Chemicals and reagents
Acetonitrile (HPLC grade) and water (HPLC grade) were purchased from Sigma-Aldrich, India. Analytical Reagent (AR) grade chemicals - Anhydrous sodium sulfate (Na2SO4), Sodium chloride (NaCl), Sodium citrate dibasic sesquihydrate (C6H6Na2O7.1.5H2O), Sodium citrate tribasic hydrate (C6H5Na3O7.xH2O) were acquired from Thomas Baker, Mumbai, India. Ultrapure water (HPLC grade) was taken from Sartorius water purification system (Sartorius AG, Goettingen, Germany). Primary secondary amine (PSA, 40 µm) was procured from Agilent Technologies, Bangalore, India.
Apparatus
Mixer Grinder (Bajaj India Pvt. Ltd., Mumbai, India), homogenizer (Heidolph 900, Germany), Mettler Toledo electronic balance (with an accuracy of 0.01 mg), vortex mixer (Geni 2T, Imperials Biomedicals, Mumbai, India), centrifuge (Kubota, Germany), microcentrifuge (Microfuge Pico, Kendro, D-37520, Osterode, Germany), Mechanical shaker and ultrasonic bath (Oscar electronics, Mumbai, India), P.P Centrifuge tubes of 50 ml capacity, Click lock centrifuge tubes of 2 ml capacity, Volumetric flasks of 10 ml and 50 ml capacity (Grade A), Volumetric pipettes of 1 ml, 5 ml and 10 ml capacity (Grade A), Micropipette (100–1000 µ1), and Micro tips HPLC auto-sampler vial (2 ml) were used.
Reference standard
The certified reference materials (CRMs) of both acetamiprid (having 99.9 % purity) and buprofezin (having 99.1 % purity) were obtained from Krishi Rasayan Exports Pvt. Ltd., India. Standard stock solutions (stored in refrigerator at -20°C) were prepared by dissolving 10 (± 0.1) mg of CRMs in 10 ml of methanol resulting in a final concentration of 1000 µg ml− 1. A working standard mixture of 10 µg ml− 1 was prepared in methanol by duly mixing the individual standard stock solutions followed by subsequent dilution, from which the calibration standard solutions of 0.01, 0.025, 0.05, 0.1, 0.25 and 0.5 µg ml− 1 were made. The matrix-matched standards of the same concentrations were made using the control rice extracts acquired from the sample preparation procedure.
Field experiment
The field (longitude: 89.386° E, latitude: 26.402° N) trial was conducted on Swarna variety of kharif paddy at Uttar Banga Krishi Viswavidyalaya, Pundibari, Cooch Behar under Terai agro-climatic zone of West Bengal, during July to November in 2018 following Randomized Block Design (RBD). The meteorological conditions and soil properties during the study period are shown in Table 1.
Table 1
Meteorological and soil physico-chemical parameters of the site of experiment
Sl. No.
|
Parameters
|
Result
|
Methods of determination
|
Meteorological conditions
|
1
|
Av. temperature (max) °C)
|
36.25
|
Modified Blaney-Criddle method (Zhan and Lin 2009)
|
2
|
Av. temperature (min) °C)
|
18.58
|
Modified Blaney-Criddle method (Zhan and Lin 2009)
|
3
|
Av. rainfall (max) (mm)
|
9.79
|
Rain gauge method (WMO 2009)
|
4
|
Av. rainfall (min) (mm)
|
1.75
|
Rain gauge method (WMO 2009)
|
5
|
Av. relative humidity (max) (%)
|
93.27
|
Psychrometer method (Gorse et al. 2012)
|
6
|
Av. relative humidity (min) (%)
|
43.53
|
Psychrometer method (Gorse et al. 2012)
|
Soil properties
|
1
|
Sand (%)
|
16.77
|
Hydrometer method (Bouyoucos 1962)
|
2
|
Silt (%)
|
44.12
|
Hydrometer method (Bouyoucos 1962)
|
3
|
Clay (%)
|
39.25
|
Hydrometer method (Bouyoucos 1962)
|
4
|
pH (soil: water ratio 1: 2.5)
