed each time when samples were analyzed by using
147 external standards. HPLC procedure was linear in the range 0.01-100 µg mL1- at 225 nm with
4
148 regression coefficient of 0.994 (± 0.02) (n = 12); the detection limit was 0.02 µg mL1- and limit of
149 quantification was 0.18 µg mL1- .
150 2.7 Data analysis

151 In the soil, the photolytic decline of a pesticide slows down with time, either due to the adsorption of
152 pesticide to soil or its movement out of a photic zone. Thus the Langmuir–Hinshelwood (L–H) kinetics
153 has been suggested for the photodegradation of pesticides. This model is based on the following
154 equation:
155 r = dC/dt = (kKC/2) + KC
156 Here r represents the rate of mineralization of pesticide, C = pesticide concentration, k = rate constant,
157 and K = pesticides adsorption coefficient. Under the conditions of smaller initial concentration (C0) i.e.
158 in ppm range, many researchers have approximated this L–H kinetics to first-order expression just to
159 obtain the parameters involved in the L–H equation easily33.
160 However, when lag phase was involved, the Hockey-stick model was used for the evaluation of
161 kinetics. This model is based on two sequential first-order curves. The pesticide concentration initially
162 declines according to first-order kinetics with a rate constant k1. At a certain point in time (referred to
163 as the breakpoint), the rate constant changes to a different value k2. Mathematically, this model is
164 described as:
165 dM/dt = -k1M for t ≤ tb
166 dM/dt = -k2M for t > tb
167 where
168 M = Total amount of pesticide present at time t
169 M0 = Total amount of pesticide applied at time t=0
170 k1 = Rate constant until t=tb
171 k2 = Rate constant from t=tb
172 tb = Breakpoint (time at which rate constant changes)
173 DTx = ln 100/100-x if t ≤ tb
174 k1
175 DTx = tb + [ln 100/100-x – t1b] if t ≤ tb
176 k2
177 The tests were carried out in triplicate and the data was expressed as average effect of the test points34.
178 3. Results and discussion

179 α-cypermethrin was chemically stable in neutral soil condition with half-life of 101 days. It was
180 microbically degraded with t1/2 of 13 weeks, but its photodegradation was only reported on soil surface
181 as thin film (Table 1). No soil incorporated photodegradation study has been reported up till now.
182 3.1 Photodegradation of soil incorporated α-cypermethrin

183 The presence of unstable groups such as isobutyl and double bonds in the structure of pyrethroids
184 renders them to degrade usually through photolysis, photooxidation and photoisomerization in the
185 natural environment35. The photodegradation data of α-cypermethrin obtained after irradiation of soil
186 samples under UV system versus irradiation time is depicted in Fig.1. The data for control samples is
187 also elaborated in the same figure for comparison. The photodegradation and photocatalysis rates of
188 pesticides on soil surfaces under UV light depend on different parameters such as temperature, soil
189 particle sizes, soil depth responsible for photodegradation and catalyst loads36.
190 The present study revealed that soil incorporated α-cypermethrin photodegraded quickly under UV
191 photoreactor with the half-life of approximately 2.3 ± 1.41 days (Table 3). Previous studies have
192 reported the half-lives of 8-16 days for the photodegradation of cypermethrin on soil surfaces37. In
5
193 sandy soils, its half life was reported to be 2-4 weeks38. It has been found that cypermethrin degrades
194 more rapidly on sandy loam and sandy clay soils than on clay soils and more rapidly in soils with low
195 organic matter 39, 40. Raikwar and Nag, (2011) reported the half-life of α-cypermethrin under UV
196 system to be 0.93 h in clay loam and 1.57 h on loam soil41. In fact only 8% of radiant solar energy is
197 comprised of UV spectrum and on reaching the earth’s surface, its intensity is further decreased. In
198 case of laboratory experiments, the source emits 100% only UV radiations with most of the intensity
199 directed on the samples that is why lower half lives were observed in the present study and in all other
200 studies carried out under laboratory UV irradiated systems as compared to the studies carried out on
201 sunlit soil surfaces.
202 Pesticides photodegradation was slow in dry soil as light was unable to penetrate deep into the
203 underneath soil and there were no chances of interaction of light with the pesticide, thus moist soil was
204 used in the present study in accordance with the findings of Graebing et al., (2004)42. α-Cypermethrin
205 is likely to volatilize as indicated by its low Henrys Law constant therefore it can move into the
206 photolytic zone of soil through evapo-condensation cycles where it degrades efficiently on irradiation.
207 Furthermore, indirect photolysis by hydroxyl radicals, singlet oxygen and other radical species were
208 believed to enhance the rate of photodegradation in moist condition42.
209 3.2 Microbial degradation of α-cypermethrin
210 Microbes also play significant roles in degrading and detoxifying the α-cypermethrin residues in the
211 environment11, 43. The large difference between reaction rate (-0.044) and t1/2 (18.18 hours) at p <
212 0.05of unsterilized and sterilized soils indicated the role of biotic degradation (Fig. 1 and
213 Table.3).When compared with the photodegradation, the t1/2 of the unsterilized treatments was
214 increased by 5 fold. Tallur et al., (2007), studied that Micrococcus sp. present in the soil utilized
215 cypermethrin as a sole source of carbon leading to hydrolysis of ester linkage to yield 3- phenoxy
216 benzoate 44. Sterilization eliminates the microbial population of the soil and thus increases the
217 persistence of the pesticide. α-Cypermethrin dissipation in the sterile soil in dark may be attributed to
218 the chemical dissipation because the possibility of photodegradation was ruled out by incubating the
219 samples in the dark 45. In the soil, the chemical dissipation of cypermethrin takes place through
220 hydrolysis whereby the ester linkage is first hydrolysed leading to the formation of 3-phenoxybenzoic
221 acid (PBA) and cyclopropanecarboxylic acid derivatives46, principally, 3-(2,2-dichlorovinyl)-2,2-
222 dimethyl cyclopropanecarboxylic acid (DCVA)43. Although, it is biodegradable pesticide but the
223 microbial release of bound residues occurs rather slowly47.
224 3.3 Effect of trace metals on photodegradation
225 The photodegradation rates of some pesticides may be enhanced in the presence of certain metals in the
226 soil by altering the enzymatic activity of soil microorganisms22, 48-51. Similarly, trace metals are also
227 known to inhibit the enzymatic reactions of microorganism by complex formation with the substrate,
228 combination with the protein-active sites of the enzymes, or reaction with the enzyme-substrate
229 complex. Thus bacterial biomass activity may also be inhibited in metal polluted soils. Kools et al.
230 (2005) have reported a positive correlation between glyphosate degradation rates and soil metal
231 pollution49.
232 3.2.1 Effect of Cu2+on α-cypermethrin

233 Photodegradation rate of α-cypermethrin was decreased from -0.299 to -0.088 when 10 mg kg1- of Cu2+
234 was added to the soil as evidenced by an increase in t1/2 from 2.32 to 7.88 ± 0.92 hours at p < 0.05
235 (Table.3). The percent photodegradation of α-cypermethrin in the presence of 25.9 mg kg1- (Co+10 mg
236 kg1-) of Cu2+ was decreased from 95.7 to 61.7 % after 8 days of continuous UV irradiation (Fig.3-c).
237 This retarding effect became pronounced when Cu2+ concentration was increased to 45.9 mg kg1-
238 (Co+30 mg kg1-), the % photodegradation was observed to be reduced to only 50.5%. Cu2+ is known to
239 enhance the photodegradation of pyrethroids in the presence of UV light1, 52. According to Sykora,
240 (1997) Cu2+ compounds may act as catalyst for photodegradation of various pollutants in irradiated
241 systems. The pollutants like α-cypermethrin may act as ligands in the coordination sphere of the Cu2+
242 and a Cu2+-Cu1+ photocatalytic redox cycle was believed to occur in Cu2+ amended solutions. This
243 catalytic effect might also arise due to secondary thermal reactions of the active species produced
244 photochemically from the Cu2+ complex53.The degradation rate of pesticides in the soil was closely
245 related to its availability to the enzymatic systems of microorganisms 54, 55.
6
246 The dissipation rates of α-cypermethrin decreased significantly at p > 0.05 in unsterilized soil during 32
247 days of incubation when compared with the dark control sterile treatments (Fig. 2-a, Fig.5-a). The half-
248 life of α-cypermethrin was observed to be increased from 10.2 ± 2.17 to 12.4 ± 2.13 hours in unsterile
249 control treatment that indicated that Cu2+ affected the activity of soil microbes involved in the
250 degradation of the pesticide (Table.3). The results of present study were compatible with the findings
251 of Liu et al., (2007)1 who reported that the persistence of α-cypermethrin was increased from 8.1 to
252 10.9 d in the presence of 10 mg kg1- Cu2+ ion in unsterile soil. The observed degradation might be due
253 to the fact that metals react with the sulfhydral group of enzymes thereby leading to inhibition of their
254 activity1. Ellis et al., (2001) and Fernandes et al., (2005) found that Cu2+-tolerant communities may
255 have replaced the soil microorganisms that were able to co-metabolize the pyrethroids 56, 57. Moreover,
256 soil microbial community was adversely affected by the presence of elevated concentrations of Cu2+.
257 Cu2+ ions have been reported to strongly inhibit the degradation of ethylthiourea (ETU)24. Different
258 initial concentrations were observed in the Fig. 3. The observation evidenced that the addition of Cu2+
259 caused the persistence of α-cypermethrin in the soil just after its addition. Fig. 4 also depicted an
260 initially high rate of degradation that was later on reduced after some days and then stalled completely.
261 This fact may be interpreted on the basis that desorption controls the biodegradation process58. The
262 sorption of the substance determines its availability for microbial degradation. The sorbed chemicals
263 are less accessible to microorganisms that utilize exclusively or preferentially chemicals in solution.
264 Thus with passage of time, the sorbed quantity of pesticide is increased and their rate of degradation is
265 reduced. It is generally accepted that sorption limits the degradation of pesticides by reducing their
266 partitioning into the soil.
267 Cu2+ ions also affected the abiotic degradation of α -cypermethrin and thus exhibited an inhibitory
268 effect. The inhibitory effect was more pronounce when the Cu2+ concentration were increased up to
269 45.9 mg kg1-. The percent degradation of α-cypermethrin was decreased from 26.5% to 20.5% (Fig.5-
270 c). These findings pointed toward the fact that α-cypermethrin dissipation in the soils containing low
271 concentrations of added Cu2+ was more dependent on biological dissipation than chemical dissipation,
272 but when high concentrations of added Cu2+ was present in soils it depended on chemical dissipation.
273
274 3.3.2 Effects of Zn2+on α-cypermethrin
275 Zn2+ addition decreased the degradation of α-cypermethrin but the inhibitory effect was less severe than
276 for Cu2+ (Fig. 1-c, Table 3). The photodegradation of α-cypermethrin was decreased from 95.69 to
277 79.5% after 8 days of continuous UV irradiation. The degree of inhibition was increased with an
278 increase in the soil Zn2+ concentration from 16.9 mg kg1- to 36.5 mg kg1- (i.e. from Co +10 mg kg1- to
279 Co +30 mg kg1-).
280 Zn2+ also inhibited α-cypermethrin degradation in unsterile dark incubation. The rate of reaction was
281 observed to be increased from -0.024 to -0.040 h1- resulting in an increase in t1/2 from 10.19 ± 1.92 to
282 21.66 ±1.10 (Table. 3). Different initial concentrations were observed in the Fig. 3, evidencing that the
283 addition of Zn2+ in the soil caused the persistence of α-cypermethrin just after its addition. The %
284 dissipation was decreased from 64.55 to 57.26 % after 32 days of continuous incubation (Fig.4-b). This
285 might be due to a change in the functional diversity of the microbial community. Under the Zn2+ stress
286 i.e., high Zn2+ concentrations, some soil microbial populations were shifted from sensitive to less
287 sensitive areas, and hence soil microbial population was affected thereby leading to their weakened
288 activities45. Kamitani et al. (2006) reported that there was a positive correlation between available Zn2+
289 content of soil and soil metabolic quotient, and a negative correlation between available Zn2+ content
290 and microbial biomass, carbon microbial biomass, nitrogen and the microbia1 quotient59. No significant
291 differences were observed in the dissipation of α-cypermethrin under the dark sterile conditions at p >
292 0.05 in the presence of soil Zn2+ load. It was therefore suggested that microorganisms are the major
293 agents that are involved in the dissipation of pyrethroids in the soil environment60.
294 3.2.3 Effects of Cd2+ on α-cypermethrin

295 The reaction rate of α-cypermethrin in soil was observed to be decrease from -0.299 to -0.871 at p <
296 0.05. This resulted in an increase in persistence of α-cypermethrin from 2.32 ± 1.41 to 3.20 ± 2.01
297 hours under UV- irradiation system. In fact, when the concentration of soil Cd2+ was increased from 0.7
298 to 30.7 mg kg1- (Co+ 10 mg kg1- to Co+ 30 mg kg1-), the % degradation was decreased from 95.68 % to
299 65.9 % after 8 days of continuous UV irradiation. On the contrary, the successful elimination of the
300 harmful pesticide (methomyl) was reported previously by using a Cd2+ based photocatalyst under the
7
301 sunlight radiation within a very short time with a removal capacity being 1,000 mg pesticide per gram
302 of the photocatalyst61.
303 Cd2+ decreased the half-life of α-cypermethrin in dark unsterile conditions from 10.19 ± 1.92 to 7.15 ±
304 0.43 days (Fig.4-c). Similarly, t1/2 in sterile conditions was decreased from 28.88 ± 1.53 to 23.90 ±
305 0.591 hours (Fig.5-c). Although, there are certain pesticide degrading strains of bacteria that are
306 extremely sensitive to Cd2+ and Cd2+ decreases their degrading activity even at low concentrations24 but
307 in fact both the biotic and abiotic dissipation of pyrethroids occur in soil simultaneously62. Under sterile
308 conditions in soils, chemical dissipation becomes more important in the presence of high
309 concentrations of added Cd2+.
310 3.2.4 Effects of Fe2+ on α-cypermethrin

