Agrisept-L Testing as an Algeacide

[deleted-112020]

Hello, This is my second letter, not sure if the first went through. I would like to test the fungicidal qualities of agrisept-l. I would purchase live algae then run an experiment testing the algae growth vs the quantified addition of agrispet-l. Does this sound like an experiment that is appropriate for 10th grade. I hope so because it seems very interesting to me. Bobbyo

[deleted-106702]

Sounds interesting to me too!
I'd like to let you know that if you find something interesting, you should definitely pursue it and research it further. There is no "10th-grade appropriate" type of science fair project. The best projects are the ones that are extremely well-done and interesting. Also, I have never seen this project before in any science fair I've been to, so it seems unique enough to me!
If you still don't think it is "appropriate", you can keep the same concept/materials and expand on it. You could do a project relating to algae growth, but one that tests a more complex hypothesis.
Let me know if that helps!

[deleted-71536]

Hi Bobbyo,

You have a very interesting and feasible project idea! I just wanted to clarify something for you: algae are not fungi. If you want to test the fungicidal properties of Agrisept-L, which appears to be used to kill all kinds of microbes, you would need to test it with a fungus, like yeast. Yeast is easy to grow on petri dishes, and you could definitely do a project with yeast. That said, it is perfectly interesting to test whether Agrisept-L kills algae; just make sure you do not refer to algae as fungi.

Please post again if you have more questions.

Heather

[donnahardy2]

Hi Bobbyo,

Snobs3747 and heatherL have give you some really good ideas for getting started on this very interesting and challenging project. I have some additional suggestions:

There are 3 project ideas on the Science Buddies website that describe experiments for measuring the antimicrobial effects of agents on various microbes. Although none of these projects describe how to grow algae, they should give you inspiration for designing your own experiment. Please note that it is important to test the dose response when doing this type of testing.

Hhttps://www.sciencebuddies.org/science-fair-pro ... p014.shtml

https://www.sciencebuddies.org/science- ... p013.shtml

https://www.sciencebuddies.org/science- ... p016.shtml

There is one project idea that involves testing the effect of fertilizers on algae growth on this website and the experimental set-up might work for your project:

https://www.sciencebuddies.org/science- ... p054.shtml

Here is a summary of a project on algaecides that should give you useful background information and an idea of how to do an experiment in a larger body of water:

http://www.pacificorp.com/content/dam/p ... tStudy.pdf

You should also do an internet search and look for more information on algae and algaecides. Your goal should be to become an expert on your topic.

I have a couple of questions also. What type of algae are you going to use? And, what will you use for a control?

Donna Hardy

[deleted-112020]

Thank you so much for replyging. I will be testing algae soon sometime in November. I will get back to you when things more evolved. Thanks again!

[donnahardy2]

Hi,

Thanks for your reply. It sounds like you are making progress.

At this point, you should be defining your hypothesis , and designing a carefully controlled experiment. Be sure to review the guidelines on the Science Buddies website for a hypothesis and designing an experiment before you start.

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

Do try to plan ahead so you will have time to do your experiment twice.

Donna Hardy

[deleted-112020]

Hi Again,
After further investigation, I have decided to test agricept-l and its proposed aglaecide quality. I will be using the algae species ankistrodesmus. I can obtain the algae and pipet 1ml quantities into glass test tubes. I will use a fluorescent lighting. I think the best way to quantify algae is to gauge the "greeness" from daarkest green to the least green. My question is what percent solutions of the agricept should I use? I was thinking 1%-5% solutions. But should I make it more of a 5%, 10%, on up to 50% solution. I'm concern oof the cost factor of this testing. Also how many times should I repeat each test, 5 times, 10 times, etc. I need to know before ordering my algae. Thanks.

[donnahardy2]

Hi,

It's good that you are finalizing the details of your experiment.

Do you have access to a spectrophotometer so you can measure the density of the green color at 550 nm? This would give you a quantitative measurement of your results. Or, do you have access to a microscope? I believe you could also count cells in a hemocytometer. However, measuring absorbance is much easier.