|
5.57
|
Digital pH meter (consisting of glass and calomel electrodes) (Jackson 1973)
|
5
|
EC1:2 (dS m− 1) at 25°C
|
0.10
|
Conductivity meter (Richards 1954)
|
6
|
Organic Carbon (%)
|
0.51
|
Wet oxidation method using K2Cr2O7 (Walkley and Black 1934)
|
7
|
Available N (Kg/ha)
|
206.40
|
Kjeldahl method/ alkaline potassium permanganate oxidation method (Subbiah and Asija 1956)
|
8
|
Available P (Kg/ha)
|
8.17
|
Olsen’s sodium bicarbonate method (Olsen et al. 1954)
|
9
|
Available K (Kg/ha)
|
177.80
|
Neutral normal ammonium acetate method (Stanford and, English 1949)
|
10
|
S (Kg/ha)
|
32.63
|
Calcium chloride extraction method (William and Steinbergs 1962)
|
11
|
Zn (ppm)
|
1.07
|
DTPA extraction method (Lindsay and Norvell 1978)
|
12
|
Cu (ppm)
|
3.41
|
DTPA extraction method (Lindsay and Norvell 1978)
|
13
|
Fe (ppm)
|
9.01
|
DTPA extraction method (Lindsay and Norvell 1978)
|
14
|
Mn (ppm)
|
9.27
|
DTPA extraction method (Lindsay and Norvell 1978)
|
15
|
Bulk density
|
1.33
|
Core method (Blake 1965)
|
16
|
CEC (cmol (p+) kg− 1)
|
17.34
|
Ammonium acetate method (Chapman 1965)
|
17
|
Texture
|
Silty clay loam
|
Texture triangle hydrometer method (Bouyoucos 1962)
|
18
|
Order
|
Entisol
|
Soil taxonomy classification method (USDA 1999)
|
Acetamiprid 20% SP formulation (under Trade name SPARK manufactured by Hindustan Agro Chemicals) was applied in the paddy field in two doses: single dose [SD i.e. 20 g active ingredient (a.i.) ha− 1] and double dose (DD i.e. 40 g a.i. ha− 1) along with untreated control throughout the experiment. Similarly, Buprofezin 25 % SC (under Trade name BANZO manufactured by Biostadt India Limited) was sprayed over the rice field in dosages of 200 g a.i. ha− 1 as single dose (SD) and 400 g a.i. ha− 1 as double dose (DD) in addition to untreated control. Each experiment was conducted in 20 m2 plots in triplicates. These two insecticides were applied thrice since the start of tillering to panicle initiation stage at an interval of 3, 7 and 15 days. The residues of the pesticide formulation were investigated in paddy (grain and straw) and soil.
Sampling
To investigate the dissipation of pesticide formulation (acetamiprid 20% SP and buprofezin 25% SC) in paddy (grain and straw) and soil, pesticide formulations were sprayed once in the tillering stage of rice. In order to achieve valid detection of residues even at 10–15 days after spraying, the treatment dosages for the dissipation study were set as 20 g a.i. ha− 1 and 40 g a.i. ha− 1 for acetamiprid, and 200 g a.i. ha− 1 and 400 g a.i. ha− 1 for buprofezin. Sufficient quantities of paddy grain, straw and soil (about 500 g each) samples were collected from 10–12 randomly chosen sampling points in each plot at 0 (2 hours after spraying), 1, 3, 5, 7, 10 and 15 days after spraying following a modified sampling plan (Oulkar et al. 2009). The soil was randomly sampled to a depth of 0–15 cm in each plot using a soil-sampling apparatus. All samples were stored in a deep freezer (-20°C) until analysis.
Sample preparation and analysis
The soil samples were made free from stones and weeds, pulverized thoroughly and passed through 2 mm sieve to get fine powder. Rice straw samples were chopped to small pieces, and grains were detached by a threshing machine followed by grinding by a vegetation disintegrator to make coarse powder.