311 Iron is one of the major elements present in the soil mostly in the forms of hydroxides/oxides/chlorides.
312 Generally the most dominant oxidation state is Fe3+and when reducing conditions (like subsurface
313 environment) are prevailing, iron exists as Fe2+ 63. Fe2+ is known to accelerate the photolysis of
314 pesticides through photosensitizing effect 64-67. Rafique et al., (2014) evidenced that a 3-fold increases
315 in percent degradation of imidacloprid was observed in moist soils by the catalytic addition of Fe2+ to
316 soil22. The present study also evidenced an accelerated photodegradation of α-cypermethrin under UV
317 chamber by the addition of Fe2+. The half-life was observed to decrease from 2.32 ± 1.41 to 0.37 ± 2.07
318 hours. These results are in agreement with the findings of several other authors who reported that Fe2+
319 catalyzed the photodegradation of several pesticides 22, 68, 69. The degradation of α-cypermethrin in soil
320 was more efficient when soil Fe2+ levels are enhanced. It degraded up to approximately 94 to 96% of
321 initial concentration after 4 days of continuous UV irradiation in the presence of Fe2+ (Co +10 mg kg1-
322 and Co + 30 mg kg1-) as compared with control of 74%. This enhanced effect was the result of direct
323 Fe2+ catalyzed photodegradation or indirect photolysis due to the reaction of Fe2+ with OH- radicals
324 from moist soil. Different initial concentrations of pesticide were observed in the Fig. 3, before and
325 after the addition of Fe2+ in the soil samples that evidenced the fact that the addition of Fe2+ in the soil
326 caused the instant degradation of α-cypermethrin in the soil.
327 Soil microbes are more efficient in degrading the α-cypermethrin in the presence of higher Fe levels
328 (CFe+30 mg kg1-) as evidence by % degradation that was increased from 71.2 % to 96% in the presence
329 of 30 mg kg1- Fe2+after 32 days of incubation. The zero valent iron (Fe0) has already been used as
330 remedial tool to enhance the degradation of HCHs and DDX in soil70. The soil Fe2+ levels also affected
331 the abiotic dissipation of α-cypermethrin in soil i.e. a reduction in half life α-cypermethrin was
332 observed from 28.88 to 21.66 days (Table.3). The % degradation was enhanced from 26.6 to 46.1%
333 after 32 days of incubation in dark at 25 oC. Singhal et al., (2012) reported the degradation of malathion
334 by zero-valent Fe nano-particles71. When it was added to the soil under anaerobic conditions, corrosion
335 (oxidation) of the iron might be effectively coupled to reductive dechlorination and nitro group
336 reductio72.
337 Conclusions
338 It is concluded that photodegradation of α-cypermethrin was retarded in the presence of elevated
339 concentrations of Cu2+, Zn2+ and Cd2+. Cu2+ was evidenced to possess slightly greater inhibition effect
340 than Zn2+ and Cd2+and increased the t1/2 from 2.3 hours to 7.9 hours. Cu2+, Zn2+ and Fe2+ also retarded
341 the microbial degradation of α-cypermethrin while Cd2+ and Fe2+ accelerated the abiotic dissipation by
342 decreasing the t1/2 from 28.88 h to 23.90 h and 21.66 h respectively.The proliferated soil Fe levels
343 however enhanced the photo and microbial degradation of α-cypermethrin.
344 Acknowledgement
345 The authors are highly thankful to the financial support provided by the Higher Education commission
346 of Pakistan under the Indigenous 5000 Ph.D. Fellowship scheme. The authors are also thankful to Dr.
347 Matten Abbas and Abdul Muqeet khan for providing the support for HPLC analysis.
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12
Figures
Fig.1Photodegradation of soil incorporated α-cypermethrin
Fig. 2Effect of concentration of α-cypermethrin on its photodegradation

Fig. 3 Effect of metal concentration on Photodegradation of α-cypermethrin

Fig. 4 Effect of metal concentration on microbial degradation of α-cypermethrin

Fig. 5 Effect of metal concentration on abiotic degradation of α-cypermethrin

Page 17 of 19
Environmental Science: Processes & Impacts


Tables
Table.1. Elementary properties of the pesticide along with its degradation prolife in soil
Pesticide Mol. Solubility in Henry Vapor Applic Chemical stability in Aerobic Phototransformat Stable
wt water ( mg L1-) constant Pressu ation dark /anaerobic ion soil surface Degrada
(g) re rate transformation tion
(g Ha1- in soil products
) in soil
α- 416. 3.97 (pH=7), 2.5x10-7
5.1 x 10-
Very stable in neutral 2–8 weeks/ UV; 56.08 min on 3- PBA73
Cypermethrin 3 25oC atm-m3107- 15g/ha
and acidic media, 63 days 73,76 clay loam, 94.07
C22H19Cl2NO3 1.25(DDW) µg /mol74nPa at .
hydrolyzed in strongly min on loam,
L1- at 20oC73 70 oC75 alkaline media,DT50 sunlight 2.24 d on
4 x 10- (pH 4, 50oC) stable over clay loam, 3.14 on
8 mm 10d, (pH 7, 20oC), 101d loam41
o 73
Hg at (pH 9, 20 C) 7.3d
70 oC74
Tomlin 200473, Hayes and Laws , 199074;Kidd and James, 199175; US Department of Agriculture,199076; e Raikwar and Nag 201141
Table 2.Physico-chemical properties of the studied soil
Soil Type Soil texture M. C O.M pH CEC Trace metals
% % (1:2) (mmol/ kg) (mg/kg)
% clay % sand % Silt Fe Zn Cd Cu
Sandy 4.5 87 8.5 2.34 4.62 7.4 8.3 863 6.9 0.7 15.9
Table. 3 Dissipation statistics of degradation of α-cypermethrin


Pesticide Environment Trace Model k1(h-) r2 tb (days) k2 (h-) r2 t1/2 (days)
metal
UV HS -1.078 0.686 1 -0.236 0.916 0.64 ± 1.41*
Dark unsterile HS -0.151 0.99 8 -0.046 0.747 33.63 ± 1.92
dark sterile** HS -0.024 0.994 32 - - -
UV Fe2+ HS -1.175 0.94 2 -0.024 0.663 0.59 ± 2.07*
Dark unsterile HS -0.925 0.989 2 -0.035 0.855 54.03 ±1.4
dark sterile** HS -0.032 0.936 32 - - -

α-Cypemethrin
UV Zn2+ HS -0.978 0.945 2 -0.017 0.935 0.71 ± 1.2*
Dark unsterile HS -0.701 0.955 1.2 -0.021 0.697 41.6 ± 2.9
dark sterile** HS -0.022 0.96 32 - - -
UV Cd2+ HS -1.397 0.813 1 -0.088 0.734 0.49 ± 2.01*
dark sterile** HS -0.029 0.991 32 - - -
UV Cu2+ HS -0.147 0.937 8 -0.067 0.944 4.7 ± 1.04*
dark sterile** HS -0.023 0.996 32 - - -
* DT50 was in hours.
** (tb not reached till 32 days of study period So, DT50 was not possible to calculate accurately by HS model)
Environ Sci Pollut Res (2016) 23:4473–4480
DOI 10.1007/s11356-015-5655-4
RESEARCH ARTICLE
Cu2+ and Fe2+ mediated photodegradation studies

