1. PRESENTATION ON
DEVELOPMENT OF SUITABLE
METHOD FOR THE DETECTION OF
CHLORPYRIFOS IN SOIL, WATER
AND VEGETABLES
Course No. – Env. 411
Presented by
Examination Roll: 101396
Registration no.: 28854
Session: 2009-2010
2. Objective of the study work
Though Bangladesh’s elite is becoming increasingly concerned
about the adverse effects of pesticide on the environment, country’s
resources, and people’s health, a very little scientific research has
been done to address the issue. For this reason we selected our
study objective to develop a suitable method for the detection of
chlorpyrifos in soil, water and vegetables which could be used as
secondary data for further research on pesticides determination in
our country.
3. Interest on chlorpyrifos insecticide as
research objective
Though 35% of crop producing areas in Bangladesh treated with
organophosphates, a very little information available on the levels of
pesticide residues on fruits, vegetables and other crops harvested in
our country (Chowdhury et al. 2012).
These compounds can cause harm to the environment and humans.
Some of these toxic effects include acute neurological toxicity,
neurodevelopment impairment, disturbances in the immune system,
disturbances in the reproductive and endocrine systems, cancer,
chronic kidney diseases and several other diseases (Guan et al.,
2010; Hercegova et al., 2007; Sinha et al., 2012).
Chlorpyrifos (O, O-diethyl O-(3, 5, 6-trichloro-2-pyridyl)
phosphorothioate) is one of the most widely used OP pesticides
effective against a broad spectrum of insect pests of economically
important crops and widely used in Bangladesh (Shahnaj, 2010;
Singh, 2008).
4. Again extensive use of chlorpyrifos contaminates air, ground water,
rivers, lakes, rainwater and fog water. The contamination has been
found up to about 24 kilometers from the site of application.
The half-life of chlorpyrifos in soil is usually between 60 and 120
days, but can range from 2 weeks to over 1 year, depending on the
soil type, climate, and other conditions (Bhagobaty et al., 2006).
5. Experimental Design
In our study work, we considered Hexane & Diethyl ether (4:1) solvents as
‘X’ method, Ethyl acetate & dichloromethane (4:1) solvents as ‘Y’ method
and Ethyl acetate solvent as ‘Z’ method.
We used ‘W’, ‘V’ and ‘S’ for water, vegetables and soil samples
respectively.
For water sample extraction method, three samples were spiked by
chlorpyrifos and one kept as control. Hexane & Diethyl ether (4:1) solvents
were named as WX method, Ethyl acetate & dichloromethane (4:1) as WY
and Ethyl acetate as WZ method.
For vegetables (Cucumis sativus) extraction, these three different methods
were also experimented with specific solvent mixing ratio. Hexane &
Diethyl ether (4:1) were named as method VX, Ethyl acetate &
dichloromethane (4:1) as method VY and only with Ethyl acetate as method
VZ. Each method had one control sample and three spiked samples.
The same three methods were followed for the soil sample extraction
naming SX, SY and SZ methods sequentially.
12. Solvent Purification in the Laboratory
Commercially purchased solvent may contain
impurities which can interfere with the test
result.
So the main purpose of solvent purification is
to remove impurities and other interfering
compounds.
To remove the impurities present in the
commercially purchased n-Hexane solvent
distillation was carried out at its boiling
temperature 65 degree celsius.
Following being in contact with continuous
water supply, it was concentrated under
cooling condition and collected in round
bottom flask connected to stand.
By using this method, the organic solvents
were made single and then to double distilled
form.
Figure : Distillation of n-Hexane
13. Clean-up procedure
Chlorpyrifos pesticide residues in different samples may present in
low or very low concentrations. Hence the residues need to be
partitioned to organic solvents before analysis.
During the process of extraction of chlorpyrifos pesticide from a
specific sample, some constituents of the sample also got extracted
into the solvent and often interfered with assay. This is called
“Matrix effect”.
It is very difficult to separate the desired chlorpyrifos pesticide alone
when it was present in much lower concentration then interfering
compounds.
Therefore, an additional clean-up step was performed to eliminate
many of the interfering species prior chromatographic analysis.
14. Clean-up procedure continued
To separate non dissolved materials, the
combined extracts were filtered.
Again to avoid the co-extraction from the
extract, the florisil column chromatography
was performed.
Photodiode array (PDA) detector of High
Performance Liquid Chromatography
(HPLC) was very sensitive to co-extractives
and would otherwise contaminate the
detector.
Figure: Sample before clean up
Figure : samples after clean up
15. Florisil Preparation
100 gm of florisil (Activated Magnesium Silicate) was heated at 300 degree celsius for 4
hours in a oven for activation and then cooled in a desiccator.
The freshly activated florisil was then partly deactivated by the drop-wise addition of 2%
distilled water (200 µl in each time with micropipette) at 40 degree celsius with constant
stirring and it became deactivated.
Column preparation
Finely washed and rinsed fresh chromatographic glass column was set with a stand and
the column was rinsed with the solvent which was used as eluent.