Here's a website that describes various methods for measuring algae:

http://www.botany.wisc.edu/courses/bota ... ations.pdf

Do you have time to order a small culture of the algae and grow it in a stock container so you will have fresh and rapidly growing algae to start your experiment? Results for experiments like this depend on having cells that are at the same phase of growth, and algae that you receive by mail may take a while to recover and start growing. And you would only have to pay for the initial small culture.

What concentration of algaecide is recommended on the product label? You want to test a range of concentrations, perhaps 5-6 concentrations, that are below, at, and above the recommended concentration. Also, you need a control with no algaecide.

I recommend doing a pilot experiment with a control and one concentration of algaecide to test your method and find out about any experimental problems you will encounter. Then plan to do your experiment in duplicate, or better, triplicate. Using a higher number of replicates will allow you to do statistical analysis of the results, so more replicates would be better. However, each sample will require a lot of work.

Donna Hardy

[deleted-112020]

Hello Again,
I have doing a lot of research and have made a hypothesis. I will be testing agrisept and its individual ingredients for their algaecidal properties. Agrisept is made up of grapefruit, tangerine, lemon, and lime seed extracts. I could buy the agrisept and the extracts. I was wondering about the extracts. when you purchase the extracts, they are in a glycerine medium. I could make my own by grinding the seed, non juicy pulp and whites of the fruits and just add them to a basic spring water. they can be quantified and the spring water is best for growing my green algae. Would you recommend buying the seeds extracts or making them myself? I know have cellometers and a great microscope. My hypotheses is that the agrisept will be 4x algaecidal then the single fruit seeds. Then grapefruit will then be 3x greater then the other. Following lemon, lime, then tangerine. This is based on research. I will be dealing wiTh serial dilutions. I will contact you more about that! Algae will be arriving in two weeks. Wanted to make sure everything is ready. Thanks-Bobby

[donnahardy2]

Hi Bobbyo,

It sounds like you are making great progress. You can either purchase the individual extracts or prepare them yourself. Either would be scientifically valid as long as you explain exactly what you did for your experiment.

If you prepare the extracts yourself, you would want to make sure you have some way of measuring the extract so that anyone reading your project would be able to replicate your results.

If you purchase the extract, I would recommend checking with the supplier first and find out what they do to test the product to ensure reproducibility from lot to lot. Ask if you can obtain a certificate of analysis for the batches that your purchase.

If you can’t obtain any information at all from the manufacturer, then you might decide it would be best to make your own. If the manufacturer can supply certificates or other helpful information, then you might decide to purchase the extracts.

Testing a series of dilutions for each sample is a great idea. Are you going to be measuring algae growth using a hemocytometer?

Please post again and let me know about your progress.


Donna Hardy

[deleted-112020]

Hey again, I contacted the distributor and asked suggested questions. Awaiting reply. I have cellometers which are like the hemocytometers to do my counts. I have to more questions. 1. Is this a strong hypotheseis? If ankistrodesmus is introduced to varing concentrations of Agrisept or it's individual componets, then the algae will die quicker with the Agrisept than each individual component. 2. I listed myself under botony, should I be under biochemistry? Thanks!

[donnahardy2]

Hi Bobbyo,

Your hypothesis is very good and you have the equipment to obtain quantitative results. This is definitely a botany project, but it could also be entered under biochemistry.

Good luck! Let us know what happens.


Donna Hardy

[deleted-112020]

Dear Miss Hardy,
I am a little frustrated. I am ready to go but I haven't had any responses from the people I have contact( 2 separate places) for questions you suggested asking from the distributor. I did follow up phone calls but still haven't has responses. I don't want to wait too long because I need to start experimentation .
I do have 2 question regarding my serial dilutions. I know how to serial dilute my algaecide from 1:10 to 1:1000. I plan to introduce a quantity of algae to these dilutions separately. I then plan to centrifuge my algae/diluted algaecide to see how may live alge cells I have. I will be using a cellometer that requires a 20 ul amount of suspension.
1. Should I centrifuge( hand made hand-held centrifuge) or just mix algae with diluted algaecIdes?
2. The cellometer instructs to multiply the counts by 10 to 4th power than multiply by dilution factor. Does that mean if I count 50 cells at the 1:10 dilution of agrisept X 10 to 4th = 5,000,000 cells per ml? I think I'm missing something about the quantity of algae I add to the algaecide dilution. Where does that come into play. For instance, if I pipette 100 ul of algae into all the different dilutions of algaecides do I times that somewhere in the calculations? I'm getting confused- think I need a break!!! Thanks Bobby

[donnahardy2]

Hi Bobbyo,

I’m sorry you are not getting the information you need. Some companies are good about answering questions, and some are not. I would go ahead and start your experiment and not wait for additional information.