of soil-incorporated chlorpyrifos
Nazia Rafique 1 & Saadia R. Tariq 1 & Karam Ahad 2 & Touqeer Taj 2
Received: 11 June 2015 / Accepted: 20 October 2015 / Published online: 28 October 2015
# Springer-Verlag Berlin Heidelberg 2015
Abstract The influences of Cu 2 + and Fe 2 + on the studied trace metals also affected the abiotic degradation of
photodegradation of soil-incorporated chlorpyrifos were in- the pesticide in the order Cu2+ >Fe2+.
vestigated in the present study. The soil samples spiked with
chlorpyrifos and selected metal ions were irradiated with UV Keywords Photodegradation . Soil . Chlorpyrifos . Cu2+ .
light for different intervals of time and analyzed by HPLC. Fe2+
The unsterile and sterile control soil samples amended with
pesticides and selected metals were incubated in the dark at
25 °C for the same time intervals. The results of the study Introduction
evidenced that photodegradation of chlorpyrifos followed
the first-order kinetics. The dissipation t0.5 of chlorpyrifos Intense and massive agricultural applications led to the accu-
was found to decrease from 41 to 20 days under UV irradia- mulation of high concentrations of pesticides in the soil that
tion. The rate of chlorpyrifos photodegradation was increased may ultimately become hazardous to the various forms of life.
in the presence of both metals, i.e., Cu2+ and Fe2+. Thus, In the soil, the accumulation and dissipation of pesticides de-
initially observed t0.5 of 19.8 days was decreased to 4.39 days pend on the nature of pesticides and the soil characteristics
in the case of Cu+2 and 19.25 days for Fe+2. Copper was found (Liu et al. 2007; Morillo et al. 2000).
to increase the rate of photodegradation by 4.5 orders of mag- Chlorpyrifos (O,O-diethyl O-(3,5,6-trichloro-2-
nitude while the microbial degradation of chlorpyrifos was pyridyl)phosphorothioate) is one of the most frequently used
increased only twofold. The microbial degradation of chlor- organophosphorus insecticides that is effectively used against
pyrifos was only negligibly affected by Fe2+ amendment. The various pests and insects of important cash crops. It is also
frequently used to control the termites, flies, mosquitoes, and
various household and veterinary pests (Zhang et al., 2011). In
Responsible editor: Philippe Garrigues soils, its t0.5 usually ranges between 60 and 120 days, but differ-
ent soil conditions such as pH, soil type, and temperature may
* Nazia Rafique extend it over 1 year. The frequent use of chlorpyrifos has been
[email protected] reported to contaminate the soils and water as well as destroy the
Saadia R. Tariq non-target organisms (EC 2005; Mugni et al. 2012). Moreover,
[email protected] its abundant use in agriculture has been linked to various health
Karam Ahad problems that may emanate from dietary exposure to the resi-
[email protected] dues of chlorpyrifos present in the food (Mugni et al. 2012). Its
Touqeer Taj high acute toxicity may affect the cardiovascular system, respi-
[email protected] ratory system, and the central nervous system (Dam et al. 2000).
1
Department of Chemistry, Lahore College for Women University, The environmental fate of chlorpyrifos is governed by both the
Lahore, Pakistan biotic and abiotic processes, such as chemical hydrolysis,
2
Ecotoxicology Research Institute (ERI), Department of Plant and microbial degradation, and photolysis (Sreekumaran and
Environment Protection (DPEP), NARC, Islamabad, Pakistan Pradeep 1999; Zalat et al. 2014).
4474 Environ Sci Pollut Res (2016) 23:4473–4480
A study evidenced that if chlorpyrifos is applied continu- the data obtained by using the photo-Fenton reaction and
ously to the soil for 360 days, the soil contained 22 % 3,5,6- TiO2 Degussa P25 (Guzsvany et al. 2010)
trichloropyridinol (TCP), less than 8 % of 3,5,6-trichloro-2- Thus, the present study was formulated to determine the
methoxypyridine, and 27–88 % of CO2 [Walia et al. 1988]. influence of Cu2+ and Fe2+ ions on the persistence/dissipation
Chlorpyrifos degrades in the soil by photoinduced reac- of soil-incorporated chlorpyrifos. The importance of the study
tions. Three different photochemical reactions are reported stems from the fact that enhanced levels of certain metals
to take place under UV irradiation involving dechlorination, present in the soil may trigger the photodegradation process
hydrolysis, and oxidation (Walia et al. 1988). During of pesticides. Thus, the problems arising from repeated and
photoirradiation of soil, different products are formed by excessive applications of pesticides to various crops and their
dehalogenation and oxidation of chlorpyrifos that are further subsequent accumulation in the soils may be overcome to
photolysed to yield O,O-diethyl phosphorothioic acid and some extent.
chloropyridinols. The rate of hydrolysis of oxon is greater than
that of chlorpyrifos so it does not accumulate in the soil. UV
irradiation is also known to cause a decrease in the levels of Materials and methods
chlorinated pyridinols thereby confirming their mineralization
[Jabeen et al. 2014]. Soil collection, pretreatment, and characterization
Sewerage sludge and organic wastes are frequently used as a
source of organic material in agricultural soils. However, some For the present study, bulk soil was obtained from the botan-
studies have evidenced that the addition of nitrogenous and ical garden of National Agricultural Research Center (NARC)
phosphate fertilizers and organic manure can alter the rate of Islamabad at a depth of 0–15 cm and placed in zip-mouthed,
degradation of pesticides in soils (Caracciolo et al 2005; Han high-density, polythene bags. No pesticides or fertilizers had
et al. 2003; Topp et al. 1996; Zhou et al. 2008). The strong previously been used on the sampling site for 10 years. Prior
binding of pesticides with clay minerals and soil organic matter to use, the soil samples were homogenized periodically and
may cause them to persist in the soil as well as to disperse sieved to obtain particles of less than 2 mm mesh sizes. A
slowly. Thus, the potential of the pesticides to cause long- water holding capacity (WHC) of 75 % was maintained for
term effects on beneficial soil microorganisms as well as aquat- the soil samples at 0.33 bars by using the method of Frank
ic species is increased (Van Zwieten et al. 2003; Liu et al. 2007; et al. (2002)). Then, these soil samples were stored in the dark,
Gaw et al. 2003). Moreover, these materials contain large quan- at 20 °C until analysis. The texture of soil was determined by a
tities of hazardous heavy metals that may accumulate in the soil hydrometer (Bao 2000). Soil physico-chemical properties
upon successive application of sewage sludge and thus cause (Table 1) were determined by using the standard method of
the problems arising from high contents of heavy metals. Ryan et al. (2001). For bioactive control experiments, soil
Heavy metals play dual roles in the degradation of pesti- samples were autoclaved in capped flasks for 2 h at 121 °C
cides in soil. Enhanced levels of some pesticides in the soil for sterilization (100 mL capacity). An atomic absorption
such as Cu2+ and Zn2+ may reduce the dissipation of delta- spectrophotometer was used for the estimation of metals in
methrin and cypermethrin while some other metals like Fe soil samples before use.
may enhance their rate of dissipation and thus reduce their
t0.5. This was explained in terms of reduction in the activity Materials
of bacterial biomass (Liu et al. 2007; Zheng and Ye 2001;
Rafique and Tariq 2014). The persistence/dissipation of pesti- Chlorpyrifos (99 % purity) standard and HPLC-grade
cides in the presence of metals has also been explained on the acetonitrile were obtained from Sigma Aldrich (Germany).
basis of their influence on the growth of bacterial populations The solutions of chlorpyrifos at a concentration of 5 mg L−1
involved in pesticide degradation (Vágvölgyi et al. 2010; were prepared in acetonitrile and stored in a freezer at 4 °C.
Škrbić et al. 2010). Cu2+ ions have the potential to retard the Analytical grade FeSO4·7H2O and CuSO4·5H2O were pur-
dissipation of ethylenethiourea produced on degradation of chased from Merck, Germany. Analytical grade reagents and
ethylene bis-dithiocarbamate fungicides. On the other hand chemicals were used without any further purification. Highly
2,4D degradation was highly accelerated by Variovorax in pure distilled water as prepared by a Milli-Q system of
the presence of Cu2+ ions. Manganese, zinc, and copper Millipore Waters Co. (Bedford, MA) was used during these
(20–50 mg L−1) were also found to accelerate the carbendazim experiments.
and diuron degradation (Vágvölgyi et al. 2010; Škrbić et al.
2010). Guzsvany et al. studied the potential of AlFe-pillared Spiking procedure
clay and Fe-ZSM-5 catalysts for the removal of aqueous
imidacloprid under UV irradiation or visible light. The The spiking solution of chlorpyrifos at a concentration of
comparison of the obtained results was also provided with 0.5 mg/mL was prepared by diluting the stock solution
Environ Sci Pollut Res (2016) 23:4473–4480 4475
Table 1 Physico-chemical properties of the studied soil
Soil type Soil texture M.C. % O.M. % pH (1:2) CEC (mmol/kg) P N K Cu2+ Fe2+
% Clay % Sand % Silt (mg/kg)
Silty clay loam 4.5 87 8.5 2.34 4.62 7.4 8.3 5.8 0.52 130 20.3 245.8
appropriately with acetonitrile. A 10.0-g portion of pre-dried the accuracy of data obtained by AAS, standard reference
soil was mixed thoroughly with water (7.5 mL) to prepare the material SRM 2711BMontana Soil^ was run with the sam-
soil slurries. This slurry was uniformly spread on the petri ples. The results of the analysis were considered to be
plate in the form of thin films of 2 mm depth and then added reliable if analysis error for repeat samples was less than
with pre-requisite concentrations of pesticide standards. Sub- 5 %, and an analytical precision of ±10 % was obtained for
sequently, these soil slurries were added with appropriate con- replicate samples.
centrations of two metals, i.e., Cu2+ and Fe2+, separately. The
final concentrations of these metals spiked with the soil are Photochemical experiments
provided in Table 1. Microsyringe was used to evenly dis-
pense the spiking solution across the soil surface. The soil The soils were irradiated with a UV tube of 8 W (Atlas,
samples were then thoroughly mixed and uniformly spread Linsengericht, Germany) equipped in a self-designed
on the plate. In the case of control experiments, no addition photoreactor. The vessels used for irradiating the samples
of metal and pesticides was made. All the samples were were provided with quartz lids at the top. In order to control
prepared in triplicate. After irradiation, the controls and the temperature, water was kept circulating beneath the sam-
samples were removed from the photoreactor. ples, throughout the floor of the photolysis chamber. A 3.0-V
electric fan was installed inside the photoreactor to allow the
Metal speciation studies in soil sample headspace to be purged continuously. The vertical
distance between spiked soil samples and UV tube was main-
Metal speciation studies were performed by using the mod- tained at 23 cm. The reference comprised an unspiked soil
ified method of Tessier et al. (1979) that distributed the sample that was irradiated in a UV photoreactor for a time
metals into six operationally defined fractions. According interval equal to the sample. In order to maintain the WHC
to the method, a 1.0-g portion of air-dried, homogenized at 75 %, the moisture content of soil samples was regularly
soil sample was sequentially treated with deionized dis- monitored, initially after every 60 min and later on after every
tilled water, MgCl 2 (1.0 M), sodium acetate solution 12 h. DDW was used to maintain the initial moisture content
(1.0 M), hydroxylamine hydrochloride (0.04 M), nitric ac- and weight of each soil sample after every sampling, where
id (0.02 M)+30 % H2O2, and HNO3+HClO4, to obtain the necessary. Subsequently, these soil samples were irradiated in
respective fractions such as water-soluble (S1), exchange- the UV photoreactor for different time intervals, i.e., 0, 4, 24,
able (S2), carbonate-bound (S3), Fe–Mn-oxide-bound (S4), 48, 96, 192, 384, and 762 h.
organic-bound (S5), and residual fractions (S 6). (Salbu
et al. 1998) For each extraction, the mixture was centri- Control test experiments
fuged for 30 min at 3000 rpm; the supernatants were re-
moved with pipette, filtered with Whatman filter paper, The control test samples of two different types were prepared
and analyzed for metals by using atomic absorption spec- including unsterilized and sterilized ones. The unsterile dark
troscopy (AAS). Before starting the next extraction step, control samples were prepared by adding a 10-g portion of soil
the sample was shaken for 30 min with 8 cm3 of double sample with prerequisite concentration of chlorpyrifos and
distilled water (DDW) water and centrifuged, and the wash two metals. These samples were incubated in the dark at
solution was discarded. All extractions were conducted in 25 °C for 0, 1, 2, 4, 8, 16, 32, 64, and 128 days. Before
triplicate. spiking, a WHC of 75 % was maintained for each of the soil
The calibration curve method was adopted for the quan- samples. For sterile control experiments, 10 g soil samples
tification of results while using the standard solutions in were autoclaved thrice at 121 °C (at intervals of 24 h) for
concentration range of 2–8 mg L−1 and recording their 30 min in air-tight flasks. A water content of 75 % was
corresponding absorptions. At least four standard solutions achieved by adding deionized water to the autoclaved soil
were run on the instrument covering the absorption range samples. These samples were then added with solutions of
of samples. The precision of quantitative results was en- chlorpyrifos and metals and incubated for 0, 1, 2, 4, 8, 16,
sured by running the triplicate samples. In order to check 32 64, and 128 days respectively in the dark, at 25 °C.
Pesticide extraction and analysis If the initial concentration of pesticide is in the range of
parts per billion or parts per million, then L–H kinetics may be
The chlorpyrifos-spiked soil samples were extracted after approximated to first-order kinetics according to the equation:
irradiation according to the procedure of Graebing and
Chib (2004). In brief, UV-irradiated samples were extract-
r ¼ dC=dt ¼ k’C ¼ kKC
ed three times with (90:10) mixture of acetonitrile and
water (10 mL) containing 1 N H3PO3. During extraction,
the contents were vortexed thoroughly for 1 min and son- The integrated forms of equation are:
icated for 10 min in an ultrasonic bath (35 kHz, 50/60 Hz,
D-7700 Singen/HtW T-700, Elma, Germany). Subsequent- Ct ¼ Co e−k’t orlnðCo =Ct Þ ¼ − kKt ¼ −k’t
ly, these samples were centrifuged for 10 min. The extrac-
tion of residue was carried out twice by using the same where k=first-order rate constant (time1) [Konstantinou and
mixture. The combined soil extracts were concentrated to Albanis 2003). The t0.5 of pesticide is determined by the
5 mL on a rotary evaporator at 40 °C, cleaned thrice with equation=(1/k) ln 2.
10 mL CH 2Cl2, and then evaporated again to 0.5 mL.
These extracts were then dissolved in a 2-mL volume of
acetonitrile, cleaned by passing through a 0.45-μm
polyethersulfone-based filter membrane present in a sy- Results and discussion
ringe, and reduced to 1.0 mL at ambient temperatures, un-
der N2 atmosphere. The extracts obtained were analyzed Chlorpyrifos is degraded in the soil on exposure to chem-
by HPLC. Chlorpyrifos recoveries obtained by this method ical reactions, sunlight, and microorganisms (Reddy et al.
were in the range of 79.9 %±2.05 for 0.5 mg kg−1 to 94 % 2013; Luebke and Hum 2002). Its half-life has been
±1.19 for10 mg kg−1. recorded to range from 48–190 days in the dark, but it
The method of Zalat et al. (2014)) was used for the deter- may exceed 1 year depending on its formulation, rate of
mination of chlorpyrifos. The analyses by HPLC were carried application, soil type, climate, etc. pH of soil was found to
out by using a reversed-phase C18 column with an internal have a profound influence on the degradation of pesticide.
diameter of 250×4.6 mm and a 5-μm particle size. The instru- On increasing the pH from 7 to 8, an increase in rate of
ment was equipped with a binary LC Pump 250 PE Nelson degradation was observed with a corresponding decrease
900 Series Interface UV/Vis Detector for separation. The mo- in t0.5 from 100 to 1.5 days.
bile phase consisted of a mixture of glacial acetic/water/ace- The microorganism-assisted degradation of chlorpyrifos
tonitrile (0.1 v/10 v/90 v) that was used at a 1 mL/min flow rate is a good technique for its dissipation, but incomplete and
and an injection volume of 20.0 μL. A wavelength of 290 nm slow degradation are the major constraints that limit this
was used for the detection of chlorpyrifos at the retention time process [Walia et al. 1988; Roberts and Hutson 1999]. In
of 6.7 min. The adopted HPLC method was observed to be water, the photodegradation t0.5 of chlorpyrifos was found
linear in the concentration range of 0.05 to 50 μg mL−1 (with to be 3–4 weeks. So far, no studies have been reported
R2 =0.999±0.04). The LOD and LOQ obtained for this meth- focusing the metal-assisted photodegradation of chlorpyri-
od were 0.05 and 0.15 μg mL−1, respectively. Calibration of fos in the soil.
the instrument was performed by using external matrix-
matched standards, every time before the analysis of samples
and the linear regression analyses were used for
quantification.
Photocatalytic degradation kinetics
The data for the photodegradation of pesticides in the soil was

studied by fitting the Langmuir–Hinshelwood (L–H) model
according to the equation:
r ¼ dC=dt ¼ kKC=2 þ KC
where C represents the pesticide concentration, k=rate con-

stant, r=rate of pesticide mineralization, and K=adsorption
coefficient of the pesticide. Fig. 1 Photodegradation profile of chlorpyrifos in soil
Table 2 Dissipation statistics for

chlorpyrifos in soil amended with Pesticide Trace metal Cultivation environment K (day−1) t1/2 (days) r2
trace metals
Chlorpyrifos (50 mg kg−1) No metal UV 3.5×10−2 19.80±2.31 0.988
Dark unsterile 2.3×10−2 30.13±1.13 0.992
Dark sterile 1.7×10−2 40.76±0.96 0.997
Cu2+ UV 1.58×10−1 4.39±1.23 0.950
Dark unsterile 5.3×10−2 13.07±2.1 0.998
Dark sterile 3.7×10−2 18.73±1.09 0.993
Fe 2+
UV 5.6×10−2 12. 37±1.38 0.998
Dark unsterile 2.8×10−2 24.75±2.04 0.979
Dark sterile 2.6×10−2 26.65±1.82 0.995
Photodegradation studies of soil-incorporated the soil and interrupts the degradation of soil organic mat-
chlorpyrifos ter. As is mostly applied aerially, it accumulates in the top
soil where it affects the soil biochemical properties. It also
On exposure to sunlight, the degradation rate of chlorpyr- retards the growth of nitrogen-fixing symbiotic bacteria
ifos in the soil was observed to be 3.5×10−2 day−1 with t0.5 and adversely affects the enzyme activity of soil that is
of 19.8±2.3 days (r=0.988). The natural logarithmic de- the degradation index for organic matter in the soil
cline corresponding to the photodegradation of soil- [Laksmikantha 2000].
incorporated chlorpyrifos on UV irradiation verses irradia- The data furnished in Table 2 shows that in comparison
tion time is provided in Fig. 1. The photodegradation with photodegradation, the t0.5 of chlorpyrifos in unsteril-
curves for dark unsterile and sterile controls are also cast ized treatments was enhanced from 19.84 to 30.13 days.
in this figure for comparison. In moist soil, chlorpyrifos These results agreed well with those of Singh et al.
photodegradation followed the first-order kinetics. It was (2002)) who reported the t0.5 of chlorpyrifos to be in the
found that the photodegradation of chlorpyrifos in soil was range of 34–46 days (Singh et al. 2002). TCP has been
quite rapid under UV irradiation with half-life being re- reported as the antimicrobial degradation product of
duced to 19.8 days from an initial half life of 40.8 days chlorpyrifos that is accumulative in the soil and slows
(R2 of 0.988, Table 2). The reaction rate was observed to be the rate of microbial degradation (Robertson et al. 1998).
varied from 1.7×10−2 to 3.5×102 per day, at a p value of Under sterilized conditions, the soil microbial popula-
<0.05. tion was eliminated and thus the pesticide became persis-
The microbial degradation and abiotic hydrolysis are tent. The degradation of chlorpyrifos was very slow in the
responsible for transforming the chlorpyrifos. The signifi- dark sterile soil conditions with a rate constant being 1.7×
cance of biotic degradation of soil-incorporated chlorpyri- 10 −2 and a t 0.5 double than that under UV irradiation
fos was indicated by a large difference in degradation rates (Fig. 1). Hydrolysis was the main process that caused the
of pesticide in unsterilized and sterilized soils, i.e., 6 × dissipation of chlorpyrifos in sterile soil kept in the dark
10−2 day−1 and t0.5 of 25 days (Fig. 1 and Table 2), thus because the possibilities of microbial dissipation and
evidencing the significant role of microbes in dissipating photodegradation were eliminated by incubating the
and detoxifying the residues of chlorpyrifos in the soil samples in the dark. Racke et al. (1996) also reported a
(Jabeen et al. 2014; Rokade and Mali 2013). Chlorpyrifos small degradation of chlorpyrifos in soils with high pH,
also inhibits the growth of bacterial populations present in under sterile conditions (Racke et al. 1996).