About two-third of the column was filled with DD-Hexane and 10gm of deactivated
florisil was slowly poured into the column by gently tapping the tube with a sterile glass
rod to avoid formation of any bubble in the column packing. Glass wool was placed at
the inner bottom of the column
Then florisil of the column was covered with appropriately 2 cm layer of anhydrous
Na2SO4 remove the water (if any) from the extracts and the stopcock of the column was
opened to drain out hexane up to 1 cm above sodium sulfate layer.
Clean-up procedure continued
16. Elution process
All of the extracted solution placed in
chromatographic column and allowed the
solution to percolate to a level around 1-2 ml
above the top of sodium sulfate in the column.
Each vial was rinsed with small portion of
eluting mixture and was allowed to percolate.
The eluting mixture was 75 ml solution
composed of 2% diethyl ether in DD- Hexane
for vegetables (cucumber), and soil samples.
The column was eluted with eluting mixture at a
flow rate 1ml/min and the eluted extract was
collected in a stopper flask.
The eluent was dried up by nitrogen blower and
volume to 1 ml with acetonitrile after
evaporation and the solution is ready to assay.
Figure: Elution process
Clean-up procedure continued
17. Chromatographic process
The samples were normally introduced in the column using micro-
liter syringe via a rubber septum in an injection port at the end of the
column.
The injector, column and detector were in close proximity to each
other and all are electrically heated to a suitable temperature.
When the components elute, they pass into a detector.
Assuming they are above a threshold level, their presence was
detected and the produced electrical signals were amplified to usable
level and displayed as chromatogram.
18. Pesticide Chlorpyrifos
Mobile phase (ml) 70% Acetonitrile in
distilled water
Injection Manual by micro
syringe
Volume 20 µl
Column C18 (Nova Pack)
Apparatus Shimadzu, Japan
Detector Photo Diode Array
(PDA)
PDA absorbance range 200-800 nm
Table: Operating Condition of HPLC (PDA)
mode
Figure: High Performance Liquid Chromatography (HPLC)
system
Chromatographic process
19. Figure: Typical HPLC chromatogram of chlorpyrifos (CPF) standard injected at 1000 ppm
(RT = retention time 8.843 min)
Chromatograms detected during study work
20. Figure: HPLC chromatogram of vegetables (Cucumis sativus) control (Ctrl) sample
showing the absence of chlorpyrifos (CPF).
Chromatograms detected during study work
21. Figure: HPLC Chromatogram of spiked Cucumis sativus sample showing the presence of
chlorpyrifos (CPF) with 90% recovery using DD-hexane with Diethyl ether solvent. (RT =
retention time 7.985)
Chromatograms detected during study work
22. Figure: HPLC Chromatogram of spiked soil sample showing the presence of chlorpyrifos
(CPF) with 86.50% recovery using DD-hexane with Diethyl ether solvent. (RT = retention
time 8.782).
Chromatograms detected during study work
23. Results
In our study, higher recovery percentages indicate that
retention times of spiked samples are closer to the retention
time of chlorpyrifos standard.
During the study, the highest recovery percentages 83.90%,
92.00% and 86.50% were found for solvent method hexane
and diethyl ether (4:1) (X method) that are indicated by WX,
VX and SX respectively.
Again the highest recovery percentages 66.60%, 75.20% and
71.20% were found for solvent method ethyl acetate and
dichloromethane (4:1) (Y method) that are indicated by WY,
VY and SY.
For ethyl acetate solvent method (Z method), the highest
recovery percentages were 56.40% for WZ, 61.50% for VZ
and 55.50% for SZ respectively.
24. Summary of the study
Chlorpyrifos is an organophosphate (OP) insecticide extensively
used in our agricultural and domestic settings.
Though the half life of the chlorpyrifos is relatively small than the
other pesticides and its concentrations decreasing with time, it still
contaminates air, ground water, rivers, lakes, rainwater and fog
water.
For this a laboratory-based study was undertaken to identify the best
detection method of chlorpyrifos in soil, water and vegetables.
Based on solvent extraction, Hexane & Diethyl ether (4:1), Ethyl
acetate & Dichloromethane (4:1) and Ethyl acetate were used as
extraction solvents for three different method experiments.
For each sample triplicates as well as control were used. Analysis
was carried out using high performance liquid chromatography
(HPLC).
25. For water samples, the mean percent recoveries for chlorpyrifos
were 83.90%, 66.60% and 56.40% with hexane & diethyl ether
(4:1), ethyl acetate and dichloromethane (4:1) and ethyl acetate,
respectively.
For vegetable (Cucumis sativus) samples, the mean percent
recoveries for chlorpyrifos were 92%, 75.20% and 61.5% with
hexane & diethyl ether (4:1), ethyl acetate and dichloromethane
(4:1) and ethyl acetate, respectively.
For soil samples, the mean percent recoveries for chlorpyrifos were
86.50%, 69% and 55.50% with hexane & diethyl ether (4:1), ethyl
acetate and dichloromethane (4:1) and ethyl acetate, respectively.
26. Conclusion
Recovery studies were performed to examine the efficacy of
extraction and clean up.
Recovery percentage indicates the effectiveness of the method in
extracting the pesticides from the sample matrices.
So it can be concluded that, the highest recovery percentages found
from the solvent method Hexane and Diethyl ether (4:1) and so that
it was our best identified suitable method for the detection of
chlorpyrifos pesticides during the study period.
