Course – AC-699 Thesis Seminar

Studies on the effect of Flonicamid residues on enzymatic

activities and microbial population in New Alluvial soil of West
Bengal

Speaker: REESHAV PAUL

M.Sc.4th semester
Department of Agricultural Chemicals
Bidhan Chandra Krishi Viswavidyalaya
Mohanpur, Nadia, West bengal

Chairman Seminar Co-ordinator

Prof. Hemanta Banerjee Dr. Sankhajit Roy
•Soil is a crucial resource supporting life and agriculture.
•Soil enzymes and microbes maintain soil fertility and nutrient cycling.
•Flonicamid, a low-pollution insecticide, is gaining popularity in agriculture.
•Little is known about its effects on soil enzymes and microbial
populations.
•This study aims to fill the knowledge gap and understand the impact of
flonicamid residues on soil health.
•Findings will contribute to sustainable pest management and informed
agricultural practices.
 Assess the Effect of Flonicamid on
Soil Enzymes, Microbial Population,
and Physicochemical Properties

Objectives

Residue Analysis of Flonicamid in

Soil
General Information on Flonicamid
Item Information on Flonicamid

IUPAC name N-cyanomethyl-4-(trifluoromethyl)-nicotinamide

Trade names Ulala, Teppeki, Carbine, Turbine, Mainman, Takashi,

Molecular formula C9H6F3N3O

Molecular weight 229.16 g/mol

pH 4.5 (at 25 °C)

Solubility Acetonitrile (132.8 g/L)

Types of
WG/WDG, SG, DF, G, WP
formulation

Chemical group Pyridinecarboxamide

IRAC group 29

Highly effective against pests of vegetables and

Use
ornamentals. (Morita et. al., 2007)
 Review of Literature

Impact of Pesticides on Soil Enzymatic Activity and

Microorganisms
Fungicides were found to negatively impact dehydrogenase activity,
while some triazole fungicides showed stimulation at low concentrations
(Kenarova and Boteva, 2023).

Phosphatase enzymes were found to be pH-dependent, with concentrations

showing stimulating and weak inhibitory effects (Kenarova and Boteva,
2023).

Risks associated with 15 pesticides on soil biota were observed in the

North China Plain, with some showing chronic risk exposure to fauna (Mu
et.al., 2023).
 Review of Literature
Pesticides Effects on Soil Health and Respiration

Insecticides treated soils were found to emit less CO2, indicating reduced soil
respiration (Subarna et.al., 2023).

Pesticides can disrupt soil microbial diversity, enzymatic activities, and

physicochemical properties (Sahid and Khan, 2022).

Continuous pesticide application may lead to changes in microbial catabolic

capabilities and alter soil ecosystem dynamics (Roman et.al., 2021).

Some pesticides showed a positive correlation with bacterial counts but

decreased fungal and actinomycetes populations (Bam et.al., 2021; Kalia and
Gosal, 2011).
 Review of Literature

Zhang et.al. (2022):

Validated QuEChERS
method analyzing
Zhao et.al. (2021): flonicamid residues
Flonicamid undergoes in crops,ranged from
biodegradation by 84.3-99.3% at 0.01
Microvirga flocculans mg/kg, with a half-
Flonicamid involving amidases life of 2.28-9.74

Ambily and George

Residue into intermediate
compound N-(4-
days.

(2020): In paddy Analysis trifluoromethyl

nicotinyl)–glycinamide
crop, Flonicamid
Sahu et.al. (2020): residues reached and (TFNG-AM), and
Developed a new method
for determining
below 0.05 mg/kg in
3 days in green Persistence lastly to TFNG.
flonicamid residues in
cucumber, tomato,
foliage of paddy,
and no residues
in Soil
bottle gourd, soil, and
water the recovery detected in harvest
percentage ranged from samples.
95-99%, with the LOQ
at 0.025 ppm.
 Materials and Methods
Sites: Jaguli
Instructional Soil Preparation:
Farm, BCKV, Samples air-
Mohanpur, dried at room
Nadia and
temperature and
Soil Sampling Central Research
Farm, BCKV, sieved through a
and Physical 2 mm mesh
Gayeshpur,
Parameters Nadia. sieve.