different concentrations of Pesticide concentration (mg/kg) Cultivation environment K (day−1) t1/2 (days) r2
chlorpyrifos
25 UV 5.41×10−2 12.81±1.82 0.993
Dark 1.95×10−2 35.53±1.01 0.975
50 UV 3.50×10−2 19.8±2.31 0.988
Dark 1.71×10−2 40.76±0.96 0.997
100 UV 2.44×10−2 28.40±1.32 0.826
Dark 9.42×10−3 73.72±1.61 0.864
Fig. 2 Degradation profile of chlorpyrifos in Cu2+-amended soil Fig. 3 Degradation profile of chlorpyrifos in Fe2+-amended soil
Effect of initial pesticide concentration Metal speciation studies in soil

on photodegradation
The form of the metal in soil decides its behavior in the soil.
An increase in initial concentration of chlorpyrifos in soil was Thus, the metal speciation analysis in the soil was performed
observed to decrease its rate of photodegradation. The data in after spiking the soil with metals according to the modified
Table 3 evidenced that on increasing the initial concentration Tessier’s scheme. The results of the study pertaining to the
of soil-incorporated chlorpyrifos from 50 to 100 mg/kg, a distribution of the metals in various operationally defined frac-
decrease in its rate of degradation to 2.4×10−2 took place. tions in surface soil evidenced that Cu and Fe mostly belonged
Simultaneously, the t0.5 of pesticide was enhanced from the to Fe–Mn-oxide-bound fraction and organic fraction. The ap-
initial half-life of 19.74 to 28.40 days at p<0.05. Further con- preciable concentration of Cu was also present in residual
firmation was obtained by reducing the chlorpyrifos concen- fraction. The least amount of the two metals was present in
tration to 25 mg kg−1, where t0.5 was significantly reduced to water-soluble and exchangeable fractions, respectively.
12.81 days. It was also depicted by an increase in degradation
rate constants from 3.51×10−2 to 5.41×10−2. Thus, the deg-
radation rate of chlorpyrifos was dependent on its initial con- Effect of Cu2+ on degradation of soil-incorporated
centration. In fact, UV sources produce photons. On increas- chlorpyrifos
ing the initial concentration of chlorpyrifos, the competition
between TCP and chlorpyrifos for UV photons was increased When the soil-incorporated chlorpyrifos was spiked with Cu2+ at
which led to a reduction in dissipation of chlorpyrifos. a concentration of 20 mg kg−1, the rate of its photodegradation
The rate of degradation of chlorpyrifos in the dark was also was increased from 3.5×10−2 to 1.6×10−1 day−1 (p<0.05,
found to be dependent on the initial pesticide concentration in Fig. 2). A sudden reduction in chlorpyrifos t0.5 was also observed
the soil (Table 3). A four-time increase in chlorpyrifos con- from 19.84 to 4.4 days (Table 2). In fact, Cu compounds are
centration (i.e., from 25 to 100 mg kg−1) led to a twofold known to catalyze the photodegradation of various pesticides
increase in t0.5. Singh et al. (2003)) have reported that even under UV irradiation (Tariq et al. 2014). An increase in Cu2+
after repeated applications of chlorpyrifos, no chlorpyrifos- concentration from C0 +20 mg kg−1 to C0 +40 mg kg−1 caused
degrading microbial population was developed in the soil an increase in the rate of photodegradation from 1.58×10−1 to
(Singh et al. 2003). 1.65×10−1 day−1. Thus, a clear reduction in the dissipation half-

chlorpyrifos in soil amended with Pesticide Trace metal Metal conc K (day−1) t1/2 (days) r2
different Cu2+ and Fe2+ levels
under UV irradiation Chlorpyrifos (50 mg kg−1) Cu2+ 20 1.58×10−1 4.38±1.23 0.997
40 1.65×10−1 4.20±2.16 0.999
80 1.71×10−1 4.05±0.74 0.991
Fe2+ 20 5.6×10−2 12.6±1.38 0.998
40 6.6×10−2 10.5±0.91 0.997
80 7.6×10−2 9.12±1.31 0.986
life of the pesticide was recorded from 105 to 98 h (Table 4). Conclusions
Figure 2 depicts that during 32 days of incubation, the rate of
chlorpyrifos dissipation was increased significantly in unsteril- The presence of selected trace metals, i.e., Cu2+ and Fe2+,
ized soil at p>0.05 in comparison with the control dark sterile caused an increase in the rate of chlorpyrifos photodegradation
treatments. with a resultant decrease in t0.5 from 19.8 to 4.39 and
In unsterile control treatments, chlorpyrifos t0.5 was re- 19.25 days, respectively. Cu2+ caused a six-time increase in
duced from 22.8 to 13.1 days that evidenced that Cu2+ influ- photodegradation rate and a twofold increase in microbial deg-
enced the activity of the microbes in the soil that degrade the radation of chlorpyrifos. Fe2+, on the other hand, negligibly
pesticide. Copper also influenced the abiotic dissipation of affected the biotic chlorpyrifos degradation. The selected trace
chlorpyrifos. Thus, the t0.5 of chlorpyrifos dissipation was metals also influenced the abiotic degradation in the order
reduced from 40.8 to 18.73 days when it was spiked with Cu2+ >Fe2+.
catalytic amounts of Cu2+ but on UV exposure, a further de-
crease in half-life was observed from 18.73 to 4.4 days. This Acknowledgments We are highly obliged to the Higher Education
Commission, Pakistan for providing the financial support under the
was explained on the basis of the photon capturing potential of
scheme of Indigenous Ph.D. Fellowship. We also acknowledge the assis-
Cu2+ ions and their subsequent efficient role in catalyzing the tance provided by Dr. Fayyaz Hussain, Principle Scientific Officer, Land
photolysis of chlorpyrifos. Resource Research Institute (LRRI), for carrying out the physico-
chemical and metal analysis of reference soil samples.
Effects of Fe2+ on chlorpyrifos degradation

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Compost Science & Utilization
ISSN: 1065-657X (Print) 2326-2397 (Online) Journal homepage: https://www.tandfonline.com/loi/ucsu20
Spent Mushroom Compost of Pleurotus ostreatus: A

Tool to Treat Soil Contaminated with Endosulfan
Saima Sadiq, M. Mahmood-ul-Hassan, Nazia Rafiq & Karam Ahad
To cite this article: Saima Sadiq, M. Mahmood-ul-Hassan, Nazia Rafiq & Karam Ahad (2019):
Spent Mushroom Compost of Pleurotus�ostreatus: A Tool to Treat Soil Contaminated with
Endosulfan, Compost Science & Utilization, DOI: 10.1080/1065657X.2019.1666067
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COMPOST SCIENCE & UTILIZATION
https://doi.org/10.1080/1065657X.2019.1666067
Spent Mushroom Compost of Pleurotus ostreatus: A Tool to Treat Soil

Contaminated with Endosulfan
Saima Sadiqa , M. Mahmood-ul-Hassanb, Nazia Rafiqc and Karam Ahadc
a
Soil Environment Lab, Department of Plant and Environmental Protection, PARC Institute of Advanced Studies in Agriculture
Affiliated with Quaid-i-Azam University, Islamabad, Pakistan; bLand Resources Research Institute, National Agriculture Research Centre,
Islamabad, Pakistan; cEcotoxicology Research Program, National Agriculture Research Centre, Islamabad, Pakistan
ABSTRACT
Composts especially spent mushroom composts (SMC) have been used for their ability to
degrade toxic organic pollutants. Due to extreme toxicity, endosulfan (C9H6Cl6O3S) is catego-
rized as a Category 1 pollutant by the U.S. EPA because of its well-reported carcinogenicity.
This study was done to monitor the biodegradation potential of SMC against this pesticide.
For this purpose, bioreactors (BRs) system was designed to mimic the field conditions. Soil
within all four BRs contaminated with endosulfan was amended with four different treatments
of SMC. Quantitative reduction in amount of endosulfan isomers was calculated using Gas
Chromatography–Electron Capture Detector. For the monitoring of metabolites formed as a
result of biodegradation, Gas Chromatography–Mass Spectrometry was used. Maximum
attenuation was observed in BR1 (fresh SMC and soil). In BR2 when fresh SMC was added in
sterilized soil, rate of removal was declined as compared to BR1. In another bioreactor BR3,
where unsterilized soil was used with sterilized SMC, total reduction in quantity of endosulfan
was less than BR1 and BR2. BR4 (abiotic control) showed the least reduction suggesting the
role of SMC and soil microbes. Degradation was well described using simple first-order kinet-
ics which revealed that the active microcosm of BR1 manifested least DT50. Denaturation of
either SMC(BR3) or soil(BR2) or both (BR4) resulted in less biodegradation than BR1.
Introduction Endosulfan, an organochlorine pesticide, is a

