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Discussion Starter · #1 ·
I'm currently a Chem major at the University of Pittsburgh. In my instrumental lab this year, we're given three four hour lab sessions to carry out an independent experiment of our choosing. As I am an avid aquarist (word?), the first thing that came to mind was something involving aquaria.

I have access to some pretty advanced instruments including ICP-AES, Mass Spec., GC, and ATR-FTIR among others.

I was wondering if anyone here would have any ideas on an experiment or set there-of regarding aquaria that would contribute to the knowledge base. I could waste this opportunity to measure iron concentration in tap water or something elementary like that, but I'd much rather use it to contribute something meaningful.

One idea I had was to find concentrations of nitrogen, potassium, phosphate, and various other trace elements in various aquarium plants. Probably a per volume deal. The idea would be to see what plants are actually using for growth, what they aren't using, and some basic inferences as to what might be able to do differently regarding fertilizing and such. One idea would be to (and I would have to start this soon as my time in the lab would be in November) have various small tanks with equal amounts of lighting, CO2, substrate (probably inert, as I don't have a lot of time to carry this out) and dose each tank differently, as well as a tank that isn't dosed at all (control). Finding the concentration per volume of the various fertilizers in the plant tissue would give a rough idea of how much of each fertilizer was actually used and how much was wasted. There are various kinetics and processes that probably can have a big impact on these variables, (flow past the plant, plant movement, lighting cycles,) But that would be beyond the scope of what I would be able to accomplish.

I'm not a biologist, and the chemistry behind these processes can be simple or complex, but I'm willing to give it a try.

The point of the post, which is long overdue, is to get feedback and perhaps other ideas on how I could use this time. Is there a specific problem regarding the chemistry of aquaria that would benefit the community as a whole, or even a curiosity of yours that would be interesting to explore? Something regarding algae, lighting conditions, impact of fauna on a planted system, CO2 chemistry regarding dissolved organics, fertilizer composition, whether wet or dry, commercial or home made, DIY yeast CO2 versus pressurized, all are fair game.

Fellow members, the search for the ultimate truth may very well be upon us; "Why is algae such a jerk?"

Thoughts, ideas, criticisms and snarky comments are all encouraged and appreciated.
 

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I like your idea, i'd say go with that. You'll have to get things setup quickly though as you said your lab time is Nov.

I'd use the same commonly found plants in all the tanks. Amazon sword, some type of crypt, anubias, camboda, ludwigia, etc
 

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How about?... The effects of tapwater pollutants on aquarium fish reproduction.
not sure he would have time to test that in just one month. to do a thorough test you'd need tap water from various water providers as well.
 

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It would be interesting to see a comparison of what N/P/K bearing compounds are present, and in what proportions, in a non-planted vs. a planted tank, both with fish. Might give some insight into why plant growth suppresses algae, even when all these nutrients are present in non-limiting amounts. I've long suspected that only specific nutrient forms, produced from organic waste, act as algal growth triggers; while the ferts we add do not.
 

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If you're testing anything to do with plant growth you'll need all fast growing specie. I do like DC's idea of non-organic vs organic (natural) but not sure that can be accomplished in the time frame you have available.

Also with the time limit at hand I assume you need to be sure you can reach conclusive results for the project to be a success. (good grade)
 

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Get a planted tank growing good, with CO2 and good fertilizing, and appropriate lighting. Then add measured ppm of nitrates, phosphates, potassium, and a trace element mix plus chelated iron. Measure the concentration of each of those elements vs time for 2-3 days or more. Repeat a few times. That should give you data on how fast the nutrients are used up by the plants, even though it would be applicable only to the plants you use. (That might make it best to limit the tank to one type of plants, or use two tanks, each with one kind of plant.)

This stuff has been done before, but rarely with plants that we are really interested in, like HC, hairgrass, vals, etc.
 

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Beam different spectrums of t5 lights at plants/algae and measure the change in carbohydrate levels in their tissues.

I know this has been done, but maybe if you post your results we can put this whole idea that you need a specific spectrum for plant growth out to pasture.
 

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Ask Tom Barr. He does this sort of thing at UC Davis with aquatic weeds.

I think your question could be interesting. Most of the others that have been suggested are less chemical and more horticultural, and would be hard to do in a lab like yours. Stick to questions best answered with the fun fancy tools you have :)
 

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Discussion Starter · #10 ·
A lot of great food for thought here, thanks!

