Bronxville High School Page 1 of 10 Environmental Science Blackworm Toxicity Lab Mrs. Bastone/Mr.

Ippolito
Name: Alexandra Adrian
INTRODUCTION
In this lab we are investigating the toxicity of two substances. We will be focusing on the effects that these toxins have on California Blackworms. These organisms, also known as Lumbriculus Variegatus, live in shallow marshes, ponds or swamps and feed on the microorganisms and other organic material found in their habitat. Native to North America and Europe, blackworms are known for their quick reflexes. Each adult worm has about 150-250 segments that can regenerate an entirely new worm if the segment is separated from the body. This method is their primary way to reproduce. Although it is hard to tell, the worms have anteriors and posteriors. The anterior has a slightly green pigmentation and it is the location of the curious reflex escape mechanism. As I mentioned before, the worms have very fast reflexes especially when they are touched. Depending on where the are touched they behave differently; if you touch the posterior the worm will swim in a cork screw fashion but if you touch the anterior the worm will reverse its body and swim the other way. The posterior is also where the organism carries out necessary functions and it is where photoreceptors are. Photoreceptors are crucial for the worms to avoid predators. When the worm lifts its end at a 90 degree angle out of the water it will exchange oxygen and Co2 which is necessary for the worm to live. If the photoreceptors sense shadows or movement during the exchange of oxygen and Co2, the worm will quickly retreat. With this background about the California Blackworms we can test the toxicity of alcohol and caffeine on the organisms.

HYPOTHESIS
If the California blackworms behave similarly to how humans behave when humans exposed to caffeine then I would suspect that the drug would make the worms more energetic and jittery as well. As for the alcohol, I think that the worms will be able to handle it to a certain extent. But, due to the fact that any substance has the potential to be deadly, if the worms are exposed to too much caffeine or alcohol their small bodies will not be able to absorb and dispose of the toxins fast enough so I think that they will die if we give them too much of each concentration.

PROCEDURE
Our goal is to determine how different concentrations of two toxicants (caffeine and alcohol) affect the worms behavior. We will only be able to do this if we know how the worms act naturally, without any toxins. We can observe their swimming and crawling behavior and rate their activity level in our data packet. After we record what they are doing and how they react to being touched, we can begin to test the toxic potential of the substances. With a few worms in separate dishes we can add different amounts of each toxin to individual dishes and observe what happens to the experimental group of worms after being exposed for a duration of about ten total minutes. We can determine the acute and chronic exposure and effects of the concentrations on the worms. To do this we will need to see if there are any longterm (chronic) effects that may appear more slowly and less intensely. Depending on how the worms react to the concentrations, we may discover the lethal dose (LD50) if the toxins cause the worms to die. With the information that we gather from this experiment, we can apply our findings to the rest of the world and hypothesize how humans would behave under the same circumstances.

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Bronxville High School Page 2 of 10 Environmental Science Blackworm Toxicity Lab Mrs. Bastone/Mr. Ippolito
DATA/OBSERVATIONS DATA/OBSERVATIONS

Swimming Behavior Control 0 minutes Notes:

Alcohol Toxicity Data Low Conc Medium Conc High Conc 0 Dead

3 minutes Notes: 6 minutes Notes: 10 minutes Notes:

2 Swimming/ Clumped Together/ One red end/when touched they swim away 2 Swimming/Clumped Together 2 Swimming/Clumped Together 2

2 1 Swimming/Some Barely move slowly move/Slowly died

2 One died

1 Slowly died

0 Dead 0 Dead 0
Dead

2 0 Less movement Slowly died 2 0 Slowly died

Swimming/Clumped Less movement Together

Rate Activity as 0 (Not Active) 1 2(normally active) 3 4 (very active)

Swimming Behavior Control 0 minutes Notes:

Caffeine Toxicity Data Low Conc 4 4 Twisting No Change around/ in behavior Clumped together 4 3 No Change in No Change behavior in behavior 4 3 No Change in No Change behavior in behavior 4 3 No Change in No Change behavior in behavior Medium Conc 4 No Change in behavior High Conc 4 No Change in behavior

3 minutes Notes: 6 minutes Notes: 10 minutes Notes:

3 No in 2 No in 2 No in

Change behavior Change behavior Change behavior

1 Little movement 0 All dead 1

Moving again

Rate Activity as 0 (Not Active) 1 2(normally active) 3 4 (very active)

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Bronxville High School Page 3 of 10 Environmental Science Blackworm Toxicity Lab Mrs. Bastone/Mr. Ippolito
Swimming Behavior Control 3 minutes Notes: 6 minutes Notes: 10 minutes Notes: R R R P R R Alcohol Toxicity Recovery Low Conc Medium Conc N P P High Conc D D D

Rate Activity as R (complete recovery) P (partial recovery) N (no recovery)

Swimming Behavior

3 minutes Notes:

6 minutes Notes: No behavio r change 10 R minute s Notes: No behavio r change

No behavio r change R

Caffeine Toxicity Recovery Control Low Medium High Conc Conc Conc R R P P No No Still Still behavio behavio swimmin swimmin r r g g sloly change change slowly R R R R Swimmin Swimming normal again g normal again R R

No behavio r change

Swimmin g normal again

Swimming normal again

Rate Activity as R (complete recovery) P (partial recovery) N (no recovery)

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Bronxville High School Page 4 of 10 Environmental Science Blackworm Toxicity Lab Mrs. Bastone/Mr. Ippolito
Class Data for Alcohol:

Class Data for Caffeine:

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Bronxville High School Page 5 of 10 Environmental Science Blackworm Toxicity Lab Mrs. Bastone/Mr. Ippolito
Exposure Bar Graph for Alcohol Activity Rating 4 3 2 1 0 minutes 0 3 6 10 0 3 6 10 0 3 6 10 0 3 6 10 Control Low Medium High Exposure Bar Graph for Caffeine Activity Rating 4 3 2 1 0 minutes 0 3 6 10 0 3 6 10 0 3 6 10 0 3 6 10 Control Low Medium High
RESULTS
Answer all of the questions from the handout here.

1. For our investigation the toxicants were alcohol and caffeine. The exposure frequency would be once because the worms came into contact with the toxins only one time but they stayed in the boat for ten minutes whiel we observed their activity level every three minutes. The exposure duration was 10 minutes in total and the exposure concentration differed for each of the plastic boats. We had 5 boats filled with 40mL of distilled water, 2 of which were the control and the other 3 which served as the recovery baths for the worms after being exposed to the toxicants. The high caffeine concentration was filled with 40mL of caffeine, the medium caffeine concentration was filled with 13.3mL of caffeine and 26.7mL of distilled water, and lastly the the low caffeine concentration was filled with 3mL of caffein and 37mL of distilled water. For the alcohol setup there were 5 boats filled with 40mL of distilled water, just like the caffeine setup. The high alcohol concentration was filled with 40mL of alcohol, the medium alcohol concentration was filled with 10mL of alcohol and 30mL of distilled water, and lastly the low alcohol concentration was filled with 1mL of alcohol and 39mL of distilled water.

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Bronxville High School Page 6 of 10 Environmental Science Blackworm Toxicity Lab Mrs. Bastone/Mr. Ippolito
2. _Based upon the two types of tests that can be performed, our tests were both acute toxicity tests. This is because the worms were exposed to the toxicant (alcohol or caffeine) for a brief duration but in high concentration baths. The benefit of performing an acute toxicity test is that you get results faster because the experiment is extended for a short amount of time. The benefits of performing a chronic toxicity test is that the organism has more time to adjust to the toxicant and we would be able to observe more precisely what amount of each toxicant is the LD50. We would also discover what the long term effects of the toxicants which we could apply to humans and possibly discover what the potential long-term dangers are for people who ingest large doses of these substances.

3. Our assigned toxicant was caffeine and if we had the oportunity to design a chronic toxicity test I would use more plastic boats and make the increase in concentration of caffeine in each boat more gradual. By this I mean that the worms would not be shocked by an extremely higher concentration of the toxicity so we will be able to zero in on exactly what amount kills half of the california blackworm population. I would hypothesize that the worms would be able to handle the concentrations better because they can adapt to the baths. If someone jumped into an ice cold bath on a hot summer day their body will be more shocked than if they had jumped in a medium temperature pool before entering the bath because their body would adjust to the colder temperature.

4. The exposure pathway for the caffeine would be skin contact because the worms were effected by the toxicant by swimming in it so it seeped through their skin and into their blood stream. This was proved when we witnessed another groups worms bleed out through their skin which shows that the toxicant entered the body thorugh the same way that the blood exited their body. They also could have ingested it if they drank the water.

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Bronxville High School Page 7 of 10 Environmental Science Blackworm Toxicity Lab Mrs. Bastone/Mr. Ippolito
5. Taking into account the different intrinsic factors for my toxicant, caffeine, I think that the age of the worm would effect the worms ability to handle the toxicant. I would predict that a younger worm would be able to metabolize the caffeine better than a baby worm or an old worm because their internal organs not be as developed and quick. In addition, I would think that a small worm would not be handle the caffeine as well as a larger worm. Just as a 5 foot person may experience a greater change in behavior when drinking the same amount of caffeine as a 6 foot person, I suspect that worms react similarly. A larger body has more physical volume to disperse the toxicant and metabolize it. Genetic differences would also effect the worms reaction to the toxicant because different mutations in genes can change the chemical structure of a gene which can pass on a characteristic to further generations of worms. A worms DNA or combination of chromosomes are what contribute to variation among people and organisms.