Mushroom production is the biggest solid-state combination of the two isomers, a and b
fermentation industry in the world (Moore and (Shivaramaiah and Kennedy 2006). Bioaccumulation
Chiu 2001). Spent mushroom compost (SMC) is of endosulfan in the food chain is mainly because
the bulky waste byproduct left after mushroom of its high solubility in lipids (Weber et al. 2010).
production/harvesting. Pleurotus ostraetus (Oyster Endosulfan transforms to endosulfan sulfate in soil
Mushroom) accounts for almost 25% of world- (Aktar, Sengupta, and Chowdhury 2009) which
wide edible mushrooms production (Chiu et al. remains there as significant residue for long
2000). For the production of every kilogram of (Berntssen et al. 2008). Endosulfan sulfate is more
mushrooms, 5 kg of SMC is generally generated toxic than its parent isomers. This pesticide is well
(Semple, Reid, and Fermor 2001). Exploitation of known for its toxicity to most fish species causing
this compost is carried out for various purposes massive mortalities (Naqvi and Vaishnavi 1993).
but the most important is treatment of sites con- Despite the ban imposed on use of this pesticide in
taminated with organopollutants (solid phase bio- agriculture sector years ago, it is still detected in
degradation) (Semple, Reid, and Fermor 2001). surface water, air and soil (Siddique et al. 2003; Sun
Because of the presence of residual nutrients et al. 2006). In Pakistan, no consideration has been
(Marın-Benito, Sanchez-Martın, and Rodrıguez- given to implementation of pesticides policy
Cruz 2016) and immobilized enzymes in it (Akhtar et al. 2014) which made the situation even
(Tsang 2004), SMC has emerged as one of the worse. Due to its history of continuous use in coun-
effective ways to treat polluted sites. try, this pesticide is widely distributed from the
CONTACT Saima Sadiq [email protected] Mailing Address Soil Environment Lab, Department of Plant and Environmental Protection,
PARC Institute of Advanced Studies in Agriculture Affiliated with Quaid-i-Azam University, Islamabad, Pakistan
ß 2019 Taylor & Francis Group, LLC
2 S. SADIQ ET AL.
locations where it was actually applied (Anwar, (anhydrous) and sodium acetate trihydrate
Ahmad, and Tahir 2012). In a market-based survey, (NaAc.3H2O) used during extraction of pesticides
this pesticide was detected in various fruits. were purchased from Merck, Millipore. All other
In the past, biodegradation strategies involved chemicals used for enzymes analysis i.e. veratryl
batch enrichment cultures using the compound alcohol (3,4-Dimethoxybenzyl alcohol), Hydrogen
of interest as a substrate, followed by isolation of peroxide, d-tartaric acid, o-dianisidine, manga-
pure culture, to grow on pesticide as a sole nese sulfate, sodium tartrate and 2,20 -azino-di-[3-
source of carbon and energy (Bollag and Liu ethyl-benzo-thiazolin-sulphonate (ABTS) were
1990). Biodegradation of endosulfan was tested also of reagent grade.
under lab conditions using several bacterial and
fungal cultures either directly in soil or after Preparation of fresh spent mushroom compost
extracting certain microbes from soil which are
later on utilized for this purpose (Hussain et al. Pure culture of P. ostreatus (WC-814) was shipped
2007, Kullman and Matsumura 1996; Sharma from Mushroom Research Program, Pennsylvania
et al.2013; Shivaramaiah and Kennedy 2006) In State University, USA. Spawn was grown on wheat
soil, viability of tested microbial inoculum (either grains and inoculated medium was incubated at
in liquid extracts or under solid state fermenta- 25 C for 28 days. Subsequently, wheat straws were
tion) is a point of concern as many microbial chopped (2–3 cm), moistened (70%), autoclaved
consortia cannot acclimatize natural field condi- at 120 C under 15 Psi for 2 h and were used as
tions. Endosulfan adheres to clay particles and growth substrate. The spawn was placed in poly-
ethylene bag containing chopped straws (com-
persists in soil for several years because of being
pletely sterilized). Generation of fruiting bodies
readily water-insoluble. Goal of any biodegrad-
occurred at day 15 (humidity level 80%) which
ation work should be to apply it under natural
continued for 2 months. After harvesting all the
field conditions. Therefore, a strategy which
fruiting bodies, the fresh waste colonized mush-
forces this pesticide to be bioavailable for further
room medium was called as SMC.
biodegradation is needed. Remotely, biodegrad-
ation of organochlorines using SMC gained atten-
tion and has been well documented in DDT Physicochemical properties of soil and SMC
degradation (Purnomo et al. 2010, 2014), but no Uncontaminated soil having no previous history
work has been done on biodegradation of endo- of endosulfan use for the last 5 years (surface
sulfan contaminated soil using SMC so far. soil: 0–15 cm) was collected from Organic
Evaluation of SMC as an agent to carry out bio- Farming Orchard, National Agriculture Research
degradation of endosulfan-contaminated soil Center, Islamabad, Pakistan (73.127855 E, and
under aerobic BR conditions was done in this latitude 33.666042 N). The soil was dried under
study. This research work will be helpful for the shade for 48 h and then passed through a
treatment of sites which are highly contaminated stainless steel sieve (<2 mm). A portion was
with OCPs especially endosulfan. obtained from the 2 mm fraction and plant roots
were removed. Soil was ground again to obtain
Material and methods <200 mm fraction which was later used to deter-
mine its physicochemical properties and other
Chemicals and regents
batch degradation experiments. The physical and
Analytical grade a-endosulfan and b-endosulfan chemical parameters of soil were measured fol-
were purchased from Sigma Aldrich. All the sol- lowing the standard procedures described by
vents like n-hexane, acetone, acetonitrile used in (Sparks et al. 1996). The soil was classified as
extraction and analysis were of pestanalV R grade. sandy-clay loam (52.53% sand, 25.32% clay,
The stock solution was prepared mixing a-endo- 22.55% silt) with moisture content of 35%. Loss
sulfan and b-endosulfan in equal ratio in n-hex- of ignition test was used to calculate the organic
ane. Reagent grade Magnesium sulfates matter content of the soil which was found to be
COMPOST SCIENCE & UTILIZATION 3
Table 1. Physicochemical properties of soil and SMC used Table 2. Lignolytic enzyme activities of Pleurotus ostreatus
during study (n ¼ 5). SMC used during biodegradation studies (n ¼ 3).
Properties Soil SMC Activity of enzymes
pH 8.04 7.25 Enzyme analyzed Sterilized SMC Fresh SMC wheat Straw
EC (mS/cm) 2.49 n.d
Organic matter (%) 0.901 30.66 Laccase (U/ml) 0 ± 0.00 4.87 ± 2.67 1.34 ± 0.59
Total organic carbon (%) n.d 14.62 MnP (U/ml) 0 ± 0.00 9.75 ± 8.12 4.93 ± 1.4
Total nitrogen (%) n.d 1.036 LiP (U/ml) 0 ± 0.00 Nil Nil
Total protein (%) n.d 6.48
C:N ratio n.d 14:01
CaCO3 (%) 3.15 n.d It€avaara 1990) using ABTS [2,20 -azino-bis-(3-ethyl-
benzothiazolino-6-sulphonic acids)] as a substrate.
0.9%. SMC was alkaline in nature. Total organic Changes in absorbance were monitored at 436 nm
content (TOC), total nitrogen and total proteins (e ¼ 29,300 M1 cm1) (Table 2).
were also determined by following the method of
Marın-Benito, Sanchez-Martın, and Rodrıguez- Design of aerobic BR and experimental microcosms
Cruz (2016) (Table 1).
Standard method of solution preparation was
used to prepare the stock solution of Endosulfan
Lignolytic enzymes assays
(100 mg ml1). Since some fungi are sensitive to
Fresh SMC was added to sodium citrate buffer toxicity of Endosulfan (others can tolerate up to
(pH 5.0) and suspension was agitated for 3 h at 400 mg L1 (Bhalerao and Puranik 2007). It is
speed of 200 rpm at 4 C. The suspension was fil- always important to select the dose because high
tered through Mira cloth (22–25 lm) and filtrate concentration of endosulfan poses an adverse
was later on centrifuged at 10,000 rpm and 4 C effect on enzymes activities and production. High
for 25 min. The absorption of supernatant was dose of this pesticide sometime denatures the
measured using ultraviolet–visible spectropho- enzymes which are considered to be the most
tometer (Spectramax 250 Microplate) at different important factor for these types of biodegrad-
wavelengths (mentioned below). Activity was ation. Therefore, an optimum dose of endosulfan
recorded in (U ml1; where 1 U ¼ 1 lmol min1). was selected. For this purpose, predetermined
The estimation of oxidized substrate was made quantity of endosulfan was added in 20 g soil and
using the formula: 5 g of SMC to give an initial loading of 12.5
DC DA mgkg1 of endosulfan (on dry weight basis).
¼ (1)
t eDt:L Self-designed BR was used to study SMC-
where C is concentration, A absorbance and e mediated biodegradation of Endosulfan in soil.
extinction coefficient (Mm1 cm1). Bioreactor was designed by modifying the BR of
Reaction mixture for measuring activity of Gallego et al (2001) (Figure 1). Two holes were
Lignin peroxidase(LiP) consisted of 2 mM veratryl drilled on the top of a sealed reactor. One for
alcohol, 0.4 mM H2O2, 50 mM tartaric acid and supply of air to ensure aerobic conditions using
air pump (ResunV Air 8000) and the other for
R
extract from SMC containing enough LiP to give
an absorbance change of 0.2 min1 at 310 nm inserting agitation unit in main body of reactors
(e ¼ 9300 M1cm1) (Tien and Kirk 1988). to allow complete mixing of soil and chopped
Manganese peroxidase(MnP) activity was deter- straw in Reaction tank. Reactors were placed at
mined by the oxidation of o-dianisidine at 460 nm water bath (BenchmarkV R , BS-SB0012) to ensure
(e ¼ 29,400 M1 cm1) The reaction mixture con-

the temperature at 22 C. The study was con-
tained 0.1 mL of 1 mM manganese sulfate, 0.2 mL ducted for 35 days. Soil moisture was retained
of 0.5 M sodium tartrate, (pH 5.0), 0.1 mL of periodically after weighing the BRs to avoid evap-
1 mM H2O2 (Prepared fresh) and 0.1 mL of 1 mM orative water losses.
o-dianisidine. (Paszczynski, Crawford, and Huynh Following four different types of treatments
1988). Laccase activity was assessed by following were used to see the overall effect of SMC on
protocol described by (Niku-Paavola, Raaska, and biodegradation of endosulfan in soil.
4 S. SADIQ ET AL.
Figure 1. Schematic diagram of bioreactors used to monitor the dissipation of mixture of endosulfan isomers by using SMC where
(A) aeration pump, (B) agitation unit, (C) reaction tank, and (D) water bath.
BR1 (Active microcosm): unsterilized soil ino- 10 mL water in 50 mL centrifuge tube for 30 min.
culated with unsterilized fresh SMC. Later on 10 mL acidified acetonitrile (mixture of
BR2: Double sterilized soil inoculated with acetonitrile and acetic acid mixture – 99:1 v/v)
unsterilized fresh SMC. was also added and content was vortexed for
BR3: Denatured SMC in fresh soil (unsteril- 5 min. 4.0 g magnesium sulfate, anhydrous
ized soil inoculated with double sterilized 1.0 g ± 0.05 g sodium chloride, 1.0 g ± 0.05 g triso-
fresh SMC) dium citrate dehydrate and 0.5 g ± 0.03 g diso-
BR4: Double sterilized soil inoculated with dium hydrogen citrate sesquihydrate were added
double sterilized fresh SMC. for drying and buffering and were centrifuged at
SMC was sterilized twice using autoclave to 5000 g for 5 min. Acetonitrile layer was separated
denature all the enzymes and to kill all from aqueous layer and concentrated to approxi-
the microbes. mately 5 mL in rotary evaporator (Buchi-
Samples were periodically removed for analysis of Rotavapour R-210). The concentrated extract was
residual Endosulfan from sampling outlet. All the mixed with water (2 mL) and n-hexane (10 mL)
experiments were replicated thrice. The samples and swirled for 1 min. The mixture was allowed
were stored at -20 C before extraction and analysis. to stand for few more minutes, an aliquot of
8 mL of the upper n-hexane layer was again
reduced to 1.4 mL in amber glass vials using
Extraction and analysis
rotary evaporator. Samples were stored for chro-
Quick, easy, cheap, effective, rugged, and safe matographic analysis. The above-mentioned
(QuEChER) with cleanup extraction method pesticide recovery method was developed in
(Rashid et al. 2010) was used with slight modifi- endosulfan-free soil mixed with SMC. Recovery
cations for the extraction of endosulfan from was in accordance with performance acceptability
matrix. Samples (10 g) were hydrated by adding criteria set in SANCO’s procedure (2007).
Gas chromatograph (7890 B Agilent) equipped Statistical analysis