Soil Sampling Soil Collection: 5 kg Physical Parameters:

samples collected • Bulk Density (BD):
Location: New in a zigzag fashion
Alluvial Zone, Determined using core
at 10 cm depth sampler method.
Nadia, West from each location. • Water Holding Capacity:
Bengal.
Measured using Keen
Rackzowski box method.
• Soil Textural Analysis:
Carried out using the
Bouyoucos hydrometer
method.
 Materials and Methods
Chemical Parameters:
• Soil pH: Determined by
soil-water suspension
method.
• Electrical Conductivity Chemical Parameters and
(EC): Measured after
allowing soil-water
suspension to stand for 24
hours.
• Organic Carbon: Analyzed
using Walkley and Black
method.
Soil Respiration:
Determined by acid-base
titration method.
• 40 g soil incubated at 30
degree C in sealed jar with
5 ml 0.5 N NaOH.
• NaOH transferred to flask
Soil Respiration and titrated with 0.5 N HCl
at different time intervals
(0, 3, 6, 9, 12, 15, 18, 21
days).
 Materials and Methods
Calculation of Flonicamid
Doses and Methodology
Recommended Dose (RD) for
Paddy Crop: 75 g a.i./ha,
applied 4 times.
Methodology for Spiking Soil with Flonicamid:
• Mother stock solution: Dissolve 20 g of Flonicamid 50% WG in 1L of
distilled water (10000 ppm).
• Prepare a 25 ppm solution by adding 0.25 ml of mother stock
solution to 99.75 ml of water.
• Calculate the required amount for each dose and add it to 96.80 ml of
water.
• Apply the solutions to soil-filled containers and keep them at room
temperature.
Three Doses Selected for the Experiment:
• Half of Recommended Dose (HRD): 40 µg a.i./200 g
soil.
• Recommended Dose (RD): 80 µg a.i./200 g soil.
• Double of Recommended Dose (DRD): 160 µg a.i./200
g soil.
Treatments and Soil Sampling Locations

Soil Sampling Locations:

• Jaguli Instructional Farm, Mohanpur (JI)
• Central Research Farm, Gayeshpur (CRG)

Control (No Pesticides):

• JIC: Control at Jaguli Instructional Farm
• CRGC: Control at Central Research Farm

Flonicamid Doses:
• JIH: Half of Recommended Dose (HRD) at Jaguli Instructional Farm
• JIRD: Recommended Dose (RD) at Jaguli Instructional Farm
• JIDRD: Double of Recommended Dose (DRD) at Jaguli Instructional Farm
• CRGH: Half of Recommended Dose (HRD) at Central Research Farm
• CRGRD: Recommended Dose (RD) at Central Research Farm
• CRGDRD: Double of Recommended Dose (DRD) at Central Research Farm.
 Materials and Methods
Isolation Procedure of Total Bacteria, Fungi, and Actinomycetes from Soil:

Ten grams of sieved soil were mixed with 90 ml of sterile water in a conical flask and serial dilutions (10-1 to
10-6) were prepared by shaking vigorously.

Fungi were isolated from dilutions 10-3 and 10-4, actinomycetes from dilutions 10-4 and 10-5, and bacteria
from dilutions 10-5 and 10-6.

Transferred to sterilized petri plates, and growth media were poured into the plates. Petri plates were incubated
at 28°C.

Sterile petri plates were taken, and soil suspension was aseptically transferred to each plate with hot media.
Plates were rotated to evenly spread the suspension and media.

The petri plates were labeled, inverted, and incubated at 30 ± 1°C for 7 days.

Microbial colonies were counted after 3, 5, and 7 days of incubation.

