PESTICIDES IN FRUITS AND VEGETABLES: NATURAL VS. ORGANICALLY GROWN FOOD

PRE-LABORATORY ASSIGNMENT:

Before the lab experiment begins, you must bring in a naturally and organically grown fruit or vegetable of choice.

OBJECTIVES:

To investigate the content of pesticides in fruits and vegetables.
To compare the effects of pesticides on organic and naturally grown food.
To apply gas chromatography analysis with an ECD detector.

INTERDISCIPLINARY ASPECTS:

biology, agriculture, organic chemistry, food industry, toxicology

BENCHMARKS:

The origin of pesticides in fruits and vegetables.
Environmental effects of the pesticides on food.
Principles of chromatographic separations.

PESTICIDES

A pesticide is any substance or mixture of substances intended to prevent, destroy, or mitigate any pest. Pests can be insects, mice, other annoying animals, unwanted plants (weeds), fungi, or microorganisms, like bacteria and viruses. Under United States law, a pesticide is also any substance or mixture of substances intended for use as a plant regulator, defoliant, or desiccant. By their very nature, pesticides are designated to kill or otherwise adversely affect living organisms. Thus, they are harmful to humans, animals, or the environment. Simultaneously, pesticides are useful to society because of their ability to kill potential disease-causing organisms, as well as, control insects, weeds, or other pests. Pesticides have also helped the farming industry become more stable by increasing crop production to meet the demands of the worlds population. Pesticide, herbicide, insecticides, and fungicides have been widely used since the Post-World War II era up until the present.

DDT (dichlorodiphenyltrichloroethane) is one of the most well known pesticides. It was banned from use in the early 1970s for its effect on bird eggshells. DDT was once very useful in the production of bigger and juicer fruits. It's positive effects are preventing pests, killing bugs and making homes safer for kids, and killing horn flies and other pests, allowing cows to grow fatter. Other banned organochlorine pesticides are aldrin, dieldrin, and heptachlor. Endrin was banned in 1992. All isomers of BHC are also banned due to their potential to cause cancer. Today, some of those banned pesticides can be detected in fruits and vegetables because they are not easily biodegradable and are insoluble in water.

During the last 30 years, an increasing number of pesticides present in the atmosphere and rainfall have been reported and well documented. It is the result of drift during applications, volatilization from treated surfaces, and wind erosion of soil.  Recent research conducted by Heberer and Dunnbier ( Environ. Sc. Technol. 1999; 33, 2346-2531) have proved that DDT is distributed globally by atmospheric transport. Once the pesticides enter the atmosphere, they can be transported for a certain distance away from its application site. This is especially true for organochlorine pesticides; they have been detected in remote regions such as the North and South Poles. They have also been found in arctic mammals which eat the contaminated fish. DDT residues are extremely persistent in the environment and degradation strongly depends on the environmental conditions. It was demonstrated that forms of DDT, or its metabolite, form residues in seawater, surface water, ground water, or drinking water (Kumari et al. Environ. Contam. Toxicol, 1996, 57, 787-793). Therefore, it may also be found in fruits, vegetables, or seafood, even though there is no direct use of those banned pesticides on our food.

Pesticides may harm a developing child by blocking the absorption of important food nutrients necessary for normal healthy growth (Raloff and Pendick, Science News 1993, 1444, 4-5). Another time pesticides may cause harm to humans is during the development of the child's excretory system. Under such conditions, the body may not be able to fully remove pesticides. There are "critical periods" in human development when exposure to a toxin can permanently alter the way an individual's biological system operates. It is important that pesticides are easy to remove by washing and have short half-lives, so that its harmful effects will not affect humans who eat fruits and vegetables..

"Organically grown" food is processed using no synthetic fertilizers or pesticides. Pesticides derived from natural sources (e.g. biological pesticides) may be used in producing organically grown food. Increasingly, some consumers are purchasing organically grown food as a way to reduce their exposure to synthetic pesticides or fertilizers. Organic products sell for a higher price, and they are usually smaller in presence. They may also contain some bugs. Consumers may question whether it is worth it to buy less "attractive" organic products at higher prices. Elimination of pesticides, of course, would benefit our health, but economically, it will cause many negative effects. For example, low production yield, but higher selling price would disturb the global economy. Moreover, as it was mentioned above, there are many pesticides which cannot be avoided due to the atmospheric transport. A study by Zalom and Strand (Calif.Agric. 1990, 44, 16) showed that there are no substitutes for 30 % of the pesticides.