with micro-electron capture detector (G3440B) To determine the effect of each treatment on the
having capillary column HP-5(30 m 0.25 lm biodegradation of endosulfan, a factorial design
i.d 0.320 mm) was used to analyze Endosulfan with four treatments and three experimental blocks
in the samples. Other operational conditions (five repetitions per block) was applied (Steel and
were: carrier (nitrogen) flow rate 2 mL min1, James 1960). Least significance difference (LSD)
makeup flow rate 2 mL min1. Injector (split less was used to determine statistical significance of
mode) and detector temperatures maintained endosulfan removal between treatments. Data was
during study were 225 C and 280 C, respect- considered to be significantly different among
ively. Initial oven temperature of 80 C (0.5 min), value p (f) < 0.05. An analysis of variance and
increased to 15 C min1, then to 180 C (hold means separation was performed using the statis-
for 10 min) and finally at 15 C min1 to 250 C tical program Statistix 8.1.
(holding 10 min). 10 lL Hamilton syringe was
used to inject 1 mL of extracted sample by the
Results and discussion
solvent flush injection technique. Peaks appeared
at 17.186 min and 19.315 min were identified as Composting strategy is responsible for biodegrad-
a-endosulfan and b-endosulfan. Metabolites mon- ation of many toxic compounds (Duah-Yentumi
itoring was done using gas chromatograph (GC- and Kuwatsuka 1980) due to the presence of
7890B Agilent) mass spectrometry (MSD-5977A active micro-flora, lignolytic enzymes, and several
Agilent) GC-MSD. 99.9% Helium gas was used as other factors. High levels of residual nutrients
carrier gas at flow rate (2 mL min1) with col- and enzymes present in SMC make it an ideal
umn DB-5 Ultra inert (30 m 0.25 mm i.d tool to treat many organopollutants. SMC after
0.25 lm). The oven temperature was programed application in soil helps to maintain pH, mois-
to increase from 60 C (0.5 min) to 170 C ture content, soil structure and also acts as a
(3 min) at 20 C min1 and finally increased at nutrient source, thereby improving the condition
rate to 295 C (5 C min1). RTLPEST3 was used of contaminated soil for indigenous or intro-
as matching library. duced microbial degradative activity (Semple,
Reid, and Fermor 2001).
For this study, four different treatments were
Kinetic studies
applied in designated BRs (Figure 1). When
The best-fitted model suggested by the software endosulfan contaminated soil was amended with
R (version 3.0.3) with active kinfit software pack- fresh SMC, reduction was different for both the
age for biodegradation data with time was simple isomers in all four BRs. Reduction data obtained
first-order kinetics (SFO). Half-life (DT50) of was fitted in different kinetic models for good-
endosulfan was calculated by formula (2) men- ness of fit. The experimental data and regression
tioned below in line with FOCUS guidelines coefficient (r2) obtained for endosulfan incuba-
(Slana and Sollner-Dolenc 2016) for each set of tion experiment after statistical analysis using a
laboratory conditions was used. formal approach proposed by (Timme and
0:693 Frehse 1980) showed that SFO model fitted good.
DT 50 ¼ t1=2 ¼ (2) Therefore, for the description of the persistence
k1
For determination of rate constant following of endosulfan under different conditions, this
model was applied by using Microsoft Excel
equation was used (Yang, Wang, and Chen
Solver as reported by (Ma et al. 2004). Figure 2a
2014):
and b showed the reduction of both the isomers
lnC ¼ a þ K1 t (3)
of Endosulfan under four different BRs. In BR1
Where t is time given to compound for deg- (active microcosm) (fresh soil and SMC), reduc-
radation, LnC is natural logarithm of concentration of a-endosulfan was 58% of applied concen-
tion and K1 is first order rate constant. tration while b-Endosulfan was removed almost
6 S. SADIQ ET AL.
(a) 60
BR1 BR3
50 BR2 BR4
40
% relative reduction of β-Endosulfan
30
20
10
0
0 5 10 15 20 25 30 35 40
Time(Days)
BR1 BR3
(b) 60 BR2 BR4
% relative reduction of α-Endosulfan
50
40
30
20
10
0
0 5 10 15 20 25 30 35 40
Time(Days)
Figure 2. Relative reduction of endosulfan isomers (1:1) (a) a-endosulfan (spiked double) and (b) b-endosulfan.
48%. About 49% of total a-endosulfan reduction reduction of applied extractable a-endosulfan and
was observed during first week while the remain- b-endosulfan, respectively.
ing reduction occurred during next 3 weeks. This Earlier findings suggested that various enzymes
could be attributed to degradation in the first and indigenous microbes present in the soil
phase of rapid decomposition during composting amended with SMC degrade organic pollutants
mechanism (Megharaj et al. 2011). Dynamic (Ntougias et al. 2004). DDT, a notorious OCP
equations for the description of dissipation are was already documented to be degraded by SMC
presented in Table 3. Rate of dissipation of of Pleurotus ostreatus but this degradation was
a-endosulfan in BR1 was 0.024 per day associated with intracellular enzymes (Purnomo
(K1 ¼ 0.024) which resulted in DT50 equal to et al. 2010). Lignolytic enzymes, Laccase, MnP
28.69 days while in case of b-endosulfan it was and LiP of SMC were also monitored (Table 2).
36.81 days (K1 ¼ 0.018). In comparison to BR1, In the past, role of Laccase was found not to be
BR4 (abiotic control) resulted only 17 and 10% of correlated with biodegradation of Endosulfan
Table 3. Kinetic dissipation of endosulfan isomers in different BRs using various treatments of SMC in soil.
Isomers Treatments Kinetic equation K1 (day1) DT50 DT90 r2
a-Endosulfan BR1 lnC ¼ 4.52 0.02t 0.02415 28.69 95.32 0.95
BR2 lnC ¼ 4.61 0.0125t 0.0125 55.45 184.198 0.99
BR3 lnC ¼ 4.59 0.01t 0.01 69.18 299.82 0.98
BR4 lnC ¼ 4.61 0.005t 0.005 138.29 459.38 0.99
b-Endosulfan BR1 lnC ¼ 4.53 0.018t 0.0188 36.81 122.31 0.94
BR2 lnC ¼ 4.60 0.0102t 0.01019 67.96 225.76 0.99
BR3 lnC ¼ 4.59 0.007t 0.007 96.85 321.71 0.98
BR4 lnC ¼ 4.61 0.0034t 0.00338 204.79 680.3 0.98
(Ulcnik, Cigic, and Pohleven 2013). No LiP was a-endosulfan was 30% (K1 ¼ 0.01% day1) result-
detected but reduction occurred showed that there ing in DT50 of 69 days. b-Endosulfan was reduced
is no considerable role of this enzyme in reduction only 23% of applied concentration showing the
of endosulfan. In BR1, maximum reduction of slowest rate of dissipation (0.007 day1) and
both the isomers of endosulfan is consistent with manifested DT50 of 97 days. Since in this BR
earlier findings where soil enriched with SMC has effective role of enzymes and microbes in SMC
the ability to degrade complex toxic pollutants was eliminated during double sterilization
(Eggen 1999) because of enzymes present in SMC (Kunjadia et al. 2016), removal was less than BR1
(not essentially lignolytic) and indigenous microbes and BR2. Nonetheless, the sterilized SMC could
(Hussain et al. 2007). only serve as carbon amendment in soil and
In another finding, Pleurotus pulmonarius has increased the amount of microbial biomass in
been tested for biodegradation potential against soil that might remove endosulfan through their
endosulfan. A degradation of 95% during its sub- degradative capacities (Perez-Piqueres et al. 2006)
strate colonization phase and 99% during fruiting and/or adsorption processes (Ulcnik, Cigic, and
phase was attributed to enzymatic loads present in Pohleven 2013).
this fungi (Hernandez-Rodriguez et al. 2006). The difference in reduction at each time interval
However, another study conducted in the past asso- was significant in case of a-endosulfan (P < 0.001).
ciated the reduction in amount of endosulfan with However, in case of b-endosulfan there was no sig-
mycelial adsorption (Ulcnik, Cigic, and Pohleven nificant difference in biodegradation at most of
2013). The reduction could also be due to the abil- time interval where sampling was done to find the
ity of SMC and other compost to decrease the rate. In treatment BR4 (abiotic control condition),
compactness of soil which resulting into more aer- a very low reduction could be due to physical and
ation which favors the bacterial growth in the soil. chemical adsorption on either soil mineral or SMC
Increased metabolic activities of bacteria in conta- (Megharaj et al. 2011). The large difference in
minated soil results in faster degradation of pesti- reduction of a-endosulfan between BR2 (unsteril-
cides (Abioye, Agamuthu, and Abdul Aziz 2012). ized SMC) and BR4 (unsterilized soil and SMC)
In BR2 (fresh soil in sterilized soil), total demonstrate the important role of external carbon
reduction was 36 and 29% of applied a-endosul- source on soil microbes for their potential to bio-
fan and b-endosulfan, respectively. The rate of degrade pollutants. The amendment of soil with
reduction of a-endosulfan resulted in DT50 of SMC reduces the persistence or DT50 of endosul-
55 days. Calculated DT50 for b-endosulfan was fan isomers to varying degrees (Table 3). Based on
67.96 Days (Table 3). This less degradation than results, addition of SMC is recommended for the
BR1 was associated with the absence of activity of remediation of hotspots located throughout the
soil inhabitant microbes. Total removal of both country containing huge piles of obsolete pesticides
the isomers of endosulfan was possibly due to the including endosulfan (Syed, Malik, and
role of SMC, not only as source of enzyme and Muhammad 2014) because it degraded endosulfan.
indigenous microbes but also as carbon amend- Biodegradation of endosulfan in all the BRs
ment (Meynet et al. 2012). tested showed that b-isomer of endosulfan was
In BR3 (fresh unsterilized soil and doubly steri- reduced in less quantity in all BRs than a-isomer.
lized denatured SMC), total relative reduction of The results are in agreement with earlier findings
8 S. SADIQ ET AL.
(a)
(b)
Figure 3. Dissipation kinetics using simple first-order equation (a) a-endosulfan and (b) b-endosulfan. (Lines showed the fitted sim-
ple first-order equation.
where the solid phase biodegradation in the soil another reason of why the degradation of
showed the rate of b-endosulfan slower than a-endosulfan was more than b-endosulfan.
a-isomer and was attributed to its higher parti- Metabolite monitoring done at GC-MSD
tion coefficient on the soil (Tiwari and Guha showed that two main metabolites were detected
2013). Degradation rates of the a-endosulfan in in first three BRs. One was endosulfan sulfate
soils is generally higher than b-isomer (Hwang, and the other was endosulfan lactone (Figure 4).
Lee, and Kim 2015) (Figure 3). Inter conversion No other metabolites were detected at any phase
of both the isomer is also well reported in the of study. This agrees with the fact that the activ-
past (Miles and Moy 1979) especially conversion ities of microbes helped in formation of endosul-
of b-isomer to a-isomer which is irreversible. It fan sulfate by an oxidative way while the alkaline
has also been reported that rate of conversion of nature of both soil and SMC (Table 1) favored
a-endosulfan to endosulfan sulfate is more than hydrolysis which resulted in formation of endo-
b-endosulfan (Singh et al. 1991). This could be sulfan lactone. In the soil, formation of some
Figure 4. Identification of endosulfan sulfate and endolactone as breakdown metabolic products of endosulfan on GC-MSD (Peak
a: endolactone, Peak b: a-endosulfan, Peak c: b-endosulfan, Peak d: endosulfan sulfate).
other metabolites such as endosulfan diol, endo- and Moy 1979; Kullman and Matsumura 1996)
sulfan hydroxyether and endosulfan dialdehyde is however none of the aforesaid metabolites were
well reported during hydrolytic mechanism (Miles detected in this case. Under abiotic control in BR4,
10 S. SADIQ ET AL.
Figure 5. Endosulfan pathways (based on two detected metabolites). Compounds enclosed in box and with solid lines were those
appeared after application of SMC in this study. Dotted lines indicate the proposed pathways based on study using Phanerochaete
chrysosporium (Kullman and Matsumura 1996) which were not detected during this study.
no metabolite was detected. It is indicated that hotspots of OCPs, waste dumps, industrial efflu-
degradation occurred along with interconversion ents and water environments.
and adsorption/absorption. However, under all
other three BRs (BR1–BR3) endosulfan sulfate and
endosulfan lactone were formed. Extent of forma- Conflict of interest
tion of metabolites was higher in BR1 followed by The authors declare no conflict of interest.
BR2 and BR3. At day 7, endosulfan sulfate
appeared in BR1 which began to decline afterwards
and endosulfan lactone appeared at the end of Acknowledgments
third week. The identified peaks of endosulfan lac- We are very thankful to Dr. Ming Tien, Department of bio-
tone and endosulfan sulfate are shown in Figure 4. chemistry and molecular biology, Pennsylvania State
The proposed pathway after monitoring of two University for his technical support in lignolytic enzyme
metabolites using SMC in soil is given in Figure 5. analysis and Shengzhong Su of Statistical Consultation
Center, Pennsylvania State University for his guidance in
statistical analysis and handling with Kinetics software.
Conclusion
ORCID
SMC-mediated biodissipation of endosulfan is
Saima Sadiq http://orcid.org/0000-0002-0711-4946
reported in this study. Soil amendment with
SMC positively enhanced the rate of biodegrad-
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Photocatalytic degradation of imidacloprid using

Cite this: RSC Adv., 2023, 13, 19326
Ag2O/CuO composites
Open Access Article. Published on 26 June 2023. Downloaded on 5/29/2024 11:29:08 AM.
Saadia Rashid Tariq, *a Zunaira Niaz,a Ghayoor Abbass Chotana, b

Dildar Ahmadc
and Nazia Rafiqued
Imidacloprid is one of the most commonly used neonicotinoid pesticides that has been identified as
a neurotoxin for various non-target organisms. It binds to the central nervous system of organisms,
causing paralysis and eventually death. Thus, it is imperative to treat waterwaters contaminated with
imidacloprid using an efficient and cost effective method. The present study presents Ag2O/CuO
composites as excellent catalysts for the photocatalytic degradation of imidacloprid. The Ag2O/CuO
composites were prepared in different compositions by adopting the co-precipitation method and used
as a catalyst for the degradation of imidacloprid. The degradation process was monitored using UV-vis
spectroscopy. The composition, structure, and morphologies of the composites were determined by FT-
IR, XRD, TGA, and SEM analyses. The effect of different parameters i.e time, concentration of pesticide,
concentration of catalyst, pH, and temperature on the degradation was studied under UV irradiation and
dark conditions. The results of the study evidenced the 92.3% degradation of imidacloprid in only 180
minutes, which was 19.25 hours under natural conditions. The degradation followed first-order kinetics,
with the half life of the pesticide being 3.7 hours. Thus, the Ag2O/CuO composite was an excellent cost-
Received 31st March 2023
Accepted 21st June 2023
effective catalyst. The non-toxic nature of the material adds further benefits to its use. The stability of
the catalyst and its reusability for consecutive cycles make it more cost effective. The use of this material
DOI: 10.1039/d3ra02109b
may help to ensure an immidacloprid free environment with minimal use of resources. Moreover, the
rsc.li/rsc-advances potential of this material to degrade other environmental pollutants may also be explored.
leads to the death of insects.3,4 Fleas on domestic pests can also