Thornton’s agar media, Martin’s Rose-Bengal Media and Jensen’s Agar media was prepared for isolation of
bacteria, fungus and actinomycetes respectively
 Materials and Methods

Measure the
Take 1 gram Add 10 mL of
pink-colored
of air-dried methanol to
supernatant
soil and add the incubated
Incubate at (Triphenylfor
Dehydrogenas 0.2 mL of 3% solution,
28 ± 0.5°C for mazan, TPF)
e Assay TTC solution shake
24 hours. formed at 485
and 0.5 mL of vigorously, let
nm using a
1% glucose it stand for 6
spectrophoto
solution. hours.
meter.
 Materials and Methods

1 gram of soil and add 0.2
Phosphatase mL of toluene, 4 mL of
modified universal buffer
Assay (MUB), and 1 mL of p-
nitrophenyl phosphate.
Add 1 mL of 0.5 M
Incubate at 37°C for 1 CaCl2 and 4 mL of 0.5
hour. M NaOH to the
suspension and filter.
Measure the released
p-nitrophenol in the
filtrate at 420 nm
using a
spectrophotometer.

Calibration Curve Preparation:

1.For Dehydrogenase Assay: Prepare Triphenylformazan (TPF) solutions of 100 ppm, and dilute to obtain 2, 4, 6, 8, and 10 ppm
concentrations. Use ethanol as a blank sample.
2.Measure absorbance at 485 nm and plot absorbance versus concentration to create the calibration curve.
3.For Phosphatase Assay: Prepare p-nitrophenol solutions of 1, 2, 3, 4, and 5 ppm concentrations. Use distilled water, CaCl2, and NaOH as
blank samples.
4.Measure absorbance at 440 nm and plot absorbance versus concentration to create the calibration curve.
 Materials and Methods
Flonicamid Residue Analysis in Soil - Sample Preparation and
LC-MS/MS
Take 10g of treated and control homogenized soil from each location in
separate 50 mL centrifuge tubes.
Add 10 mL of water and vortex for 2 minutes. Allow it to stand for 15
minutes.

Add 10 mL of 1% acetic acid in acetonitrile, vortex for 2 minutes.

Add 5g of activated MgSO4 and 1.0g of NaOAc, vortex for 2 minutes,

and centrifuge for 8 minutes at 5500 rpm (Step-1).
Transfer 1.5 mL of supernatant from Step-1 to a 2 mL micro centrifuge
tube.
Add 50 mg PSA sorbent, 150 mg anhydrous MgSO4, and 25 mg florisil to
the micro centrifuge tube.

Vortex for 2 minutes and centrifuge at 6000 rpm for 6 minutes.

Transfer 1.0 mL of supernatant to the auto sampler vials using a 0.22

µm syringe filter for LC-MS/MS analysis.
 Materials and Methods

Flonicamid Residue Analysis in Soil - Identification,

Quantification, and Residue Calculation

Use analytical grade flonicamid solution as an external standard and record retention time.

Inject 2 µL of each cleaned up treated and control soil samples into the LC-MS/MS.

Identify residues by comparing retention time and qualifying MRM transitions from Table 1.

Use Mass Lynx 4.2 software for regression equation, R2, and calibration curve.
 Results and Discussion

Results of Soil Physicochemical Parameters and Soil Soil pH

Respiration
0 days 21 days
•Soil pH: Minimal changes observed between treatments and control in
7.5
both locations (JI and CRG). CRG soil showed a decrease, while JI soil 7
had a slight increase on day 21. 6.5
6
•Electrical Conductivity (EC, dS.m-1): CRG soil exhibited higher EC JIC JIH D RD
C H RD RD
JIR CR
G G
values. Significant increase in EC on day 21, indicating potential ion JID CR C RG G
D
CR
accumulation.
•Organic Carbon (%): CRG soil had higher organic carbon content. No
Soil EC
significant changes on day 21.
•Soil Respiration (mg-CO2/40g soil): Negligible respiration on day 0. 0.6

0.4
Initial surge on day 3 for all treatments. Fluctuations from day 6
0.2
onwards. Increase on day 18 and 21, reaching highest values. Some
0
treatments showed higher respiration, suggesting differences in JIC JIH JIRD JIDRD CRGC CRGH CRGRD CRGDRD

microbial activity. 0 days 21 days

 0.9
0.8
Control: Increased dehydrogenase activity over time, more 0.7 absx + 0.0402380952380952
f(x) = 0.0780857142857143
in CRG soil. 0.6 R² = 0.985102132195178
0.5
0.4
0.3
Half dose: Initially stimulated activity (JI: 7 days, 0.2
CRG: 14 days), then decreased. Recommended dose is 0.1
0
similar. 0 2 4 6 8 10 12