If you have time, you could try calling the company’s toll free number and explain why you need the information.

It would be good to get a count of the algae before you introduce the algaecides. This will be your time zero count. Then add the algaecide to the samples at time zero. The concentration of algae at the beginning of the experiment is one of your controlled parameters.

You are correct! If you make a 1:10 dilution, then you would multiply the number of cells counted by 10 to the 4th x 10. So if you count 50 cells, the number of cells would be 5,000,000 per mL.

Donna Hardy

[deleted-112020]

Thanks for all your support. Today I have a company who was very willing to send me a certificate of analysis with the items I purchased. I will ask my algae specialist at Carolina Biological more about how much ankistrodesmus would be good to introduce to each dilution of algaecides and approximate time I should wait ...I figure a half hour at first. This may be a trial and error pretesting run to figure times and amounts. All will be logged in notebook.

Would my controls be considered the algae in its spring water medium at a 1:10,1:100, 1:1000? And at o time read for each run and than the wait times (maybe 30minutes)? A control should be read along with the material be tested right?

Ready to run. Bobby O

[donnahardy2]

Hi,

The best place to start is to check a standard method for testing algaecides. Here is a link to a site that includes lots of information about testing disinfectants in swimming pools; there is no specific information on algae or algaecides, but the general information should be useful to you. I was looking for a standard EPA or AOAC method, but I could not find one for algaecides.

. http://www.oecd.org/chemicalsafety/testing/46053426.pdf

The 30 minute time of exposure seems reasonable, but you may want to try a pilot experiment and also do a shorter and longer time. Hopefully the algae specialist at Carolina Biological will have a good answer for you.

It's good that you will recording everything in a lab notebook.

I'm not sure why you are doing dilutions of the algae for the control. The control should be the identical sample with no algaecide. If your samples have a different concentration of medium and algaecide, then there are two variables, and you would need a control for each dilution. Please post your proposed protocol if I have not understood what you are trying to do.

Donna Hardy

[deleted-112020]

Good Afternoon,
I have been doing more thinking. Here is my project protocol. I am going to have 1:10, 1:100, and 1:1000 dilutions of algaecides . I will place 1ml of each dilution sample with 1 ml of algae. The dilution factor will just be a 1:2. My control will be a 1 ml algae sample added to a 1ml spring water . I spoke with a specialist at Carolina Biological and he thinks this would be a good dilution to read under a cellometer for Ankistrdesmus falcatus sample. So if I get a 50 count on the cellometer and x that by 10,000 ,per instructions, and x that by 2 , my results would be 1,000,000 cells per ml. I would like to see what the smallest amount of algaecide is needed to effect the algae and how much time it takes. The specialist at Carolina Biological does not know about the timing but thinks a half hr / 1 hr studies are good for several hrs. I might have to leave it overnight and continue testing. I would just have to make note of it in my studies. As for the 1 to 1 dilution and times, I may have to do some trial tests as you have suggested.
Summed up like this.....
1:10 1:100. 1:1000 dilutions of spring water and Agrisept (contains all oils listed below)
1:10 1:100. 1:1000 "grapefruit seed oil extract
" " lemon seed oil extract
" "lime seed oil extract
" " tangerine seed oil extract

A ml sample of each placed with 1 ml of algae. These are the variables.
Control will be 1ml algae placed into 1 ml spring water.

Does it seem a go to you? Bobby O

[donnahardy2]

Hi Bobby O,

Thanks for sharing your protocol. It sounds excellent! . I like your use of dilutions, and it sounds like you have gotten the best possible advice on the timing for a trial run. Be sure to keep all other factors as controlled as possible, for example:

1. The algaecides should be diluted in spring water.
2. The algae for each experiment should all be from the same master sample.
3. Try to use algae that are rapidly growing, not old dying cultures.
4. Try to keep the temperature and light the same for all experiments.

