Urgent help needed - Daphnia- water toxicity in my town

[deleted-62614]

I have received the Daphnia and have the experiment ready to start. I am using the Water Toxicity Experiment on this site except, I am testing various water sites in my town for water toxicity. I collected the water from eight places (near the dump, in a neighborhood, at a busy parking lot, near an auto mechanic lot with a pipe leading into the pond, downhill from a farm that uses fertilizer, at the major lake for homes and recreation in our town, pond near a rural road, pond in a golf course). I am going to use five of the test sites.
1. Should I feed the daphnia during the experiment?
2. How long will they live in the container that we received without doing anything to them, except loosing the jar lid so they get oxygen?
3. Is it important or necessary to check the acid levels with ph paper?
4. The experimental procedure on SB says to count them every 8 hours. I was thinking I would do every 4 hours. I am afraid they all might die before that and then I won't have good results to analyze. Is it better to test at the beginning every 15 minutes? I am wondering if I need to get up every four hours or more in the night to count live and dead daphnia?

[donnahardy2]

Hi,

This is an excellent project:

https://www.sciencebuddies.org/science- ... p043.shtml

Here are some answers to your questions:

1. This is a really good question. Feeding your Daphnia should be one the controlled parameters in your experiment, and it is probably best to add some algae or yeast to the sample so that the only difference between your control samples and the experimental samples if the source of water.

2. The following website suggests storing Daphnia for up to 3 days in the refrigerator if you don’t have time to start feeding them. Please also note that it’s possible to overfeed Daphnia, so you should add algae or yeast, and wait until the water is clear before you feed them again.

http://buylivefishfood.com/culture_live_daphnia.html

3. If you measure the pH and notice a toxic effect of a water sample, then the pH measurement that is outside of the optimum range could help explain the reason for the toxicity. I would also recommend measuring the temperature, although this should be a controlled parameter in your experiment. Here is a website that includes the optimum conditions for growing Daphnia. The experiment would be perfectly good without pH and temperature measurements, but having additional measurements would add to the information you would have available when analyzing the results.

http://www.caudata.org/daphnia/


4. You definitely need a reading at the beginning of the experiment, and it might be helpful to add a reading at 4 hours, but I would not recommend doing more frequent readings. This is going to be a lot of work and you are going to have lots of data in a short amount of time. I would definitely recommend setting up duplicate samples and be sure to include a control sample. You should schedule your readings so you don’t have to get up in the middle of the night. You will be graphing number of Daphnia over time, so it is OK if a reading is not exactly at 12 hours, for example. If you notice unusually results with one of more samples, and if you have time, it would be very helpful to set up the experiment a second time to find out if your results are reproducible.

Please note that you should use proper names in science projects. Daphnia is the genus of this organism and should be underlined or italicized on your project board. The classification of Daphnia species is complex, so you may not be able to identify the species you are working with, however, it would be wonderful if you could say you are working the Daphnia pulex, for example, but there are over 150 different species in North America alone, so don’t worry if you can’t.

http://en.wikipedia.org/wiki/Daphnia

If you have time and interest to learn more about scientific names, here is a general description with the rules for naming:

http://en.wikipedia.org/wiki/Binomial_nomenclature

Good luck with the experiment! I am very interested in knowing about your results.

Donna Hardy

[deleted-62614]

It took over an hour to measure 100 ml of the 6 waters +1 control three times. My mom says I take too much time to be too exact. On Monday, it took me three hours to use a pipette and gather ten daphnia for each water sample & control. My dad and I made a light stand so I could use a flashlight to shine through the containers. It works well.
On Tuesday, I started throwing up at school and continued at home. Still trying to count daphnia this evening so the test won't be hindered. So glad it doesn't have to be an exact time table! I am cold and all I want to do is sleep in my bed. My mom wondered if I got some polluted water on my hands....and it made me sick. There is a something going around at school, so maybe not.

[donnahardy2]

Hi,

I am sorry that you got sick and I hope you are feeling better by now. It is likely that you had a rotavirus or norovirus infection that is highly contagious and spread from person to person. If something is going around at school, then it’s likely that you picked up the infection there. It is much less likely that the water was the source of the infection, however, I hope you did not collect samples that were downstream of a sewage plant or that could have been contaminated with raw sewage. Where did you collect your samples? Determining the source of your infection could be an interesting study in epidemiology.

It sounds like you did a lot of careful work in setting up your experiment and I think that you did an excellent job; the light stand is a good idea. It is always much harder and more time consuming to set up an experiment, especially the first time. If you do set up the experiment again, you will find that it will take less time because you will have gained experience.