Introduction be treated by the use of imidacloprid. The water solubility of
The environmental pollution caused by industrial and agricul- imidacloprid coupled with its excessive and unwise use leads to
tural waste is continuously affecting the global climate. Pesti- its emissions into water bodies there by adversely affecting the
cides are widely used in the agriculture sector to control pests aquatic organisms as well as human health. In humans, it can
and crop damaging insects. These pesticides are persistent cause loss of consciousness and severe respiratory failure.5–7
under normal environmental conditions and thus stay in the Therefore, it is imperative to nd a cost-effective method for the
environment for a very long time, thereby causing many abatement of hazardous pollutants like imidacloprid from
hazardous effects on human health and the environment. water bodies. A number of methods have been reported to
Water pollution caused by the excessive use of pesticides is one mitigate the imidacloprid and similar pesticides from water
of the important issues faced nowadays.1,2 bodies including bio-degradation, photolysis, ozonation,
One of the commonly used pesticides is imidacloprid that electro-fenton oxidation and ultrasound assisted
belongs to chloro-nicotinic class of pesticides. It was developed degradation.5,8–10 Bio degradation is one of the important
in 1986 and since then it has been registered in 120 countries methods to transform imidacloprid but the efficiency of this
worldwide. It works on the motor neurons of insects and creates method is very low.5 Similarly, the other methods suffer from
the over simulation of the nervous system which ultimately certain limitations such as the formation of metabolites or the
generation of waste that need further processing. In this regard,
a
photocatalytic degradation has been given due consideration
Department of Chemistry, Lahore College for Women University, Jail Road Lahore,
during recent years. This method involves the use of metal/
54000, Pakistan. E-mail: [email protected]
b
Department of Chemistry and Chemical Engineering, Syed Babar Ali School of Science
metal oxides as the catalyst to facilitate the degradation of
& Engineering (SBASSE), Lahore University of Management Sciences (LUMS), Lahore pollutants.11,12
54792, Pakistan Semiconductor metal oxide nanoparticles are considered
c
Department of Chemistry, Forman Christian College (A Chartered University), Lahore, excellent photocatalysts. The process involved mainly depends
54000, Pakistan on the transfer of electrons from the valence band to the
d
Pakistan Agricultural Research Council, Islamabad, Pakistan
19326 | RSC Adv., 2023, 13, 19326–19334 © 2023 The Author(s). Published by the Royal Society of Chemistry
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Paper RSC Advances
conduction band on the surface under illumination by a suit- were collected and dried at 80 °C in an electric oven for 30
able wavelength of light.13,14 When the light strikes the surface minutes. Finally, the calcination of dried precipitates was
of the semiconductor, the electrons get excited from the valence carried out in a furnace at 400 °C for 2 hours.21 The prepared
band to the conduction band by the absorption of photons and Ag2O/CuO composites were characterized by XRD, FTIR, SEM
create free electron–hole pair. The rate of production of active and TG analyses, and employed as catalyst for the photocatalytic
charge carrier species is directly related to the efficiency of the degradation of imidacloprid.
catalyst. To achieve the maximum activity, the energy of the
photon should be higher or equal to the bandgap of the catalyst Photocatalytic degradation of imidacloprid
material.15,16
Metal/metal oxide-based nanoparticles show better catalytic The solutions of different concentrations of imidacloprid (10,
activity than bulk materials because they have a high surface-to- 20, 30, 40, and 50 mg L−1) were prepared to study the photo-
volume ratio as compared to their respective molecular or catalytic degradation of imidacloprid. These solutions were
atomic scale materials.17 Copper oxide (CuO), a p-type semi- stirred under UV light in the presence of Ag2O/CuO composite
conductor has gained paramount importance as a photocatalyst for specic time intervals and then analyzed by a UV-visible
due to its high stability, excellent optical and electrical prop- spectrophotometer (LABOMED, INC, model no: “UVD-3200”)
erties as well as low cost of formation.13,18 at 268 nm for determining the le over concentration of pesti-
To enhance the photocatalytic properties of metal oxide cide aer degradation. The same process was also performed
semiconductors, they are doped with other metal ions exhibit- under dark. The blank experiments were carried out in the
ing different bandgap values that create an interface between absence of Ag2O/CuO composite. The effect of various param-
them and efficiently reduce the recombination rate of photo- eters was also studied on the degradation of the aqueous
generated electron–hole pairs. This method enhances the utility solution of imidacloprid. These included: irradiation time,
and efficiency of the catalyst to a noticeable extent.15,19 The pesticide concentration, dose of catalyst, pH, and temperature.
incorporation of noble metals has drawn the attention of many The effect of UV irradiation time on the photocatalytic
scientists due to their fascinating catalytic properties. Doping of degradation was studied by stirring the imidacloprid solution for
silver on CuO can enhance the reactivity and selectivity and different time intervals i.e., 60, 120, 180, 240, and 300 minutes in
produce an excellent photo-catalyst.20 Thus, present study the presence catalyst under UV light. The effect of the initial
focused on designing an efficient catalyst i.e., silver oxide (Ag2O) concentration of pesticide was studied by preparing different
doped copper oxide composites for the degradation of widely concentrations i.e., 10 mg L−1, 20 mg L−1, 30 mg L−1, 40 mg L−1
used pesticide imidacloprid under UV irradiations. The mate- and, 50 mg L−1 of imidacloprid solution. A 0.01 g portion of Ag2O/
rial exhibited low leachability and high stability under aqueous CuO catalyst was added to a 50 mL volume of each of these
conditions. solutions. The samples were stirred for a specic time interval
in the presence of UV light and analyzed for imidacloprid
concentration. The similar control experiments were performed
Experimental methodology in the dark. Various amounts of Ag2O/CuO composite i.e., 0.01 g,
The Ag2O/CuO composites were prepared with different 0.02 g, 0.03 g, 0.04 g and, 0.05 g were added to 50 mL volumes of
compositions and used for the degradation of imidacloprid. imidacloprid solutions to study the effect of catalyst dose on
The kinetics of photocatalytic degradation was determined by degradation under UV light for a specic time interval.
Langmuir Hinshelwood kinetic model. The effect of pH was studied by maintaining the pH of imi-
dacloprid solution at 3, 5, 7, 9, and 11 by adding HCl or NaOH
solutions, in the presence of 0.01 g of catalyst. The stirring of
Quality control and quality assurance
the prepared solution was continued for an optimum time in
All the chemicals used in the present study were of analytical the presence of UV light as well as dark. The effect of temper-
grade with a purity of 99.9%. The glassware used was properly ature on photocatalytic degradation of imidacloprid solution
washed and rinsed with distilled water to remove any impurity was studied by varying temperature of pesticide solution from
adhered to it. Aerward, it was dried for 30 minutes in an 20 °C–40 °C in the presence of 0.01 g catalyst. The reusability of
electric oven set at 100 °C. A doubly distilled water prepared by the catalyst was studied for three consecutive cycles by regen-
(Milli Q) was used throughout the study. The technical grade erating the catalyst. The stability of the catalyst was also studied
imidacloprid was provided by Ali Akbar group of industries. by carrying out the leaching experiments.
Preparation of Ag2O/CuO composites

Results and discussion
The co-precipitation method was adopted to prepare the
composites where the weight% of Ag2O in Ag2O/CuO compos- The study focused on the degradation of imidacloprid in the
ites was kept at 1.5, 3.0, and 5.0. For this purpose, different presence of Ag2O/CuO composite. The UV-visible spectra of
amounts of silver nitrate (0.0038, 0.0076, and 0.0127 g) were imidacloprid (Fig. 1) depicted its lmax to be 268 nm which is
added to copper nitrate solutions separately and dissolved consistent with literature value.22 This lmax of 268 nm was used
completely. Aerward 0.1 M ammonium bicarbonate was added throughout the study for the determination of pesticide
dropwise until no more precipitation occurred. The precipitates concentration during different sets of experiments.
© 2023 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2023, 13, 19326–19334 | 19327
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Fig. 1 UV-vis absorption spectra of imidacloprid.
Characterization of Ag2O/CuO composites

All the three Ag2O/CuO composites were characterized by XRD,
FTIR, TG, and SEM analysis. X-Ray patterns of pure CuO and
Ag2O/CuO were recorded in the 2q range of 10–80° and depicted
in Fig. 2(a and b). The diffraction lines for CuO were found at 2q
values of 32.7°, 35.4°, 36.1°, and 39.3° that agreed well with
standard diffraction values for monoclinic CuO with planes
(110), (002), (−111), (200) (JCPDS File no. 01-080-0076) [ref]. The
characteristic diffraction peaks of Ag2O were observed at 2q
values of 26.75°, 33.9°, 38.2° and 46.7° that matched well with
the JCPDS File no. 00-001-1041 of cubic Ag2O. Sharp peaks were
Fig. 3 SEM images of Ag2O doped CuO composites (a: 1.5% Ag2O/
CuO, b: 3% Ag2O/CuO and c: 5% Ag2O/CuO).
observed for Ag2O/CuO composites that evidenced the absence

of any impurity in the sample. The average particle size
observed was in the range of 20–35 nm which was determined
by Scherer calculations using X'pert Highscore soware.23,24 The
Scanning Electron Micrograph of Ag2O/CuO is presented in
Fig. 3. The gure clearly showed agglomerated spherical parti-
cles. The presence of Ag2O in Ag2O/CuO turned the samples into
more homogeneous ones. The EDX study conrmed the
formation of Ag2O/CuO composites as depicted in Fig. 4. The
peaks represent the presence of Ag followed by Cu and O.
Different elemental proportions of Ag were observed in the
analysis which conrmed the different doping concentrations.23
The IR spectrum of Ag2O/CuO composites is presented in
Fig. 5. The vibrations around 400 cm−1 to 600 cm−1 are char-
acteristic of Cu–O and Ag–O. The vibration observed at
1600 cm−1 is due to the bending vibrations of absorbed water
molecules. The vibrations observed at 1370 cm−1 are attributed
to the longitudinal phonon, which is similar to the character
commonly possessed by nanoparticles.24–26 The bands at 1160
(a) XRD patterns pure CuO nanoparticles. (b)XRD patterns of
Fig. 2
and 1100 cm−1 were assigned to C–O stretching vibrations.27
Ag2O/CuO composite.
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Fig. 6 TGA curve of Ag2O doped CuO nanoparticles.
Fig. 4 EDX of Ag2O doped CuO composites (a: 1.5% Ag2O/CuO, b: 3%

Ag2O/CuO and c: 5% Ag2O/CuO).
Fig. 7 Photo luminescence spectra of different compositions of Ag2O

doped CuO catalyst.
Fig. 5 FT-IR spectrum of Ag2O doped CuO nanoparticles.

the rate of electron–hole recombination becomes lower as
compared to CuO this rate is reduced with increase in
concentration of Ag.29,30 Thus 5% Ag2O doped CuO was better
The thermal stability of Ag2O/CuO composites was investigated
photocatalyst.
by thermogravimetric analysis as depicted in Fig. 6. Small
weight loss was observed up to a temperature of 100 °C that was
attributed to the removal of moisture present on the surface of Photocatalytic degradation of imidacloprid by Ag2O/CuO
composites. The weight loss observed in the range of 600–900 ° composite
C was due to the loss of solvent molecules trapped in the crystal The photocatalytic degradation of imidacloprid was studied by
lattice. Aer 900 °C no more weight loss was observed.28 All the optimizing various parameters such as time, catalyst dose, the
compositions followed the same pattern. concentration of pesticide, temperature, and pH in the presence
of Ag2O/CuO as a catalyst.
Photoluminescence (PL) analysis
Photoluminescence (PL) study of CuO and Ag2O doped CuO Optimum composition of the catalyst
composites are depicted in Fig. 7. The gure shows two domi- The presence of silver in Ag2O/CuO affected the degradation
nant emission peaks for CuO centered at 416 nm and 475 nm efficiency of copper oxide. The silver ions play the role of the
that are attributed to the green emission produced from elec- sink for the photo-excited electrons from the semiconductor.
tronic transitions of ionized oxygen vacancies. However, in the The separation efficiency of holes and photo-excited electron is
case of Ag2O doped CuO composites the peak at 416 nm is also improved as the silver ions also play a role in the inhibition
shied to 405 nm. The gure clearly shows a decrease in the of charge recombination.31 It was also found that photocatalytic
intensity aer doping with silver. Furthermore, the intensity of activity of Ag2O/CuO was increased by increasing the silver
peak was decreased with increase in the concentration of Ag2O. concentration from 1.5% to 5%. Aer optimum concentrations,
These results are explained on the basis of fact that presence of the degradation efficiency was reduced due to oxygen defects.
Ag in the composite changes the size of the crystallites due to its Tariq et al. 2019, calculated the degradation efficiency of Ag
amorphous nature. Therefore, for Ag2O doped CuO composites, doped ZnO with Ag doping of 3% to 7% and a decrease in
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RSC Advances Paper
So, UV irradiation slightly promoted the degradation

process. The presence of the catalyst signicantly enhanced the
degradation of imidacloprid even under dark conditions i.e.,
65% of imidacloprid degraded under dark which was increased
to 72% under UV irradiation for 3 hours. Aer 3 hours, no major

increase in the degradation of imidacloprid was observed. It is
also worth noting that the degradation pattern observed in the
presence of a catalyst was different from one observed in the
absence of the catalyst. In the absence of the catalyst, the
degradation rst increased slowly up to 2 hours, and then it
rapidly increased up to 3 hours. Aerwards, little or no degra-
Fig. 8Degradation of imidacloprid with different compositions of

Ag2O/CuO composite. dation was observed. In the presence of the catalyst, a rapid
increase in degradation was observed for up to 2 hours. Aer
this, a non-signicant increase in degradation was observed.
The main advantage of this process is the relatively lower
degradation efficiency was observed aer optimum concentra- reaction time that leads to a reduction in the construction and
tion. Fig. 8 depicted the degradation efficiency of different operating costs.33
Ag2O/CuO composites. The maximum degradation of imida-
cloprid was observed with 5% Ag2O/CuO which was found to be Effect of concentration of pesticide
92.5%.32 Therefore, this composition of catalyst was used The data for degradation of different concentrations of imida-
throughout the study. cloprid is provided in Fig. 10. It was found that the maximum
degradation of 74.3% was observed for 10 mg L−1 of imidaclo-
Effect of time prid under UV light and 67% under dark. The percentage
degradation of imidacloprid was decreased by increasing its
The appropriate time for the photocatalytic degradation of
concentration. It was attributed to the increased equilibrium
imidacloprid was determined by adding 0.02 g of 5% Ag2O
adsorption on active catalyst sites.32,34
doped CuO composite to 25 mL of imidacloprid solution and
stirring the samples for different time intervals ranging from 1–
Effect of dose of catalyst
5 hours. Fig. 8 depicts the data for the imidacloprid degradation
in the absence of a catalyst under UV light and dark. The cor- The data for degradation of imidacloprid solution (10 mg L−1)
responding data for the experiments conducted in the presence by using different amounts of catalyst i.e., 0.01, 0.02, 0.03, 0.04,
of a catalyst are also furnished in Fig. 9. The data showed that in and 0.05 g, is presented in Fig. 11. The maximum degradation
the absence of catalyst, a 21.8% degradation of imidacloprid (80.7%) of imidacloprid was observed with 0.01 g of catalyst in
was observed in dark. Under UV irradiation, the extent of the presence of UV light and 77.4% under dark conditions.
degradation was enhanced to 27.4%. By increasing the catalyst dose from optimal amount, the
degradation efficiency was observed to be reduced due to the
clustering of Ag2O/CuO particles that consequently resulted in
a decreased available sites on the catalyst surface. The increased
catalyst concentration also reduced the light penetration due to the
turbidity of the medium that also led to reduced degradation.35
Effect of pH
The data for degradation of 10 mg L−1 imidacloprid at different
pH values is depicted in Fig. 12 which showed a maximum
Fig. 9 Effect of time on the degradation of imidacloprid. Fig. 10 Effect of concentration of imidacloprid on its degradation.
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Paper RSC Advances