Yes, definitely you should proceed.


Donna Hardy

[deleted-112020]

Hi Donna,
Thank you so much. All your advise will be used. Just waiting for my algae to arrive. Carolina biological said I have about a week to run the tests before algae begin to degrade. Delivery of algae will be on a day that I am free for several days without interruptions. Thanks again, Bobby O

[donnahardy2]

Hi Bobbyo,

You have planned your experiments very well; I’m sure you will be successful.

I have one more idea.

When you receive your sample of algae, try transferring a small amount to some sterile growth medium with light and at a temperature suitable for algae growth. Here is a recipe for the growth medium that supports the growth of Ankistrdesmus falcatus:

https://ncma.bigelow.org/node/71

If you don’t have access to the special algal growth medium, then use hard tap water or collected rain water that has been boiled for a few minutes. This will give you an additional supply of freshly grown algae if you need it for this project; or a supply for your next project.

Here is an interesting reference on this plant that I used to find the optimum growth medium:

http://www.academia.edu/1815839/Ankistr ... oductivity

Good luck. Let us know about your results.

Donna Hardy

[deleted-112020]

I did my test results and came out with some interesting results. The agrisept was the strongest of them all, then grapefruit, tangerine, lemon; then lime. I was really surprised by the tangerine results. I thought that lemon and lime would come out infront of it. I worked out percentages of cells killed for each algaecide per each dillution tested. Is there anything you can think of to add to my experiment. I made charts and graphs and have my conclusion. What do you think so far...

[donnahardy2]

HI Bobbyo,

It's great to hear from you again. Your results sound really exciting.

It sounds like you have done everything for a complete project. The only other suggestions I can think of that would distinguish your experiment would be in data analysis and conclusion. Do you have any explanation for your results? Why would tangerine be different compared to lime or lemon? Is there a difference in the chemistry, or was it just a difference in concentration of the samples you used? What experiments would you do to confirm your ideas?

Also, did you run your results in duplicate or triplicate? If so, you could do further analysis and determine if the results are statistically different compared to your control. If you have any questions on this please post your raw data.

Donna Hardy

[deleted-112020]

Hi Donna,
I read your thoughts on the chemical make up of the tangerine lemon and lime seed oil extracts. I had the chemical make up for all. What stood out was tangerine has a chemical component in it called alpha -thujone while the others lack this component. Alpha-thujone is more known in wormwood and after researching it, I found it has antibacterial qualities. All oils used seem to have some antibacterial qualities, but tangerine has this extra one. Since algae and bacteria live in symbiosis, this could be a reason why it works better. Algae gives off o2 and needs co2. Bacteria gives off co2 and needs o2. COZY!!! If there were bacteria in the sample sent to me, perhaps the tangerine effected it more than the lime and lemon!!!! This would make sense

I ran the experiment twice. I couldn't run more because the counting was making me nauseas and caused me a lot of neck pain. I hope they accept this. I wanted to run more but the thought of it made me sick.

My raw data follows. The numbers have already been multiplied by 5 to account for the 1:5 dilution factor
Cells counted must still be x10 to 4th for the cellometer per manufacturer
1:10. 1:100. 1:1000
Con. 1555/1660 1465/1550. 1475/1490.
Ag. 730/770. 1075/1025. 1075/1135
Gr. 1120/1065. 1260/1220. 1280/1300
Lem. 1260/1230. 1299/1450. 1255/1220
Lim. 1225/1270. 1339/1310. 1420/1430
Tang. 1070/1165. 1295/1280. 1440/1450

I worked out the percentages of cell loss based on the averages between the two test runs. I took the average cell count of an algaecide and divided it by the average control cell count for each dilution group.. If I got 47% for Ag at the 1:10 dilution, I said there was a 53% cell loss. Does his sound right?

I have controlled variables....lighting, temp, same lot of Ankistrodesmus introduced to each sample, time for counting, all used same spring water for dilutions.