A lot of the limitations and merits of an instrumental approach to chemical analysis lies in the resolution these machines are capable of. Ppb and ppt (parts per billion and parts per trillion respectively) are common to see.

A healthy aquarium, as we all know, is an extremely complex ecosystem. Plants, fish, and all sorts of bacteria are all doing their own thing chemically speaking. A lot of these processes and reactions involve any number of intermediate reactions, meaning that at the ppt range, you're looking at possibly hundreds of organic compounds in the matrix. While I would love to take a vial of water it, pop it in the GC-Mass spec., (I may have tried this >.>,) the results are going to be muddled as I really don't know the extent of the compounds in the water.

With that said, I've noted and expanded all the ideas above and will run them by my instructor, who is a very, very competent analytical chemist, and see what she thinks.

I would love to solve the algae dilemma, but there are a ton of variables that influence growth and propagation of algae. Surface area, light, porosity of the surface etc. This really is beyond my scope and means. Focusing this down to water chemistry would be doable, but I think, in the long run, it wouldn't be very meaningful.

Regarding fish reproduction, a lot of variables in that too, and since I haven't really attempted breeding beyond random luck, I doubt I would have the expertise to make that very meaningful either.

I'll have to give the "optimum growth spectrum" idea some thought. That's something that's interested me in the past and, though it may not prove as conclusive as I may like, would add to the data pool.

I'm still leaning towards my first idea. I really think that if one was able to say, hey plant x uses y amount of z to grow n amount of leaf/stem area, it would give some interesting insight into methods of fertilization. Also, if I can manage enough combinations of ferts, we may be able to see how certain elements/compounds affect the uptake of others. I.e. plant p with x amount of iron available had g amount of potassium per tissue volume, where as plant 2p had less iron available and absorbed more/less iron. We might be able to see that if you're missing nutrient x, nutrient y is that much less usable.

Again, I'm not a biologist and I think that's the reason this may be harder than it needs to be, but I'm willing to try. The only thing their looking for grade wise is that your method and technique were sound. I could propose analyzing the amount of uranium in filter impellers, and as long as my technique was sound, they wouldn't care.
 

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Discussion Starter · #11 ·
Ask Tom Barr. He does this sort of thing at UC Davis with aquatic weeds.

I think your question could be interesting. Most of the others that have been suggested are less chemical and more horticultural, and would be hard to do in a lab like yours. Stick to questions best answered with the fun fancy tools you have :)
Hmm, perhaps I'll shoot him a random email. I'm sure he gets fifty million a day, but it's worth a try.
 

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Discussion Starter · #12 ·
I spoke with my instructor regarding this project today. She seems confident that I'd be able to carry out elemental analysis on plant matter with a high degree of success. I'm setting up the procedure as follows.

I'll be setting up any number of small tanks, (~1 gallon), each with equal amounts of light, inert gravel, and water movement. I think aeration will keep a decent enough equilibrium with the air to introduce enough atmospheric CO2 for average plant growth. If I had a CO2 rig, I would throw that parameter into the equation, but this is probably cost restrictive. I will dose each tank using different combinations of the standard fertilizers EI recommends, as well as one control, with standard EI dosing, and one reference control, with no dosing (I fully expect this tank to show extreme signs of deficiency and probably not make it through the month.) 50% water changes each week and re-dose as per the tank's specific nutrient mix. At the end of a month, a leaf and stem section will be removed from each plant and frozen.

I'm still working on the digestion method I'll be using, but that's probably something no one here is interested in. Using AES-ICP (Atomic Emission Spectroscopy using Inductively Coupled Plasma), I'll be able to see the exact amount of each atomic species that was present in the plant matter.

Now, a question to you kind folks. I currently don't have any dry fertilizer as I haven't started dosing per EI on my personal tanks. Would anybody be willing to contribute a small amount of dry fertilizers? I can pay shipping, I just don't want order a pound of each, even though I'd probably end up using it.

Also, if everyone could give me an idea of how much they dose and on what days, or a link to a standard reference that would give me the amounts suggested, it would help me get an idea of what dose amounts to choose.

Thanks!
 

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Good to see this moving ahead!

You could use DIY CO2, and it's inexpensive; but the fluctuating levels would probably introduce an unwanted variable.

