Abstract

Pesticides have helped improve the standard of living for millions of people around the world however their use has been linked to numerous ecological problems such as damaging the nervous systems of non-target organisms including humans. The nightcrawler earthworm is a species of Annelid that is distributed across North America. Earthworms are prized by farmers for the benefits they bring to soil. The purpose of this experiment is to determine the effects various environmental factors (boric acid, an alkaline buffer solution, and glyphosate) used by farmers to regulate harvest yields can have on the nervous systems of earthworms.

In order to record the effects the factors would have upon the nervous systems’ of the earthworms, the speed that the action potentials travelled at was analyzed after being exposed to the experimental factors. The action potentials were recorded through the use of an electrode recording setup. The earthworms were exposed to a .1 mole solution of boric acid, an alkaline buffer solution from a buffer tablet, and a 1.5% solution of glyphosate in order to simulate concentrations that may be present in the environment.

Through a series of five trials for each experimental factor, the earthworms exposed to the control solution exhibited the fastest conduction velocity at 8.41 m/s, the earthworms exposed to the boric acid solution exhibited a conduction velocity of 8.142 m/s, the earthworms exposed to the buffer solution exhibited a conduction velocity of 6.538 m/s and the earthworms exposed to the glyphosate solution exhibited the slowest conduction velocity at 6.5 m/s.

Background Information

Pesticides have been used in agriculture since approximately 2000 B.C. Originally pesticides were made from toxic chemicals such as arsenic, lead, mercury however since the Green Revolution in the 1960’s, synthetic organic compounds have become widespread. Although pesticides bring multiple benefits to humanity such as increasing harvest yields, protecting biodiversity, and decreasing prices for consumers, there are certain disadvantages to utilizing pesticides. Synthetic pesticides have been linked to nervous system damage, damage to body tissue, contaminated drinking water, eutrophication, etc.

Lumbricus terrestris or nightcrawlers belong to the phylum Annelida. They are characterized by their numerous segments that resemble “small rings”. Earthworms are a vital part of healthy soils and are prized by farmers as they burrow underground, aerating the soil; they also plant matter in soil and produce worm castings, a nutrient rich organic substance that consists of bacteria, enzymes, and remnants of plant matter. Earthworms are a model organism used in neuroscience experiments as their nervous system is similar to the human nervous system.

The nervous system of an earthworm consists of two main parts. The first part is the cerebral ganglion which functions as the brain of the earthworm. The second part is the central nerve cord containing the medial and lateral fibers that runs along the center of the earthworm.

Neuron: the fundamental building block of the nervous system. Neurons transmit electrical impulses throughout the body.

Action Potential: An action potential occurs when a neuron sends an electrical impulse. These action potentials can be seen as spikes on computer software.

Conduction Velocity: the measure of how fast neuron can transmit information. It is derived by dividing the distance between two recording electrodes by the time taken for the electrical impulse to travel between the electrodes. The unit is m/s.

Faraday Cage: a metal cage used to block out electromagnetic interferences. It causes the electrical charges in the cage’s conductive material to be distributed in such a way that they do not affect the interior. A Faraday cage was used during experimentation in order block out background electrical interferences.

Boric Acid: a widespread insecticide. It comes in a powder form and is known to cause adverse effects to the nervous system of organisms.

Buffer Solution: pH buffers are used by farmers to set the pH of surrounding land to the proper amount for the species they are growing. The pH buffer tablet I used exposed the earthworms to a pH of approximately 9.

Glyphosate: one of the most widely used herbicides in the United States. It is used to regulate plant growth and ripen fruit. It is known to bring adverse effects to the nervous system of organisms.

Topic Selection

There were a few factors that influenced me into choosing this project. The first and foremost reason why I chose this project is because I would like to become a neurosurgeon when I grow up and this project explores the field of neuroscience. The second reason why I chose this project is that it draws from my experience from Science Olympiad competition where I earned a first place at the national tournament. My event consisted of learning the field of ecology, which contributed that aspect to this experiment.