Double the recommended dose: Consistently 16

increased activity in JI soil, increased at 14 days in
CRG soil. 14

Flonicamid impact influenced by dose and soil 12

characteristics. Recommended dose stimulated activity
Flonicamid Effect on (especially in CRG soil), higher doses had more pronounced
Dehydrogenase Activity effects. 10 T1
T2
8 T3
High flonicamid concentration may decrease activity on 21 T4
days (possibly related to reduced bacterial count). T5
6
T6
T7
4
Positive correlation (0.848) between dehydrogenase activity and T8
microbial count.
2

0
Dehydrogenase activity indicates soil quality (moderate positive 0 days
correlation, 0.692, with soil respiration). 7 days
14 days
21 days
ref: Ataikiru et al. (2019), Kenarova and Boteva (2023), Kalia and Gosal (2011), AL-Ani et al. (2019), and Arora et al. (2019)
 Results and Discussion
Flonicamid Effect on Alkaline Phosphatase Enzyme
Activity
0.35
Std. curve.
0.3 f(x) = 0.0664285714285714 x + 0.00142857142857142 Linearity: Absorbance-concentration correlation (r2 = 0.994).
R² = 0.994612601324218
0.25
0.2
Control: Activity increased till day 14, slight decrease on day 21 (in both
soils).
0.15
0.1 Half dose: Consistently lower activity than control.
0.05
0
Recommended dose: Higher activity than control.
0 1 2 3 4 5 6
30 Double dose: Activity similar to control.

25 Dose-dependent impact observed.

20 T1 CRG soil had higher activity due to pH values.
T2
15 T3
Positive correlation (0.733) with microbial count.
T4
10 T5
T6
Positive correlation (0.654) with soil respiration.
5
T7 Enzyme activities, microbial count, and respiration showed positive
0
T8 relationships.
0 days
Consider pH, EC, OC interactions with enzyme activities and microbial
7 days processes, in addition to flonicamid effects.
14 days
21 days
 Results and Discussion
Flonicamid Effect on Microbial
Population
Investigated microbial population (bacteria, fungi, actinomycetes) at 0, 14, and 28 days in JI
and CRG soil with flonicamid treatments and control.

Bacterial count (CFU × 10^5/g soil) ranged from 32 to 73 at 0 days, highest in JIRD soil.

Recommended dose showed the highest bacterial count in both soils.

Fungi count (CFU × 10^4/g soil) ranged from 33 to 60 in control samples.

Half and recommended doses had the highest fungal count on 14 and 21 days in CRG soil.

Actinomycetes count (CFU × 10^5/g soil) was higher than bacteria and fungi.

Total actinomycetes population highest at 14 days in JI soil and 21 days in CRG soil.

Overall, microbial population higher in CRG soil than JI soil, except in double recommended
dose.
 Results and Discussion
Soil Fortifica Recovery (%) Mean SD RS S/N Recove
tion of Flonicamid Recove D ratio ry
Flonicamid Recovery Results level ry (%) (% factor
(ppm) )

Flonicamid concentrations (5, 10, 20, 50, 100, 250, 500, and 1000 R1 R2 R3

PPB) injected in acetonitrile.

Calibration graph generated using Mass Lynx 4.2 software.
Coefficient of regression (r = 0.998168, r2 = 0.996340) indicates JI 0.05 93 89 91 91 2 2.2 10.36 0.91

linearity. 3

14.19 0.9433
Signal-to-noise (S/N) ratio > 10 for 50-1000 PPB (0.05-1.0 PPM). CRG 0.05 98 94 91
3.5 3.7 8
94.33
1 2
Flonicamid detection limit: 5 PPB, quantification limit: 50 PPB.

Recovery (%) in JI soil: 91-94.33%, RSD: 2.2-3.72%.