You can scale down the EI dosing recommendations from the link above for 1G. You probably have accurate scales at your disposal, and we can find volume-to-weight conversions if you choose that method.

Or you can mix liquid solutions, which being more dilute, would be easier to measure. In that case, just make sure you don't mix the phosphate with the CSM+B, as the iron will react with the phosphate and precipitate out. The EDTA chelated iron will also precipitate out at high pH. To completely eliminate that possibility, use distilled water and adjust it to 6.5 pH with hydrochloric acid prior to mixing in the CSM+B.

I'll be happy to donate the necessary dry ferts. Once you get it figured out, send a PM with address and required quantities.
 

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I like your original idea and I'm glad you are going with that. I think the data might be useful, simply b/c it's unknown. I'm definitely going to be following this thread. I hope you get a good grade!
 

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I'll be setting up any number of small tanks, (~1 gallon), each with equal amounts of light, inert gravel, and water movement. I think aeration will keep a decent enough equilibrium with the air to introduce enough atmospheric CO2 for average plant growth. If I had a CO2 rig, I would throw that parameter into the equation, but this is probably cost restrictive. I will dose each tank using different combinations of the standard fertilizers EI recommends, as well as one control, with standard EI dosing, and one reference control, with no dosing (I fully expect this tank to show extreme signs of deficiency and probably not make it through the month.) 50% water changes each week and re-dose as per the tank's specific nutrient mix. At the end of a month, a leaf and stem section will be removed from each plant and frozen.

I'm still working on the digestion method I'll be using, but that's probably something no one here is interested in. Using AES-ICP (Atomic Emission Spectroscopy using Inductively Coupled Plasma), I'll be able to see the exact amount of each atomic species that was present in the plant matter.

Now, a question to you kind folks. I currently don't have any dry fertilizer as I haven't started dosing per EI on my personal tanks. Would anybody be willing to contribute a small amount of dry fertilizers? I can pay shipping, I just don't want order a pound of each, even though I'd probably end up using it.

Also, if everyone could give me an idea of how much they dose and on what days, or a link to a standard reference that would give me the amounts suggested, it would help me get an idea of what dose amounts to choose.

Thanks!
Do you have stable isotopes and do you have access to 15NO3 or 15NH4? These tend to be good jobs later also, particularly for ag and environmental work. HPLC is a good method also, sort of a PITA, but used fairly often and good method to have in your tool box, mass spect obviously........

Rather than doing a submersed plant, you might just do a nutrient solution in a flask with a tight fitting sponge around the stem to prevent air/in/out. This way you can make CO2 independent.

This way the nutrient solution is the dependent variable.

A simple test would be to use a hydroponic plant(you chose a small suitable stem plant) and add say 5 treatments: NH4, NH4 + NO3(@ 2 different ratios) and 2 controls(one with a Hoagland's solution and then one with DI water).

Then measure the chemical make up of your choosing. It could be protein, Total N, Fe, Chlorophyll, could be plant growth regulators like ABA, Auxin, you name it. Plants make a lot of chemicals. You will never run out of work there.
 

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Discussion Starter · #17 ·
It's great to hear from the plantbrain himself. :-D

Unfortunately, the only reagent grade chemicals the university will provide is what I would need for the analysis itself. I'd have to bankroll anything needed before the actual analysis, which isn't really doable. So tools like isotopic labeling is really out of the question.

In regards to the nutrient flask proposal, I was really aiming for something applicable to aquaria directly. My instincts tell me that if I were to take a stem feeder, such as the one I have in abundance (Hygrophila corymbosa ''Stricta''' I believe), and put it in an immersed situation, it's uptake would be altered. I assumed that these plant are able to uptake nutrients through leaves and stem. Without having the full area of leaves and stems in solution, I tend to feel like the uptake of these would be different from the entire plant being submerged.

I really appreciate your post. I would love to get more involved into the biology of the matter. Investigating the various variables as you presented them would interest me greatly, but it's not really within the scope of what I can reliably accomplish.

If, given the parameters in my latest post, I were to aerate each solution equally, wouldn't the [CO2] be a constant. Not to assume this rules out CO2 in the equation, but it would rule it out in the ratios of uptake of the various nutrients in each solution. So to say, the plants may grow slowly, but the rate of growth and uptake of nutrients will be limited equally by that concentration.