Independent variables are the different dilutions of the algaecdies.. Used for cellometer counting ...1ml Ankistrodesmus and 4 ml of the algaecides

Dependent variable is the amount of cell death.

Control is 1ml Ankistrodesmus And 4ml spring water to use in cellometer


It is a bit involved. Does it sound Ok?

[deleted-112020]

Hello,
I was wondering if you agree with my results based on my data? I thought a testing of alpha thujone could be done for algaecide qualities. Thanks, Bobbyo

[donnahardy2]

Hi,

I apologize for the delay in responding. Your data looks great and your calculation about the cell loss is correct. Here is an additional suggestion for data analysis, if you are interested:

Here is information on the student’s t-test. This provides a calculation to determine if there is any statistical difference between two groups of data.

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

And here is a free calculator to calculate your results:

http://studentsttest.com/?i=1607%0D%0A1 ... 1105%0D%0A

I assumed that the two values together were duplicate reading of the same sample, so I averaged those and used the 3 values for each sample.

Comparing the control data points and the Agrisept results, the p value is 0.00424. In biological systems, results are considered significant if there is a less than 5% chance that results occurred by chance (p<0.05) With this set of data (1607, 1507, 1482 and 750, 1050, 1105) the p value = .004, so there’s a 0.4% chance that results could have occurred by chance. So your results show a very significant difference in the groups.

Try this calculation with the other sets of data, compared to the control.

1607 1507 1482

750, 1050, 1105

1092, 1240, 1290

1245, 1374, 1238

1248, 1325, 1425

1117, 1288, 1445

I recommend using a bar graph to display the data. Be sure and label the graph completely.

Your controlled parameters are good; include this in your discussion section.

Your identification of the independent and dependent variables is perfect.

Great job!


Donna Hardy

[deleted-112020]

Thanks a lot for your info. I plugged in the other groups. The p- values were: Grapefruit 0.9% , Lemon 1%, Lime 3% but Tangerine 7%. Does that mean the tangerine really should be repeated? If I can't repeat the tangerine tests because I had to return the microscope, should I just note this. Do you think it will hurt my chances at the fair. I could try to get the scope back. I do have my results on a bar graph. Should I make a graph for the p - values or does one just note these? Should I also note the means and standard deviations from the calculator for the p- values?
Is the result of the tangerine a reason why it seemed rather strange? Statistics are not my strong point. I read he history of the t-test and got lost after the brewery, that was interesting. Thanks again, Bobbyo

[donnahardy2]

Hi Bobbyo,

No, you don't need to repeat your results.

You did your experiment in triplicate and counted each sample twice, so your data is good. What you can say about the 7% result is that the results are not significantly different. There is a 7% chance that the difference in results could have occurred by chance. The other results are less than 5%, so the difference is statistically significant for the other samples.

If you had time, the samples, and the microscope available, you could repeat the results and perhaps the results would be different or the same. The science fair judges will be interested in your analysis of the data and the statistical analysis will help you with the discussion.

Your research into the chemical make up of the tangerine with its unique chemical, alpha-thujone should be included in the discussion also. Your results, of course, don't confirm that this chemical is a good algaecide, but its certainly a topic worth further investigation in next year's science fair project, perhaps.

I hope this helps.

Donna Hardy

[deleted-112020]

Shout out to Donna Hardy for everything that she's done. Placed second at South Jersey Science Fair. MOVIN ON TO DEL VAL!!! WOOT!!!

[donnahardy2]

Hi Bobbyo,

Congratulations! This is great news. All of your hard work was definitely worthwhile. Thanks so much for taking the time to let me know about the results of your science fair.

I have another suggestion now that you are a winner. Did you receive any comments from the judges on how your display board could be improved? Did they seem to be confused by any of the information you presented? If so, you can take advantage of the time before the next science fair to revise your board. If not, then don’t change anything.

Good luck!

Donna Hardy

[deleted-112020]

Hi Donna,
Did not place at DelVal. The judges asked why I didn't check the pH of the water and the essential oils that made up the agrisept along with the agrisept. These could have impacted the algaecidal qualities. I guess that's the way it goes. Bobbyo