REAGENT LIST:

Acetonitrile
Sodium chloride, NaCl
Sodium sulfate (Na2SO4)
Nitrogen gas
Toluene
acetone
Pesticide standards:

Alpha-BHC; Gamma-BHC; Beta-BHC; Heptachlor; delta-BHC; Aldrin; Heptachlor epoxide; 4,4' - DDE, Dieldrin; Endrin; 4,4' - DDD, 4,4' - DDT; Endrin aldehyde

GLASSWARE AND ACCESSORIES:

-    Kitchen blender (homogenizer), centrifuge tubes, 10 mL tubes, evaporating vessel, ENVI-carb SPE tube 57094 (6 mL tubes, 500 mg packing), rotary evaporator, 15 mL tubes, 5 mL bottles with septa caps, Hamilton syringe (10 ml),

-    GC/ECD

SPBTM-608 capillary column (30 m x 0.53 mm ID, 0.50 mm film; Oven: 100 oC to 290 oC at 10 deg/min; keep at 290 oC from 30 min; carrier gas: helium, 10 ml/min, detector: ECD - 300 oC, injector: 1 mL, 250 oC.

PRINCIPLE OF THE METHOD

In this experiment 1 mL volumes of each sample extract are injected in the GC (set at the above mentioned conditions). The retention time is related to the boiling point of the species and their interactions with the stationary phase of the chromatographic column. For greater accuracy the precision, multiple injection of each sample is recommended. Peak heights, peak areas, and corresponding elution times for each sample are recorded and used in determination of pesticides Comparing their elution times to those of the standards identifies the species present in the sample.

The following steps are used in the determination procedure:

Determination of the concentration of each pesticide based on the calibration curve.
Calculation of the total mass of pesticide in the sample extract.
Calculation of the ppb of each pesticide in the sample

PROCEDURE:

I. Determination of the calibration curve

Prepare 4 calibration solutions for each pesticide.
Inject the appropriate volumes of standard to four battles with rubber septa containing 200 ml of hexane.
Your concentrations should be about: 0.05 mg/L, 0.1 mg/L, 0.2 mg/L 0.3 mg/L and 0.5 mg/L.
Inject 1 mL of each solution to GC and record the chromatogram.
Based on the peak heights construct a calibration curve for each compound.

II. Extraction of pesticides

Homogenize 50 g-chopped sample with 100 mL acetonitrile in a kitchen blender for 5 min.
Add 10 g sodium chloride and homogenize for another 5 min.
Transfer ~ 13 mL of acetonirile (top) layer to 15 mL graduated centrifuge tube.
Add ~ 3 g sodium sulfate, cap and shake well to remove water.
Centrifuge at high speed for 5 min.
Transfer 10 mL aliquot to a clean 15 mL tube.
Evaporate to 0.5 mL under clean nitrogen with water bath at 35 oC.
Transfer to ENVI-Carb SPE tube 57094 (6 mL tube, 500 mg packing).
Elute pesticides with 20 mL acetonitrile/toluene (3:1).
Using a rotary evaporate, concentrate sample to ~ 2 mL.
Add 2 x 10 mL acetone concentrating the materials to ~ 2 mL after each addition, to make a solvent exchange to acetone.
To prepare a blank, transfer quantitatively to a clean 15 mL tube. Add 50 mL internal standard, than bring volume to 2.5 mL with acetone.
Set aside 0.5 mL of final extract for GC analysis.

III. Determination of the content of pesticides in samples

Inject 5 mL of sample to GC/ECD.
Record the retention time and peak area for each congener.
Calculate the concentration of detected pesticides in the extract.
Calculate the amount of detected pesticides in the samples.

CALIBRATION DATA SHEET (for one concentration):

Pesticide

Concentration

mg/L

Retention time

[mL]

Peak height

[%]

a; b; R2

1

       

2

       

3

       

4

       

5

       

6

       

7

       

8

       

9

       

10

       

11

       

12

       

13

       

SAMPLE ANALYSIS DATA SHEET:

Detected pesticide

Name

Retention time

[mL]

Peak height

[%]

Concentration

[ppb]

1

       

2

       

3

       

4

       

5

       

6

       

REPORT:

Prepare a laboratory report containing:

    1. An introduction with an objective on the experimental work.
    2. Experimental procedures and analytical methods used.
    3. Discussion of the data, BENCHMARKS, possible sources of pollution, and environmental effects of pesticides in food.

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