I know you're probably a very busy scientist, hence me foregoing the "random email" I mentioned before, but I truly do appreciate the input from a scientist working on real problems. (I'm currently working with a research group that's investigating molecular lithography via DNA localized catalysis, completely off subject from anything this forum relates to, but I spend hours out of each day investigating and pondering the implications and possible solutions of the research.) Anywho, thank you.
 

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It's great to hear from the plantbrain himself. :-D
In regards to the nutrient flask proposal, I was really aiming for something applicable to aquaria directly. My instincts tell me that if I were to take a stem feeder, such as the one I have in abundance (Hygrophila corymbosa ''Stricta''' I believe), and put it in an immersed situation, it's uptake would be altered.
But....the point is to make the test fair and specific to nutrients correct? You will need to take CO2, since it is a dependent variable, highly so...........OUT of the equation.

Look at Ole and Troels article:

http://www.tropica.com/advising/technical-articles/biology-of-aquatic-plants/co2-and-light.aspx

Same light etc, but changes in the CO2 caused 300-400% increases in growth, if you are off just a tiny amount.........this will cause huge differences in your results.

I assumed that these plant are able to uptake nutrients through leaves and stem. Without having the full area of leaves and stems in solution, I tend to feel like the uptake of these would be different from the entire plant being submerged.
Most all plants can take up ferts through their leaves, not just aquatics.
But growth rates for submsered plants are lower than emergent plants.
CO2 and gas exchange is lower.

If, given the parameters in my latest post, I were to aerate each solution equally, wouldn't the [CO2] be a constant. Not to assume this rules out CO2 in the equation, but it would rule it out in the ratios of uptake of the various nutrients in each solution. So to say, the plants may grow slowly, but the rate of growth and uptake of nutrients will be limited equally by that concentration.
Constantly limiting.

See liebig's law in the above article.

You can lard on KNO3 to 200ppm of NO3, it will not make the plants grow faster if the CO2 is limiting.

What is limiting CO2 to a submersed plant?

Do you know?

Can you find a reference?
I'll let you look.


(I'm currently working with a research group that's investigating molecular lithography via DNA localized catalysis, completely off subject from anything this forum relates to, but I spend hours out of each day investigating and pondering the implications and possible solutions of the research.) Anywho, thank you.
Enjoy!
 

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If I might add a bit here.

ICP-AES might present problems with your analysis. Most AES techniques are only really useful for atomic analysis (generally metals). For K, Ca, or Fe this will be fine, but it won't do as well with N, C, or other atoms. You might have better luck with an ICP-MS if you have access to one.

Tom mentioned HPLC. I work with an HPLC all day long, and all I can say is leaks..... all of the time..... However, if you have access to an HPLC-MS (or a UPLC-MS :D) you can get all sorts of useful information for us, especially if you're willing to run an extraction to prep your sample. Depends on what kind of work you want to put into this/what you have access to.

I'm still not 100% sure what you want to look at though. Are you going through with your metal uptake analysis, or are you looking at anything else?
 

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If I might add a bit here.

ICP-AES might present problems with your analysis. Most AES techniques are only really useful for atomic analysis (generally metals). For K, Ca, or Fe this will be fine, but it won't do as well with N, C, or other atoms. You might have better luck with an ICP-MS if you have access to one.

Tom mentioned HPLC. I work with an HPLC all day long, and all I can say is leaks..... all of the time..... However, if you have access to an HPLC-MS (or a UPLC-MS :D) you can get all sorts of useful information for us, especially if you're willing to run an extraction to prep your sample. Depends on what kind of work you want to put into this/what you have access to.

I'm still not 100% sure what you want to look at though. Are you going through with your metal uptake analysis, or are you looking at anything else?
Yes, and you lose some product in the darn things, and getting the flow right etc. We have switched to ELISA test, much easier for plant growth regulators.

The other thing that you could measure is the trace metals for several sediment types for use in plant culture. AA might be a relatively easy method there, but does not address bio availability. For that, you'd need to measure the plant tissue analysis also(you'd planned to do this already.......so adding sediment might be an easier process than water column + sediment) and see if there is correlation.

Water column/CO2 etc adds more complexity to the experiment. I think taking this out of the variables would make the design of the test easier.

Do not make this too hard on yourself. You'll need to do a few rep's also.
This stuff will add up real quick and be tough to do and maintain.
 
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