Research Question

What is the effect of various environmental factors (boric acid, an alkaline buffer solution, and glyphosate) on the conduction velocity of earthworms?

Hypothesis

If earthworms are exposed to various environmental factors (boric acid, an alkaline buffer solution, and glyphosate), then the earthworms exposed to the control factor will have the largest conduction velocity. This is due to the fact that the boric acid, buffer solution, and glyphosate adversely effect the nervous systems of earthworms.

Variables

Independent Variables

• Boric Acid


• Buffer Solution

• Glyphosate


• Control

Dependent Variable

• Nerve conduction velocity (meters per second)

Controlled Variables

• Temperature of laboratory

• Time exposed to environmental factors


• Stretching of earthworms

Experimental Control

The experimental control in this experiment are the worms exposed to none of the environmental factors. This is due to the fact that the worms subjected to the control will serve as baseline measurements for further environmental factors.

Materials

• .59cm x .59cm Square Dowels (Five)
• 100 mL Glyphosate Solution
• 100 mL Boric Acid
• 100 mL Graduated Cylinder
• 100cm x 100cm Metal Mesh (One)
• 200 mL Beaker (One)
• 220 Grit Sandpaper (One sheet)
• 473.176 mL Plastic Cups (30)
• 4cm x 12cm Cork Board (One)
• Alkaline Buffer Tablet (One)
• Audacity Software
• Bag of Organic Potting Mix (One)
• BNC Cable (Four)
• Box of Rubber Nitrile Gloves (One)
• Distilled Water (20 Liters)
• Earthworms (20)
• 250 mL Erlenmeyer Flask (One)
• Ethyl Alcohol 200 Proof (50 mL)
• Fume Hood (One)
• Hacksaw (One)
• Indirect Vent Goggles (One Pair)
• Lab Apron (One)
• Lab Chart 7 Software
• Roll of Lab Tape (One)
• Micropipette (One)
• Pack of 300 Paper Towels (One)
• 700 mL Plastic Container (Four)
• Plastic Forceps (One Pair)
• Plastic Pipet (One)
• Plastic Weigh Boat (Two)
• Scissors (One pair)
• Sewing Pins (Three)
• Stir Bar (One)
• Stir Plate (One)

Procedure

1. Utilizing a pair of scissors, cut the sheet of metal mesh to the parameters of 20.32cm x 40.64cm
2. Utilizing the hacksaw, cut the 0.59cm x 0.59cm square dowels to a length of 20.32cm
3. Utilizing a stapler, fasten the first dowel at the top of the metal mesh, the second dowel 13.97cm down from the first dowel, the third dowel 6.35cm down from the second dowel, the fourth dowel 13.97cm down from the third dowel, and the fifth dowel 6.35cm down from the fourth dowel.
4. Shape the metal mesh into a box by folding the corner of the second, third, and fourth dowels up.
5. Utilizing the soldering iron, solder two lead wires to the middle of one sewing pin that will serve as a ground electrode
6. Utilizing a soldering iron, solder one lead wire to the middle of the second sewing pin that will serve as the first recording electrode
7. Utilizing a soldering iron, solder one lead wire to the middle of the third sewing pin that will serve as the second recording electrode
8. Connect one lead wire from ground electrode and one lead wire from the first recording electrode to a lead wire- BNC adapter
9. Connect one lead wire from ground electrode and one lead wire from the second recording electrode to another lead wire- BNC adapter
10. Connect one BNC cable to the first lead wire- BNC adapter
11. Connect the open end of the BNC cable to the input port of an amplifier
12. Connect a BNC cable from the output end of the amplifier into the first input port of the digitizer
13. Connect another BNC cable to a lead wire- BNC adapter
14. Connect the open end of the BNC cable to the input port of an amplifier
15. Connect a BNC cable from the output end of the amplifier into the second input port of the digitizer
16. Connect the USB to USB cable from the output end of the digitizer to the port in the computer.
17. Secure the cords using multiple four centimeter strips of lab tape to ensure the cords do not move during recording
18. Place the cork board inside the Faraday cage
19. Fill a graduated cylinder with 90 mL of distilled water
20. Under the fume hood, pour 10 mL of the ethyl alcohol into the graduated cylinder
21. Pour the 10% alcohol solution into one 700 mL plastic container
22. Cap the plastic container loosely

