Case study edited scientific journal on pesticide residue analysis on grapes at Karnataka

Introduction

Grape is an important fruit crop of northern parts of Karnataka, India. Grape cultivation produce profitable returns due to favorable climate conditions. One major factor responsible for the successful cultivation of grape is prudent manuring with proper pest management. An experiment was therefore conducted to assess the pesticide residues in grape fruits.

Due to heavy pest infestation, a wide range of pesticides are used for the production of harvests in India. Pesticides can bring about potentially hazardous effects on vegetables, fruits, animal resources and human health, as toxic residues of pesticides have been found in various environmental matrices due to their widespread usage. Hence, determination of pesticide residues in fresh produces (such as fruits and vegetables) and environmental commodities like soil and water have become increasingly important for consumers, producers and authorities.

An experiment was therefore conducted to test for the amount of pesticide residues in grape harvests. The aim for this experiment was to collect data which will form the base line for determining changes in residue levels of different pesticides in the future.

Materials and Methods

Sample preparation:

To obtain a representative sample, ten samples of grapes were freshly collected from grape yards in Bijapur district.

To prevent oxidation of grapes and change chemical structures of the residue compounds, samples were left in refrigerator and were only taken out just before the analysis. 

Samples were then homogenised in a high-speed blender to ensure equal concentration throughout the sample and ensure consistent linear split ratio during the Gas-Liquid Chromatography analysis.

Sample extraction and clean up:

Liquid-liquid partitioning implementation: Two immiscible phases(ethyl acetate and the homogenised sample are shaken together. Phases are then settled and seperated with gravity. This enables molecules to partition (dissolve) into preferred solvent phases (extract and raffinate).

The extract is then being cleaned up using column chromatography and standard methods are used to minimise systematic errors.

Gas Liquid Chromatography and High Performance Gas Chromatography analysis conditions: 

Analysis conditions for OC  (GLC with a ECD):

Electron capture detector, highly selective towards chlorine compounds and low detection limit 0.1pg CI/sec, were used to detect the organochlorine residues during the GLC.


Reasons for using GLC:

• able to conduct trace analysis for the pesticide residue
• High resolution
• Very sensitive with wide linear dynamic range
• OC is a thermally stable sample with high volatility

capillary column	30m × 0.32mm i.d × 0.25µm film thickness  5% diphenyl 95% dimethyl siloxane.
Column temperature	150°C initially for five minute then programmed at 8°C / minute up to 190°C for two minutes and then at 15°C / minute up to 280°C for 10 minutes Programmed temperature used to avoid general elution problem and improve peak shape.
Injector temperature	280°C (ensures flash vaporization of all components)
Detector temperature	300°C
Flow rate	nitrogen flow rate 2 ml / minute through column and split ratio 1:10.

Analysis for OP insecticides

Nitrogen Phosphorous detector was used to detect organophosphate residues during the HPLC.


Reasons for using HPLC:

• trace analysis of pesticide residues
• Sample has high solubility in organic solvents
• Organophosphate has low volatility
• Very fast analysis
• Good resolution

Column	(10m × 0.53 mm i.d × 2.65µm film thicknes) Stationary phase : polysiloxane was used. Small particles size to ensure higher resolution, faster analysis and high throughput. Small column physical parameter to support the small particles Isocratic elution -prevent compression of the peaks -prevent lower loading capacity -prevent lower productivity
Column temperature	100°C initially for one minute then increased at the rate of 10°C / minute to 200°C and was finally increased at the rate of 20°C / minute to 260°C

 Spiking is implemented by introducing pure compounds to unknown sample so as to determine the unknown analytes. Spiked samples were then processed and replicated in threes to minimise human error. Control samples were processed together with spikes ones.

Result & Discussion

Presence of  the following residues were detected: organochlorines,organophosphates,captan, chlorpyrifos, filpronil and 4-bromo-2-chlorophenol. Some of these residues have already exceeded the safety limit set by FAO/WHO for human consumption and exposure. For example, oxyfluoren which may be a human carcinogen and may cause inhalation toxicity, has already greatly exceeded the limit (  1 x 10^-6 ) mg/kg by having an average of 0.0182 mg/kg in the samples.  Hence, these levels of residues are very likely to pose a health concern to consumers.

Conclusion

Pesticide residues of the samples have found to exceed the safety maximum limits that can be ingested and exposed to the human body. To lower the amount of pesticide residue, it is recommended to either process the grapes before selling and to wash the grapes. Washing helps as most residues compounds are hydrophillic and processing helps by destroying the residue compounds by adding other chemicals or by heat.




Pujari, A. S. (2010). Multiresidue analysis of pesticides in grapes in bijapur district. In Green Pages. Retrieved from http://www.eco-web.com/edi/100623.html

HPLC intro

HPLC Introduction Higher pressure to accomadate smaller particles size. Smaller particle size meant higher surface area of interaction  between mobile phase, stationary phase and analytes. This will result to better resolution and improved peak shape on the chromatograph. Jim, Clark. " HIGH PERFORMANCE LIQUID CHROMATOGRAPHY - HPLC ." chemguide Helping you to understand Chemistry. N.p., 2007. Web. 31 Jul 2012. <http://citationmachine.net/index2.php?reqstyleid=1&mode=form&reqsrcid=MLAWebDocument>.

Brief explanation Gas Liquid Chromatography

Gas Liquid Chromatography:

Gas- inert mobile phase

Liquid: stationary phase where analytes interact with.

Sample

Minute quantities of the sample is drawn into the micro syringe.

Sample must be thermally stable and volatile.

Injection

Once sample is injected into the injector port, the sample undergoes flash vaporisation. 

Brief explanation on how GLC works:

Clark, J.. "GAS-LIQUID CHROMATOGRAPHY."chemguide helping you to understand chemistry. N.p., 2007. Web. 31 Jul 2012. <http://www.chemguide.co.uk/analysis/chromatography/gas.html>.

Introduction

In the world of fresh fruits and vegetables, there are those whose skins you eat and those you don't. One typically eats the skin of an apple or a sweet pepper, but not a banana or grapefruit. Invariably, the skin of any fruit or vegetable grown non-organically contains pesticide residue. Wash it all day long, but it will still be there – it's sort of “baked in”.

Here is the acceptable levels of different pesticides on any piece of produce defined by EPA : http://www.epa.gov/pesticides/reregistration/status.htm

The health effects of pesticides depend on the type of pesticide. For example, Acephate can cause cholinesterase inhibition in humans;  that is, it can overstimulate the nervous system causing, respiratory paralysis and death. On the other hand, over exposure to ethephon causes skin injury (irritation).

Hence, it is important to determine the presence and amount of pesticide residue on produces before being sold for consumption. In this blog, we will explore pesticide residue analysis done using Gas-Liquid Chromatography.

Todd, Heft. "Pesticide Residue In Food Poses Health Risks Author." Technorati. N.p., 10 June 2011. Web. 31 Jul 2012. <http://technorati.com/women/article/pesticide-residue-in-food-poses-health/>.

 "Pesticides: Health and Safety." Human Health Issues. EPA, 09 May 2012. Web. 31 Jul 2012. <http://www.epa.gov/opp00001/health/human.htm