What are your results so far?

Donna Hardy

[deleted-62614]

The results aren't really showing much. The daphnia are dying little by little from each water source. I don't know whether that is because it is "their time to die" or because of the water. The dump water daphnia have 5 or 6 dead in each of the three samples since I began the test on Monday. That is interesting because when I collected the water in November there were two daphnia (I think) in the water and one was still living when we opened the jars in February!
What do you think? - If the water was toxic, would they all be dead by now? How long should I continue counting??

[donnahardy2]

Hi,

Did you set up a control sample using water that was non-toxic and one that was toxic? The answer to your questions would be based on a comparison of your experimental water and a control

Daphnia live for 29 days at 28 degrees Centigrade and 108 days so their lifespan depends on the temperature. Since you have seen a gradual die-off during the week, it is most likely that the Daphnia are dying due to old age. I think if the water was toxic, the population would have died suddenly near the beginning of the experiment.

http://en.wikipedia.org/wiki/Daphnia

Why don't you graph your results showing numbers of Daphnia and time on the x-axis? If you don't have a control water sample, then you can assume, for purposes of your experiment that the sample with the lowest death rate is the control sample, and you can compare the other results to that sample. This analysis will give you a basis for your discussion section of your project.

You can conclude this experiment; if the water was toxic, you would have seen some effect on the Daphnia by now.

You just have a few days left before your project is due, so you should start writing up your board. The Science Buddies website has a very good guide for this part of the project.

https://www.sciencebuddies.org/science- ... oard.shtml

If you have the time and energy, it would be good to set up your experiment again, including control water samples and setting up each sample in duplicate and do a 2-3 day experiment. However, if this is not feasible, don’t worry because you have plenty of data to write up for your project.


Please let me know if you have any questions.


Donna Hardy

[deleted-62614]

How closely are toxicity and acidic related? I do have a control of spring water. I don't have any water that I knew for sure was toxic.
The science fair is the 19th, but I do have most of it done by Thursday bc my mom is away for the weekend. I have the display board and am ready and my titles ready.

[donnahardy2]

Hi,

The Daphnia toxicity test will test everything in a sample that can be toxic to organisms. This includes organic chemicals such as pesticides or heavy metals such as copper or cadmium, and acids and bases that can alter the pH away from the optimum 7.6 to 8 that Daphnia prefer. If the Daphnia toxicity test was positive, you would not know exactly what caused the toxicity, but you would know that additional testing would be needed. If you had measured the pH, and found it was 3 or 10, for example, then the pH results would explain why the Daphnia died and you could say that the water was acidic or basic. However, determining the reason for the toxicity would be beyond the scope of your project.

You should concentrate on writing up your results at this point. It’s very good that you have the spring water control. You should prepare a graph plotting the number of viable Daphnia on the y axis and time on the x axis for all of your samples. Put all of the results on one graph and label each line separately, making sure that the control line is clearly distinguished from the sample lines. This will be the centerpiece of your project board.

Now, compare the percentage of viable Daphnia between your control and each test water sample at 48 hours and at the end of the experiment. The analysis of water toxicity testing is based on LC50, which is an estimate of the concentration that will results in 50% mortality of Daphnia. If you observe no difference in the results, then your results would be described as “No-Observed-Adverse-Effect Concentration” or NOAEC. The statistical analysis of water toxicity results is complex, so let me know if you have questions.You will want to include a short discussion of this in your project board.

If you have any questions about preparing the graphs, please post your data and I’ll try to help. The data analysis is a very important part of your project.

http://www.agls.uidaho.edu/etox/resources/probit.pdf
http://www.ehow.com/how_6873345_calcula ... alues.html

The science fair judges will be looking to see if you understood the science behind your project, and will be checking to make sure the project is complete. Some science fairs are judged on a point basis, so you want make sure your write up is complete. Do include the following:

You should include a short abstract describing your project and the results. In your introduction/background section, you should include information about the biology of Daphnia why water toxicity testing is done, and you should report any information about toxic water problems in your city. Your hypothesis should be based on the background research information. What was your hypothesis? Your materials and methods section should be presented in enough detail that someone else would be able to repeat your experiment and obtain the same results. You can include a map of your city and mark where all of the water samples were taken. It would be helpful to include photographs or drawings of your experimental set up, and a drawing or photograph of a Daphnia. Your results section should include a short paragraph summarizing the results, in addition to the line and bar graphs.

