PRESENTATION ON DEVELOPMENT OF SUITABLE

METHOD FOR THE DETECTION OF CHLORPYRIFOS IN SOIL, WATER

AND VEGETABLES

Course No. – Env. 411Presented by

Examination Roll: 101396Registration no.: 28854

Session: 2009-2010

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.

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).

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).

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.

Experimental design at a glance

Figure: Experimental design at a glance

Experimental design for Water sample extraction method

Figure : Experimental design for water sample extraction

Experimental design for Vegetables sample (Cucumis sativus) extraction method

Experimental design for Vegetables sample (Cucumis sativus) extraction method continued

Experimental design for soil sample extraction method

Experimental design for soil sample extraction method continued

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

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.

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

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

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

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.

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

Figure: Typical HPLC chromatogram of chlorpyrifos (CPF) standard injected at 1000 ppm (RT = retention time 8.843 min)

Chromatograms detected during study work

Figure: HPLC chromatogram of vegetables (Cucumis sativus) control (Ctrl) sample showing the absence of chlorpyrifos (CPF).

Chromatograms detected during study work

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

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

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.

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).

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.

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.

Thank you….