Fig. 13 Effect of temperature on the degradation of imidacloprid by

Fig. 11 Effect of catalyst dose on degradation of imidacloprid. using Ag2O doped CuO catalyst.
degradation of 85% in the presence of UV light and 79.8% The degradation efficiency increased rst with increasing
under dark condition at a pH 11. The minimum degradation temperature. Aer an optimum temperature, there was no
was observed at pH 3 which was 64.1% under dark and 72.15% increase in degradation efficiency with temperature. Patil et al.
in the presence of UV light. studied the degradation of imidacloprid at temperatures above
The degradation of imidacloprid was increased at higher pH 30 °C and found the extent of degradation to be 12.85%,
due to the increased OH− concentration that increased the 12.69%, and 12.54% at operating temperatures of 34 °C, 39 °C,
hydrolysis of imidacloprid. A –C]N– bond couples with and 42 °C respectively.37 Our studies also showed that the
electron-withdrawing –NO2 group of imidazolidine ring degradation efficiency increases till the optimum temperature
creating a small positive charge that reacts with OH ions in is achieved. At 30 °C the extent of degradation observed was
solution due to high pH thus increasing the hydrolysis and 92%. Aerwards, no further increase in degradation was
extent of degradation. The degradation of imidacloprid was also observed.
studied by Thuyet et al. at pH 7 and 10. A faster degradation was
observed at pH 10 as compared to 7. At pH 10, the concentration
Kinetic study of photocatalytic degradation of imidacloprid
of imidacloprid dropped by 48% as compared to 12% at pH 7 in
paddy water.36 Similarly, in the present study, degradation of The imidacloprid degradation was observed for 30–300 minutes
64.1% was observed under dark at pH 3 that was increased with by following rst-order kinetics as depicted in Fig. 14. It is
an increase in pH approaching an optimum at pH 11. evident from the data that without a catalyst, the degradation
rate of imidacloprid was 0.036 h−1 under dark conditions while
it was 0.045 h−1 under UV light. Thus, the half-life of imida-
Effect of temperature
cloprid under these conditions was 19.25 hours under dark
The degradation of 10 mg L−1 imidacloprid solution containing which was reduced to 15.4 hours under the inuence of UV
0.01 g catalyst was studied at various temperatures i.e., 20 °C, light. The presence of Ag2O/CuO catalyst facilitated the degra-
25 °C, 30 °C, 35 °C, and 40 °C under optimum conditions. Each dation of imidacloprid both in the presence of UV light and
sample was stirred for 3 hours in the presence of UV light and dark. It is evident from the data that the rate of degradation in
under dark. The relevant data is depicted in Fig. 13. the presence of the catalyst under dark was 0.068 h−1 which
The maximum degradation was observed for samples yielded a half-life of 10.19 hours that was quite lower as
maintained at 30 °C both in the presence of UV light and dark. compared to that noted under similar conditions in the absence
Fig. 12 Effect of pH on degradation of imidacloprid using Ag2O doped

CuO catalyst. Fig. 14 Degradation kinetics of imidacloprid.
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RSC Advances Paper
Table 1 Effect of different scavengers on degradation of imidacloprid
Scavengers Radicals % Degradation
Without scavengers — 92
Formic acid 20 mM h+ 99
Isopropyl alcohol 1 M HOc 70
K2Cr2O7 1 mM e− 35
1,4 Benzoquinone 1 O2c− 45
mM
Fig. 15 Schematic diagram of photocatalytic degradation of imida-

cloprid by using silver oxide doped copper oxide catalyst. degradation at optimum conditions that provided maximum
degradation of 92.3%. It was then ltered, washed thoroughly
and dried at 100 °C. Aer drying the catalyst was reused at
optimum conditions and the degradation observed was 78%.
of the catalyst. UV irradiation further enhanced the process of The catalyst was reused again aer activation by using the same
degradation and reduced the half-life to 3.7 hours as the rate of procedure and the degradation observed for the third cycle was
reaction was increased to 0.187 h−1. Yari et al. (2019) degraded 63.41% as shown in Fig. 16. The stability of the catalyst was
imidacloprid using ZnO and TiO2, where the half-life was studied by leaching experiments. The catalyst was soaked in
calculated to be 4.5 hours. In the case of Ag2O/CuO composite acidic conditions for 4 hours and no concentration of Cu and Ag
(present study), the half-life observed was 3.70 hours which was detected in the solution as checked by Atomic absorption
showed better degradation efficiency35 of present catalyst. spectrophotometer. The XRD spectra of catalyst aer three
GC-MS analysis was carried out to study the possible degra- cycles is shown in Fig. 17. The XRD patterns revealed the
dation products of imidacloprid and to get an insight into its structural stability of the catalyst. The decrease in activity of the
degradation pathway. It is worth noting that no metabolites of catalyst aer three cycles is due to the agglomeration of parti-
imidacloprid were observed and the present catalyst was cles which reduces the surface area.43 Furthermore, the
capable of bringing out the complete mineralization of imida- adsorption of imidacloprid molecules within the pores of
cloprid. The schematic diagram for pesticide degradation in the catalyst is another factor which blocks some of the active sites
presence of Ag2O/CuO nanoparticles is elaborated in Fig. 15.38 and thus decreases the efficiency of the catalyst.
The CuO nanoparticles exhibit a narrow bandgap of 1.2 eV
which offers easy recombination of photo-generated electron–
hole pairs. But, the doping of silver improves the band gap to
2.6 eV which restricts the electron–hole recombination rate and
improves the charge separation.39,40
Scavenger studies
The reactive species such as electrons, holes, superoxide radi-
cals and hydroxyl radicals are responsible for determining the
mechanism of the photodegradation. The role of these reactive
species on the degradation of imidacloprid was studied by
using different scavengers for an initial imidacloprid concen-
tration of 20 mg L−1. At this concentration, the photo-
Fig. 16 Reusability study of Ag2O doped CuO catalyst.
degradation efficiency was observed to be 92% in the presence
of UV light. The data in Table 1 depicted that the degradation
efficiency was decreased in the presence of electrons and
superoxide radical scavengers i.e. K2Cr2O7, 1,4 benzoquinone
and isopropyl alcohol. While in the presence of formic acid (h+
scavenger), the degradation of imidacloprid was increased to
99%. In conclusion, h+ scavengers allowed the electrons to react
with the pollutant resulting in lowering of the recombination
rate of electron–hole charge carriers leading to better degrada-
tion efficiency.41,42
Reusability of the catalyst

The catalyst was reused for three successive cycles to determine Fig. 17 XRD pattern of Ag2O doped CuO catalyst after three reaction
its stability and activity. The catalyst was primarily used for cycles.
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Paper RSC Advances
6 R. Garg, R. Gupta and A. Bansal, Int. J. Environ. Sci. Technol.,

Conclusions
2021, 18, 1425–1442.
The present study focused on the preparation of a highly 7 K. Babic, V. Tomašic, V. Gilja, J. Le Cunff, V. Gomzi, A. Pintar,
effective catalyst Ag2O/CuO that was used for degradation of G. Žerjav, S. Kurajica, M. Duplancic, I. E. Zelic, T. V. Pavicic
imidacloprid. The maximum degradation of imidacloprid was and I. Grcic, J. Environ. Chem. Eng., 2021, 9, 105611.
observed for 10 mg L−1 imidacloprid solution with 0.01 g 8 A. Qayyum, I. A. Bhatti, A. Ashar, A. Jilani, J. Iqbal,
catalyst at pH 11 in the presence of UV light. The optimum M. Mohsin, T. Ishaq, S. Muhammad, S. Wageh and
temperature for the degradation process was found to be 30 °C. M. R. Dustgeer, Polymers, 2022, 14(2), 295.
The efficiency of Ag2O/CuO was found to be 92.3% and 84.3% in 9 B. Ruomeng, O. Meihao, Z. Siru, G. Shichen, Z. Yixian,
UV light and dark at optimum conditions. The prepared catalyst C. Junhong, M. Ruijie, L. Yuan, X. Gezhi, C. Xingyu,
was effective in reducing the half-life of imidacloprid to 3.7 Z. Shiyi, Z. Aihui and F. Baishan, Synth. Syst. Biotechnol.,
hours under UV light. The catalyst was able to be reused for up 2023, 8, 302–313.
to 3 cycles with slight reduction in activity. The catalyst was able 10 D. D. D. Nguyen, K. A. Huynh, X. H. Nguyen and
to maintain its structural integrity even aer use in three T. P. Nguyen, Res. Chem. Intermed., 2020, 46, 4823–4840.
consecutive cycles. The stability studies showed that the catalyst 11 F. S. Bruckmann, C. Schnorr, L. R. Oviedo, S. Knani,
was also stable during the reaction course as evidenced by no L. F. O. Silva, W. L. Silva, G. L. Dotto and C. R. Bohn
detectable leaching of either Ag or Cu by Atomic absorption Rhoden, Molecules, 2022, 27, 6261.
spectrophotometer. Thus, Ag2O/CuO may be used as an efficient 12 A. Massoud, A. Derbalah, I. El-Mehasseb, M. S. Allah,
material for removing the pollutants like imidacloprid from M. S. Ahmed, A. Albrakati and E. K. Elmahallawy, Int. J.
contaminated aqueous environments. Environ. Res. Public Health, 2021, 18(17), 9278.
13 E. F. A. Zeid, I. A. Ibrahem, W. A. A. Mohamed and A. M. Ali,
Author contributions Mater. Res. Express, 2020, 7, 026201.
14 A. Iqbal, A. U. Haq, G. A. Cerrón-Calle, S. A. R. Naqvi,
Saadia Rashid Tariq planned, supervised, edited and nalized P. Westerhoff and S. Garcia-Segura, Catalysts, 2021, 11(7),
the whole project. Zunaira Niaz performed experimental work 806.
and initial write up, formatting and editing. Ghayoor Abbas and 15 K. Bano, S. Kaushal and P. P. Singh, Polyhedron, 2021, 209,
Dildar Ahmed provided the instrumental facilities for material 115465.
characterization and testing. Nazia Raque reviewed the paper. 16 S. Merci, A. Saljooqi, T. Shamspur and A. Mostafavi, Environ.
Sci. Pollut. Res., 2021, 28, 35764–35776.
Conflicts of interest 17 G. Murugadoss, N. Kandhasamy, M. Rajesh Kumar,
A. K. Alanazi, F. Khan, B. Salhi and H. M. Yadav, Inorg.
There are no conicts to declare. Chem. Commun., 2022, 137, 109186.
18 V. Srivastava, E. N. Zare, P. Makvandi, X. qi Zheng,
Acknowledgements S. Iekhar, A. Wu, V. V. T. Padil, B. Mokhtari, R. S. Varma,
F. R. Tay and M. Sillanpaa, Chemosphere, 2020, 258, 127324.
The authors acknowledge the chemicals provided by the 19 M. Shkir, B. Palanivel, A. Khan, M. Kumar, J. H. Chang,
Department of Chemistry, Lahore College for Women Univer- A. Mani and S. AlFaify, Chemosphere, 2022, 291(2), 132687.
sity, Lahore to carry out this research work. We are also thankful 20 M. Lal, P. Sharma, L. Singh and C. Ram, Results Eng., 2023,
to Mr Zajif from LUMS, Lahore for carrying out the XRD and 17, 100890.
SEM-EDX analyses. 21 J. Lang, J. Wang, Q. Zhang, X. Li, Q. Han, M. Wei, Y. Sui,
D. Wang and J. Yang, Ceram. Int., 2016, 42, 14175–14181.
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Environmental

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Photodegradation of α-cypermethrin in soil in the presence of trace

Phone No. +992-42-9208201-9

1 Photodegradation of α-cypermethrin in soil in the presence of trace metals (Cu2+,

66 2. Materials and Methods

76 2.2 Soil collection and characterization

86 2.3 Photochemical experimental set up

97 2.4 Control sterilized and unsterilized soil dark samples

106 2.5 Standard solution preparations and spiking procedure

128 2.6 Pesticide Extraction and analysis

150 2.7 Data analysis

165 dM/dt = -k1M for t ≤ tb

166 dM/dt = -k2M for t > tb

173 DTx = ln 100/100-x if t ≤ tb

175 DTx = tb + [ln 100/100-x – t1b] if t ≤ tb

178 3. Results and discussion

182 3.1 Photodegradation of soil incorporated α-cypermethrin

209 3.2 Microbial degradation of α-cypermethrin

232 3.2.1 Effect of Cu2+on α-cypermethrin

294 3.2.3 Effects of Cd2+ on α-cypermethrin

310 3.2.4 Effects of Fe2+ on α-cypermethrin

Fig.1Photodegradation of soil incorporated α-cypermethrin

Fig. 2Effect of concentration of α-cypermethrin on its photodegradation

Fig. 3 Effect of metal concentration on Photodegradation of α-cypermethrin

Fig. 4 Effect of metal concentration on microbial degradation of α-cypermethrin

Fig. 5 Effect of metal concentration on abiotic degradation of α-cypermethrin

Environmental Science: Processes & Impacts Accepted Manuscript

Environmental Science: Processes & Impacts Accepted Manuscript

Table 2.Physico-chemical properties of the studied soil

Soil Type Soil texture M. C O.M pH CEC Trace metals

% % (1:2) (mmol/ kg) (mg/kg)

% clay % sand % Silt Fe Zn Cd Cu

Table. 3 Dissipation statistics of degradation of α-cypermethrin

Environmental Science: Processes & Impacts Accepted Manuscript

Dark unsterile HS -0.151 0.99 8 -0.046 0.747 33.63 ± 1.92

dark sterile** HS -0.024 0.994 32 - - -

UV Fe2+ HS -1.175 0.94 2 -0.024 0.663 0.59 ± 2.07*

Dark unsterile HS -0.925 0.989 2 -0.035 0.855 54.03 ±1.4

dark sterile** HS -0.032 0.936 32 - - -

Dark unsterile HS -0.701 0.955 1.2 -0.021 0.697 41.6 ± 2.9

dark sterile** HS -0.022 0.96 32 - - -

UV Cd2+ HS -1.397 0.813 1 -0.088 0.734 0.49 ± 2.01*

Dark unsterile HS -0.152 0.84 4 -0.042 0.918 17.7 ± 0.429

dark sterile** HS -0.029 0.991 32 - - -

UV Cu2+ HS -0.147 0.937 8 -0.067 0.944 4.7 ± 1.04*

Dark unsterile HS -0.282 0.908 4 -0.024 0.847 49.7 ± 1.12

dark sterile** HS -0.023 0.996 32 - - -

* DT50 was in hours.

Cu2+ and Fe2+ mediated photodegradation studies

Table 1 Physico-chemical properties of the studied soil

% Clay % Sand % Silt (mg/kg)

Photocatalytic degradation kinetics

The data for the photodegradation of pesticides in the soil was

where C represents the pesticide concentration, k=rate con-

Table 2 Dissipation statistics for