The conclusion section will summarize the results. I gather that your results will show no significant difference between the control and test samples, so you will be able to conclude that there are no toxins in the test water samples. You should explain what you would do in future experiments, and in this case, you could say that you would set up the experiment in duplicate, use only newborn Daphnia to set up the tests, and perhaps do long-term toxicity testing.

http://www.clt.astate.edu/rgrippo/Toxic ... g%20II.ppt

Be sure to include a bibliography section. You can use the links on the Science Buddies website for additional information.

Good luck!


Donna Hardy

[deleted-62614]

I did measure the ph of the water and none were higher than 6.8
I was wondering if I should try another test with our well water (it was tested three years ago and has a higher than should be arsenic amount) We don't drink it anymore.
When you say viable, do you mean the ones that were living when my testing was complete?

[donnahardy2]

Hi,

The pH of your water is compatible with Daphnia survival.

Daphnia are sensitive to arsenic, and you would see a lower survival rate if the arsenic concentration in the well water was high enough. Do you know what level of arsenic was found when the well water was tested?

Yes, viable refers to the Daphnia that were alive at the end of the experiment, as opposed to the non-viable, dead Daphnia.

Your project would be improved by repeating the experiment with the spring water control and the arsenic-containing well water as a possible toxic control sample and freshly collected water samples from the same locations that you used before. It would not be that helpful to run just one more sample by itself without a control. An experiment that is repeated is always preferred when doing a science fair project. However, since your project is due on Monday, you should concentrate instead on writing up your project and considering the suggestions I made in my last post to make sure your have a complete presentation.

Let me know if you have any more questions.

Donna Hardy

[deleted-62614]

How can I send you my chart with the results?

[donnahardy2]

Hi,

If you have an Excel table of your results, you can attach the file to a reply messge. You start the process by clicking on the "upload attachment" tab at the end of the reply window. If you don't have a computer file of your results, then just write out the results paragraph style, and I'll figure it out.

Donna Hardy

[deleted-62614]

It said, "The extension xlr is not allowed."

[donnahardy2]

Hi,

If you have time , just type out the data in paragraph form with a description of each sample.

Donna Hardy

[deleted-62614]

The final

[deleted-62614]

delete

[deleted-62614]

Final number DEAD out of ten in each sample
1) 6,7,6 dump.pond
3) 7,7,5 Beaver Lake (recreational and homes)
4) 7,5,5 Hood Pond (near auto mechanic shop)
5) 2,2,1 Hoodcroft (golf coursepond- fertilized)
7) 5,8,2 Farm runoff
8) 5,3,3 Flea market parking lot/pond
9)3,2,4 Control

SO can't make much of a conclusion with the number dead....except that the #3 and #4 had the most dead with the dump #1 close. So I can conclude that the water that I tested wasn't toxic enough to kill Daphnia. It seems strange that the most living were in the golf course where it seems that there would be lots of chemicals to keep the course green.

[donnahardy2]

Hi,

Thanks! Your data looks very interesting and it definitely looks like there is a difference between some samples and your control. I will review the data and send you some more comments tomorrow.

Donna Hardy

[donnahardy2]

Hi,

This is a really great project and you have plenty of data to present for your project. Remember that the judges will be interested in your analysis of the data, so I have included a few suggestions below. I’m sure you will be able to think of other points of discussion to include as well:

I have tried to find a way to calculate the statistical difference between your control and sample results, however, it’s not possible because you ran one sample with 10 Daphnia each and all of the statistical formulas require more than one replicate. Therefore, I recommend that you do a bar graph of one time point, probably the first reading, to display on your board. Then you could make a tentative conclusion and say that the samples with more than double the number of dead Daphnia compared to the control probably contain some toxic substances. So, for example, if you use the first time point with 3 dead Daphnia in your control and 6, 7, and 7 dead in the dump pond, Beaver Lake, and Hood Pond, respectively, you can say these water samples were probably toxic. For the farm runoff and flea market pond with 5 dead each, you could say the water might be toxic, and for the Hoodcroft sample, the water is definitely not toxic.

Your numbers in the second and third reading are interesting, and you should include a discussion of why the numbers of dead Daphnia decreased in some cases. Do you think there was an error in the counting method because it was difficult to count individual Daphnia, or do you think the surviving Daphnia were having babies? The science fair judges will be interested in knowing how you would explain the results.

Also, you can include a discussion about why the Hoodcroft sample from the golf course is not toxic. This sample probably contained fertilizer, which would stimulate the growth of algae, which is a source of food for the Daphnia, but it apparently did not contain significant levels of herbicides or pesticides, which is surprising.