Boric Acid

1. Under a fume hood, measure .618 grams of boric acid into a plastic weigh boat on an electronic balance
2. Pour 100 mL of distilled water 250 mL Erlenmeyer flask
3. Under the fume hood, pour the .618 grams of boric acid into the Erlenmeyer flask
4. Place the stir bar into the Erlenmeyer flask
5. Place the Erlenmeyer flask onto the stir plate
6. Set the stir plate to 340 RPM
7. Once there are no longer any particles left visible in the Erlenmeyer flask (approximately five minutes) turn the stir plate off
8. Pour the solution into a plastic container
9. Cap the container loosely

Alkaline Buffer Solution

1. Fill the Erlenmeyer flask with 100mL of distilled water
2. Under the fume hood, open one 9.0 pH buffer capsule
3. Pour the contents of the buffer capsule into the Erlenmeyer flask
4. Place the stir bar into the Erlenmeyer flask
5. Place the Erlenmeyer flask onto the stir plate
6. Set the stir plate to 340 RPM
7. Once there are no longer any particles left visible in the Erlenmeyer flask (approximately five minutes) turn the stir plate off
8. Pour the solution into a plastic container
9. Cap the container loosely

Glyphosate Solution

1. Place a new cap on the micropipette
2. Draw 0.015 mL of the pesticide from the bottle of pesticide using the micropipette
3. Pour 100 mL of distilled water into the graduated cylinder
4. Release the 0.015 mL of pesticide from the micropipette into the graduated cylinder
5. Pour the solution into a plastic container
6. Fill three plastic cups with 250 mL of distilled water
7. Fill one plastic container with the experimental solution
8. Place the plastic cups, paper towels, and containers in the arrangement shown in Figure #4.
9. Rinse one earthworm in the first cup of distilled water ensuring there is no dirt on the earthworm
10. Remove the earthworm from the distilled water and place it on the first paper towel
11. Remove any remaining dirt from the earthworm using the paper towel
12. Transfer the earthworm to the experimental solution
13. Allow the earthworm to be exposed to the experimental solution for ten minutes
14. Remove the earthworm from the experimental solution using a pair of forceps
15. Rinse the earthworm in the second cup of distilled water
16. Place the earthworm on the second paper towel and pat it dry until there is no experimental solution visible
17. Transfer the earthworm into the 10% alcohol solution
18. Allow the earthworm to be exposed to the alcohol solution for ten minutes
19. Remove the earthworm from the alcohol solution and onto the third paper towel
20. Pat the earthworm until it there is no alcohol solution visible
21. Transfer the earthworm onto the cork board
22. Place the ground electrode one centimeter away from the clitellum
23. Place the first recording electrode one centimeter away from the ground electrode
24. Place the second recording electrode six centimeters away from the first recording electrode on the same side of the earthworm as the other electrodes, if the earthworm is not large enough, place the second electrode three centimeters away from the first recording electrode. Be sure to record this distance.
25. Open the Lab Chart software
26. Set the number of channels to two
27. Change the number of recordings to 40k/s
28. Click the start button
29. Utilizing the plastic pipette lightly tap the posterior end of the earthworm ten times with an interval of two seconds between each tap
30. Click the stop button
31. Remove the electrodes from the earthworm
32. Fill a plastic cup with 200 mL of potting soil and 25 mL of distilled water
33. Place the earthworm in the third cup of distilled water
34. After one minute, place the earthworm into the plastic cup filled with soil
35. Export the Lab Chart file as a .wav file
36. Open the Audacity software
37. Import the .wav file into Audacity
38. Isolate one action potential from the first recording electrode
39. Record the difference in time between the action potential from the first and second electrode
40. Divide the distance between the two recording electrodes and the time that was found on the Audacity software to calculate the conduction velocity of the earthworm.
41. Repeat steps 78-80 from five different action potentials
42. Repeat steps 49-81for five trials
43. Repeat steps 19-22 and 49-82 for the boric acid, buffer solution, and glyphosate.