The samples of soil (10 g), grain (5 g) and straw (2 g) were kept separately into a centrifuge tube (50 ml capacity), followed by addition of ultrapure water (5 ml) and acetonitrile (10 ml for the paddy samples and 20 ml for the soil samples). The tubes were continuously shaken for 30 minutes by an oscillator (air bath). Then, NaCl (3 g) was added and subjected to oscillation by a vortex mixer for 1 min. The mixture was then centrifuged (5 minutes, 3800 rpm), and the supernatant acetonitrile layer (1 ml) was transferred into a centrifuge tube (2 ml capacity) containing PSA (50 mg). The centrifuge tube was shaken for 1 minute on a vortexer and then again centrifuged for 3 minutes at 10000 rpm. The supernatant was passed through a 0.22 µm polypropylene filter and collected into an autosampler vial for HPLC analysis.
HPLC Operating conditions
Analytical determinations were attained using high performance liquid chromatography (HPLC, Model No. Shimadzu LC-2010CHT) having Perfectsil C18 stainless steel column (250 × 4.6 mm i.d., 5µm particle size) coupled with ultraviolet (UV) detector. The standardized mobile phase was an isocratic elution of solvent mixtures comprising of equal (1:1, v/v) quantity of acetonitrile (Sigma-Aldrich, HPLC grade) and water (Sigma-Aldrich, HPLC grade) containing 0.1% orthophosphoric acid. The injection volume was 20 µl. The column temperature was maintained at 35°C and the flow rate was 0.8 mL min− 1 with the run time of 20 minutes. The wavelength (λmax value) for acetamiprid was 254 nm with retention times of 4.7 minutes and 10.7 minutes for acetamiprid and buprofezin respectively. The data were processed utilizing system software Empower 2 (version 5.1).
Method validation
Analytical method validation was performed following standardized guidelines given by the European Commission (SANTE 2017 and EURACHEM 2021).
Calibration curves and linearity
The calibration curve was prepared by establishing six concentration points with calibration standards in the range of 0.001–0.50 µg ml− 1 in solvent as well as in the extract of matrix (control). The linearity curve was plotted by the concentrations against the responses (area of the peak) (Figs. 1 and 2).
Selectivity and sensitivity
Sensitivity was evaluated by the limit of quantification (LOQ) in different matrices (soil, grain and straw).
Recovery study
Recovery was performed at 0.01, 0.02, 0.05, 0.10, 0.50 mg/kg levels for acetamiprid and at 0.02, 0.03, 0.05, 0.1 and 0.5 mg/kg for buprofezin. The matrix effect (ME) was assessed by post-extraction spiking at 0.02, 0.03, 0.05, 0.1 and 0.5 levels in comparison to solvent standard response.
Dissipation kinetics and waiting periods
Dissipation for acetamiprid and buprofezin was studied by plotting the data to the first order kinetic equation:
At = A0e−kt (1)
Where, At = the concentration at time t, A0 = the initial concentration, k = the rate constant for dissipation of insecticides, and t = the time.
Calculation of half–life (t1/2) was required to an analysis by the following equation:
t1/2 = ln2/k (2)
The Pre-harvest intervals (PHI) can be calculated in terms of the time (days) required to dissipate of the initial deposition after insecticides sprayings to below the maximum residue limit (MRL). Straw as such cannot be used for direct consumption by human beings, but it can be used for mushroom production, conversion to the sugar syrup and yeast protein, which is further consumed by human beings. Hence, determination of PHI for both the products are necessary. Each pesticide has its own PHI, which also varies from crop to crop. In first-order kinetics, it was calculated by the following equation:
PHI = [log(intercept) − log(MRL)]/slope (3)
The residue data, after plotting against time (days) were fitted to TableCurve 2D, where (1 + 1)st order kinetics were followed during calculation in the 2D curve. According to FSSAI, the MRL values of 0.01 mg kg− 1 and 0.05 mg kg− 1 for acetamiprid and buprofezin respectively are considered.
Consumer food safety assessment
The food safety of acetamiprid and buprofezin in paddy grain was determined as per the reported literature (Majumder et al. 2020). The maximum permissible intake (MPIs) were estimated by multiplying the acceptable daily intake (ADI) by the average body weight of child (approx. 16 kg). The ADI of acetamiprid and buprofezin was 0.025 and 0.01 mg kg− 1 body weight day respectively (EU 2020). Dietary exposures were calculated by multiplying the acetamiprid and buprofezin residue present in each sample (mg kg− 1) with consumption (per person) of 0.270 kg day− 1 of cereals (rice) (ICMR-NIN 2020).