Another discussion point you can include is the time period between sample collection and the testing. You collected the samples in November and did you testing in Feb/March. During this time, bacteria would have grown in the samples, and possibly used up all of the oxygen and produced metabolic end products that would have inhibited the Daphnia. This might explain why you observed 3 dead Daphnia in the control sample where you would have normally expected no mortality. Your experiment is still valid, however, because in your analysis, you are comparing results of the control with each sample.

In your conclusion section, you might want to make a statement about the implication of your results for your community. Do you think there is a significant problem with water toxicity? What is in Hood Pond and Beaver Lake that killed the Daphnia?

At the end of your conclusion section, you should include a statement about what you would do next if you were continuing the experiment. I think you should definitely plan to include duplicate or triplicate samples and work with samples that are freshly collected. You need a control sample that does not result in dead Daphnia. And, you might consider doing a long-term project to evaluate low levels of toxicity in the water.

Let me know if there are any last minute questions.

Donna Hardy

[deleted-62614]

I think I wasn't clear with my other numbers. We took 3 samples of water from each location and counted the Daphnia ten times from each container.
The numbers (6,7,6) I gave you are the final dead count from each of the 3 samples from ONE location. I thought about averaging them, but didn't. And to type all 210 live and dead counts was too much.
Does that make sense?

[donnahardy2]

Hi,

This is good news! Thanks for confirming your data. You did a lot of work and your results are statistically significant because you did samples in triplicate.

You can use the student’s t-test to determine if there is a difference in the results between your control and each sample results. The student’s t-test compares sets of data and helps determine if there is a difference in the results.

If you use the calculator in this website and select unpaired data and type in the control results in group 1 (3,2,4) and the dump pond results in group 2 (6,7,6), or the Beaver Lake (7.7.5) and select the “calculate now” button, then the P values is .0075, or very statistically significant. The Hood Pond results (7,5,5) are significantly different from your control. So you can definitely say that these samples are more toxic compared to your control.

http://www.graphpad.com/quickcalcs/ttest1.cfm

If you use the farm runoff results (5,8,2) , flea market parking lot (5,3,3) or Hoodcroft pond (2,2,1) you will find that the results are not statistically different compared to the control.

Here’s more background information on the student’s t-test and the p-values. With biological samples, if there is a less than 5% chance that two populations are the same, then results are considered statistically significant. You could include a brief explanation in your background or results section and present

http://en.wikipedia.org/wiki/Student's_t-test
http://en.wikipedia.org/wiki/P-value

I hope this helps. You might consider doing a bar graph and including the p values of each sample on the bar. This is a fantastic project with the triplicate results, and I think you will do very well at the science fair.

Donna Hardy

[deleted-62614]

I am trying hard to understand the p-value. Basically it is the probability that it could be significant? I don't understand why the .0075 (which is a smaller number) shows the probability of the water being more toxic.

[donnahardy2]

Hi,

Yes, I knew this would be hard for you to understand because you haven't had statistics yet, but the p value is basically the probability that the results obtain from two groups are not the same (null hypothesis). If there is a less than 5% chance that the results are identical, then the results are considered significantly different. 0.0075 (0.75%) is a lot lower than .05 (5%), so it's really significant. If you look at a comparison of your control (2,3,4)and the golf course (2,2,1), the p value is .11, or 11% chance that the results are different, so these results are not considered significantly different. You can see what would happen if you have a higher number of replicates. If you had done 6 sets of samples, and your control values were 2,3,4,2,3,4 and the golf course pond was 2,2,1,2,2,1, then the p value would be .01 (1%) and this would be significant.

The t-test is based on calculating the standard deviation or variation in results of two sets of data. The t-test calculation compares the distribution of the two populations to determine if they have the same distribution of values. The p value will help you evaluate your data sets to determine if there is a difference between and control and experimental results.

http://en.wikipedia.org/wiki/Standard_deviation

If you don't understand this at all, then you should not include this in your project write up, but if my generalized explanation makes a little sense to you, then go ahead and use the p values. I promise that when you have had a few more math classes, and when you eventually do take statistics, it will make perfect sense. In the meantime, if you can use the t-test calculator and understand than >5% is not significant and <5% is significant then you can use this tool to evaluate your results.

I hope this helps.