Results

Data Analysis

The earthworms exposed to the control had the highest conduction velocity at an average of 8.41 m/s. The first earthworm exposed to the control had a conduction velocity of 7.5 m/s. The second earthworm exposed to the control had a conduction velocity of 6.84 m/s. The third earthworm exposed to the control had a conduction velocity of 8.57 m/s. The fourth earthworm exposed to the control had a conduction velocity of 10 m/s. The fifth earthworm exposed to the control had a conduction velocity of 9.14 m/s.

The earthworms exposed to the boric acid had the second highest conduction velocity at an average of 8.142 m/s. The first earthworm exposed to the boric acid had a conduction velocity of 8.57 m/s. The second earthworm exposed to the boric acid had a conduction velocity of 8.57 m/s. The third earthworm exposed to the boric acid had a conduction velocity of 8.57 m/s. The fourth earthworm exposed to the boric acid had a conduction velocity of 7.5 m/s. The fifth earthworm exposed to the boric acid had a conduction velocity of 7.5 m/s.

The earthworms exposed to the buffer solution had the third highest conduction velocity at an average of 6.538 m/s. The first earthworm exposed to the buffer solution had a conduction velocity of 4.55 m/s. The second earthworm exposed to the buffer solution had a conduction velocity of 6.82 m/s. The third earthworm exposed to the buffer solution had a conduction velocity of 7.16 m/s. The fourth earthworm exposed to the buffer solution had a conduction velocity of 7.5 m/s. The fifth earthworm exposed to the buffer solution had a conduction velocity of 9.14 m/s.

The earthworms exposed to the glyphosate had the poorest conduction velocity at an average of 6.5 m/s. The first earthworm exposed to the boric acid had a conduction velocity of 5.89 m/s. The second earthworm exposed to the boric acid had a conduction velocity of 5.89 m/s. The third earthworm exposed to the boric acid had a conduction velocity of 7.33 m/ s. The fourth earthworm exposed to the boric acid had a conduction velocity of 6 m/s. The fifth earthworm exposed to the boric acid had a conduction velocity of 6.66 m/s.

There was one outlier that occurred in the data. The first earthworm exposed to the buffer solution had a conduction velocity of 4.55 m/s approximately 2 m/s less than the average conduction velocity in that trial.

Conclusion

Based on the results from this experiment, the researcher’s hypothesis was supported. The earthworms exposed to the control group had the highest conduction velocity of all trials at an average of 8.41 m/s.

This supports the researcher’s scientific reasoning that the other environmental factors have been observed to enact adverse effects upon the nervous system of organisms and therefore would result in a lower conduction velocity.

One experimental error that may have occurred during the course of the experiment is both the experimental and alcohol solutions may have become exposed to carbon dioxide in the air while being used. This may have resulted in a slight increase or decrease in the results. This error may have been prevented if the solutions were kept tightly closed throughout the experiment. Another experimental error that may have occurred throughout the course of the experiment is human error in recording the time between action potentials. This may have resulted in a slight increase or decrease in the results and may have been prevented through the use of a computer program to accurately measure the time period. A third experimental error that may have occurred throughout the course of the experiment is human error in placing the electrodes. This error may have resulted in a slight increase or decrease in the results and may have been prevented through the use of a digital caliper.

There are multiple benefits to society that this experiment could bring. One benefit to society is this experiment could aid farmers in analyzing the effects pesticides would have on organisms on their land. The results from this experiment can also be used to understand the effects these environmental factors may potentially have on the human nervous system. A recommendation for further research could be testing the effects of different concentrations of these pesticides on earthworms.

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