6. Sublethal refers to the amount of toxicant that is slightly less than the deadly dosage. A lethal dosage of a toxicant is the minimum amount of the toxicant that causes death when an organisms is exposed to it.

7. When the worms detoxified the caffiene was being removed from the worms body. The liver is the main organ that is responsible for detoxification but the kidneys do some of the work in a few cases. I think that the worms liver probably absorbed most of the caffeine and disposed of it through excretion or through the pores/cells in the skin.

8. N/A The question pertaiend to the 24 hour recovery period which we didnt do.

9. At each of the different concentrations, not all of the worms in the boat acted alike. For example, in the medium concentration boat two of the worms were barely moving but the other three were lively. I think this dissimilar behavior occurs because not all of the worms are alike. The probability of all of the worms being the same age, size, weight etc is extremely slim so we can conclude that the concentrations effected some more than others.

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Bronxville High School Page 8 of 10 Environmental Science Blackworm Toxicity Lab Mrs. Bastone/Mr. Ippolito
10. The control in our experiment were the worms that just swam in the distilled water for the entire lab. They are the worms whose behavior we considered normal and we used this batch of worms to compare to the worms in the different concentration with to see if we saw any behavior changes. The control group allows us to see how one factor effects the worms so we keep this group from changing so that we can use it to compare and contract the normal behavior of worms to the behavior of worms in the different concentrations of toxicants. Without a control, we would not know if the worms behaved differently at different concentrations because we wouldnt know how they behave without any toxicants present!

11. Some toxicants that we might test that would harm the environment and thus pose a threat to the worms would be: types of air pollutants, such as cigareete smokewhich is known for causing lung cancer in humans; gases such as carbon monoxide, nitrogen, nitrogen dioxide and hydrocarbons. These gases are typically found in the kitchen because they are used in wooden stoves or gas stoves; biological contaminants such as bacterias, molds, viruses that are common in areas with humid air and standing water, such as the swamps where blackworms live.

12. Lifestyles are an important factor in risk assesment of human health toxicants because depending on ones job, where they live, what they are exposed to, etc all effect the risk of heal problems. For example, if someone lives in the city, they are breahing in more fumes from the buildings and more smoke because their urban lifestyle brings about these consequences. Someone who lives on a farm in the south has a different lifestlye than that of a city dweller and therefore they are exposed to completely different toxicants such as mold and viruses that are foreign to city people. However, all people, reguarless of lifestlyes, can be exposed to the same toxicants but depending on the exposure duration, the risk assesment may be different for individuals.

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Bronxville High School Page 9 of 10 Environmental Science Blackworm Toxicity Lab Mrs. Bastone/Mr. Ippolito
13. We would need to do further testing in order to declare what the LD50 of caffeine is for larger living things such as humans and other vertabres. This is because the worms LD50 is going to be extremely lower than a humans do to the obvious difference in size and weight. Humans will be able to handle much more caffeine so this experiment cant help us fine a safe nor a lethal dosage. However, this experiment does inform us that caffeiene, like any substance, can be deadly if too much is taken. We would just need to find out what the dosage is in an additional experiment.

14. Refer to Conclusion

CONCLUSION
In this lab we are investigated the toxicity of certain substances and how different concentrations effect the behavior of California Blackworms. I hypothesized that the California blackworms would behave similarly to how humans behave when humans exposed to caffeine. My educated guess was disproven when the higher dosage of caffeine made our worms less energetic. As for the alcohol, I hypothesized correctly that the worms would be able to handle it to a certain extent. However, as with any experiment, we cannot make definite conclusions soley based upon this single experiment because multiple student errors could have occurred. We could have handeled the worms too roughly and caused them to be injured, we could have prematurely declared them as dead, the worms could have been defective or sick upon their arrival to the classroom, etc. Due to these possible errors, we would need to test this experiment again in order to know for sure if California Blackworms behave abnormally at different concentrations of alcohol and caffeien. But, taking into consideration the fact that any substance has the potential to be deadly, any living organisms, large or small, would be unable to dispose of the toxicants fast enough resulting in death. With this known fact, it is important to always read warning labels and be educated about how much of the substance you are handling is the ED and the LD50.

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Bronxville High School Page 10 of 10 Environmental Science Blackworm Toxicity Lab Mrs. Bastone/Mr. Ippolito
REFLECTION
Overall I enjoyed this experiment because it was fun to see how each of the worms behaved when exposed to the toxicants. No two groups had worms that behaved exactly the same which was interesting because I thought that we would find a definite LD50. This experiment went smoothly and I am very happy with the extent of my research that I did before the experiment because I knew a lot about the worms so I could draw more elaborate conclusions during the lab. Assigning the different toxicants to different group memnbers was definitely a good idea because we didnt need to rush therefoer we were very careful during the experiment which led to fewer student errors. If I were to do this experiment again, I would enjoy leaving the worms overnight and seeing if they could recover in 24 hours.

End of Lab Report Form