Donna Hardy

[deleted-62614]

two more questions
(I think I am going to leave out the P values) But that means I am not sure of the way/what to graph.... what do you suggest? I originally showed the # of dead Daphnia over the ten times I counted, but that doesn't seem like the best idea anymore.

What do you think of this as my conclusion?
My hypothesis was that 4 (Hood Pond) would be the most toxic. My hypothesis was contradicted in that 3 (Beaver Lake) water samples were more toxic than Hood Pond. Yet, when compared to 9 (control group), there was a significant difference in the number of dead Daphnia. It also seemed strange to me that 5 (Hoodkroft Golf Course) did not have a significant decrease in Daphnia. I learned that fertilizer would cause algae to grow, thus making food for the Daphnia.
I think that my tests did go smoothly. I did have to collect the water samples in November because of the coming winter and I didn’t want to have to drill through the ice.
I stored them in the cool basement (58 degrees F.) until the testing in February and March. Bacteria may have grown in the samples, used up all the oxygen, and produced end products that would hinder the Daphnia.
I do not think that my community has a big problem with water toxicity, but I do know that 3 (Beaver Lake) and 4 (Hood Pond) sometimes close their beaches during the summer because the water is contaminated by waterfowl.
If I were to continue this experiment, I would definitely use freshly collected water samples and experiment over a longer term.

[deleted-62614]

I printed all my project info. I did a bar graph showing the locations and number of dead. Looks good. I looked more at the P- values, will mention it in my log book, but that is all. Just need to glue everything on my board. Would like to send you my bar graph if you have an email address that I could do that with. Maybe a pic of me getting an award...ha! Thanks for all your help!

[donnahardy2]

Hi,

I think you are wise to omit using the p-values for your data analysis. Your data shows that Hood Pond, Beaver Lake, and the dump pond are more toxic compared to your control, but does not show that Beaver Lake is more toxic compared to Hood Pond. And it does not show that the farm runoff, flea market parking lot pond, golf course are more toxic compared to your control. The rest of your observations are excellent. I think you have already put your board together, so don’t change anything at this point. Your conclusions are very good if you don’t include the p values.

I understand now why you collected your samples in November.

I’m not allowed to share my personal e-mail address on the Science Buddies website, but thanks for offering the send the bar graph and picture. I can imagine what the graph would look like by looking at the data, and it’s really good data.

Let me know how it goes at the science fair. Good luck! This is an excellent project and you should be very proud of it.

Donna Hardy

[deleted-62614]

Tomorrow is the day and my project is all set up with all the others. Someone asked me what level of toxicity is dangerous to humans. I don't think any of the water samples are extremely dangerous. I know we shouldn't drink them,but are they dangerous otherwise?
I know the comparisons, but I can't find the answer to that- is there a ph level that is toxic to humans to be around?

[donnahardy2]

Hi,

You are correct, you should not drink the water, but your samples are not dangerous or toxic. If you are going to display them, you should make sure they are covered so young children could not get into them. From your description, it sounds like you collected water samples from your community. If you had collected samples coming from a chemical manufacturing plant or downstream from a waste treatment plant, or if all of the Daphnia in a sample has died within 24 hours, then the samples would potentially be toxic and it would not be a good idea to display them. Safety is always a concern of researchers and here is an explanation of biosafety classification:

http://en.wikipedia.org/wiki/Biosafety_level

Good luck today!!!!

Donna Hardy

[deleted-62614]

I won second place!

[donnahardy2]

Hi,

Congratulations! You did very well. I'm excited that you did so well at your science fair. Thank-you for sharing the news.

I have been thinking about your project and I have a suggestion. As a science fair judge, I have noticed many times that students will sometimes do a very good project one year, and then will continue it or do a closely related project a second year. The advantage of continuing an investigation is that you are familiar with the background research and you've figured out the techniques required to conduct the experiment, so it's possible to do a more comprehensive experiment, improve the experimental design, and do more data analysis.

Your project would be ideal for this because your results have left so many intriguing questions. Is the golf course pond water really better than the control? Does the toxicity of the dump pond, Beaver Lake, and Hood pond vary through the year? Is there any long-term toxicity? What is making the water slightly toxic for the Daphnia? Also, you could do the project during the summer and not have to store the samples.

The biggest improvement you could make in your experimental design would be to run duplicate samples. So you would run a triplicate test on each of two samples from the same pond. Since this would greatly increase the number of samples, you could reduce the number of testing sites to make the work manageable. And you could do testing over a period of time when there's no ice.

These are just some ideas for you to consider in the future. For now, I hope you are relaxing now after all of your hard work.


Donna Hardy