Recovery (%) in CRG soil: 91-94.33%, RSD: 2.2-3.72%.

Ion ratio of flonicamid within acceptable range (± 30%).

 Results and Discussion
Soil Residues* (ppm) in different days
and 0 7 14 21
dos
e

JIH 0.15 0.09 0.05 BQL <

Microbial
Flonicamid shows No detectable 0.05
Initial residues in population
promising flonicamid
JI soil: 0.15-0.68 increased with ppm
efficiency and low residues in CRG
ppm, CRG soil: time, utilizing BQL < BQL <
residual potential soil on days 7, 14, JIR 0.37 BQL <
0.17-0.75 ppm. flonicamid as 0.05 0.05
in soil. and 21.
carbon source. D 0.05 ppm ppm
ppm

JID 0.68 BQL < BQL < BQL <

Formation of 0.05 0.05 0.05
Residue analysis at Residues declined RD
metabolites ppm ppm ppm
0, 7, 14, and 21 over time in JI soil
(TFNG-AM and BQL < BQL < BQL <
days in JI and (BQL < 0.05 ppm CR 0.17
TFNG) observed 0.05 0.05 0.05
CRG soil. on day 21).
in soil. GH ppm ppm ppm
CR 0.39 BQL < BQL < BQL <
0.05 0.05 0.05
GR ppm ppm ppm
D

CR 0.75 BQL < BQL < BQL <

0.05 0.05 0.05
GD ppm ppm ppm
 Summary and conclusion

Enzymatic Studies Conclusion

1.Dehydrogenase Activity:
1. Flonicamid initially stimulated dehydrogenase activity in both soils, indicating an initial positive influence on soil
microbial activity.
2. However, with time, dehydrogenase activity decreased, suggesting a potential inhibitory effect of flonicamid on soil
microbial processes.
3. The impact of flonicamid on dehydrogenase activity was dose-dependent, and the physicochemical characteristics of
the soil played a role in this effect.
4. A moderate positive correlation between dehydrogenase activity, microbial count, and soil respiration implies that
higher dehydrogenase activity led to increased microorganism population and CO2 emissions.
2.Alkaline Phosphatase Activity:
1. Flonicamid had a slight inhibitory effect on alkaline phosphatase activity in both soils at half the recommended dose.
2. The recommended dose of flonicamid showed a stimulatory effect on alkaline phosphatase activity, with higher values
compared to the control groups.
3. The double recommended dose did not significantly alter the effects observed with the recommended dose.
4. The relationship between alkaline phosphatase activity, microbial count, and soil respiration suggests that flonicamid
did not hinder the growth of microorganisms.
 Summary and conclusion

Residual Study Conclusion

1.Flonicamid Residue Analysis:
1. Flonicamid residue was initially detected in both soils on day 0, irrespective of treatments.
2. However, on days 7, 14, and 21, no residue of flonicamid could be detected in CRG soil, indicating its
complete utilization by soil microorganisms.
3. In JI soil, residue was only detected for recommended and double the recommended dose on day 0,
suggesting potential microbial utilization as a carbon source.
4. The higher microbial count in CRG soil may account for its more efficient utilization of flonicamid.

2.Implications for Soil Health:

1. The absence of flonicamid residues in soil after 7 days suggests that it omits residual problems and can be
applied in field crops due to its better efficiency.
2. The positive role of flonicamid on enzymatic activities and microbial populations symbolizes the quality of
soil health and its potential impact on nutrient cycling.
 Future Scope of Research:

•Global challenge: Ensuring food security and soil health is a significant

challenge worldwide.
•Detrimental effects: Excessive and continuous pesticide use negatively
impacts the food web and soil health.
•Eco-toxicity risk: Pesticide bioaccumulation and bio-magnification in the
food chain increase eco-toxicity in the soil.
•Flonicamid research: Limited information on flonicamid's impact on soil
flora, fauna, physicochemical properties, and ecological risk assessment.
•Soil ecosystem data: There is a need for extensive research to understand the
impact on soil ecosystems.
•Multiple pesticides: Monitoring soil health in the context of multiple pesticide
interactions to ensure safety and guidelines.