# BBA some exploratory experiments



## dukydaf (Dec 27, 2004)

I am recently experiencing a renewed bloom of BBA in my aquarium. So now that I have the raw material I can plan the experiments. 

General details
After I pick mature BBA from the aquarium I put them into small covered glass containers. They will be monitored and photographed. I will consider them growing if they are increasing in size or new algae colonies are observable. I will consider them "dead" if the algae is white-transparent or red and does not recover after 1 week. The light will be coming from a west window ( spectrum and amount does not matter for these experiments). All the water has a starting concentration of :
NO3 15 ppm, PO4 2.7 ppm, 11.2 ppm, 0.4 ppm Mg. Tap water has a reported GH 13, KH 11.


Controls: 2 bottles with tap water exposed to light
Experiment 1: Simply keep the algae samples in the dark for 3,5,10,15,20 days. After the darkness period re-expose to normal light and observe.

Experiment 2: Put algae in pure RO, 1/2 RO-tap, GH=4 KH=0

Experiment 3:Treat with Easylife Carbo at 1x, 2x, 5x, 10x

The problems with this experiment incluse the lack of organic substances from a normal aquarium. Lack of water movement... accepting donations to buy mixers or agitators.

Do you see any problems or confounders ?
What other tests would you like to add ?


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## fablau (Feb 7, 2009)

Very interesting Mihai. I'd also try to test the samples in very high nutrients rich water (like double EI?) and in water with saturated Co2. I'd also try to put the samples in water rich of organic waste to see if it grow more than in clean water. Very curious to see the results!


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## g4search (Aug 10, 2014)

Mihai, 
it sounds good what you are planning. However, I just want you to know that I have tried several times to "transplant" BBA and make them grow in different containers. Unfortunately, I was never successful, even if I moved them within the same aquarium they came from. They just melted away. I have the feeling BBA has to grow from spores, and once they are in the growth-phase they can't be "uprooted" and grown elsewhere.


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## dukydaf (Dec 27, 2004)

Thank you very much for your support and feedback. Keep it coming. :grin2:


fablau said:


> I'd also try to test the samples in very high nutrients rich water (like double EI?) and in water with saturated Co2. I'd also try to put the samples in water rich of organic waste to see if it grow more than in clean water.


Thank you for your great ideas @fablau. The one with 2x EI water is easily done, I will try to start a sample. Saturated CO2 and water rich in organic waste are a bit harder to do and measure. Maybe after this round of experiments ends I will have some good / easy ( read lazy) solutions to conduct the experiments.



g4search said:


> Mihai,
> Unfortunately, I was never successful, even if I moved them within the same aquarium they came from. They just melted away.


Hmm, interesting input @g4search. I agree ideally I would expose established colonies. Maybe I can put some chopsticks in the aquarium and wait for the algae to colonize them. Afterwards, I can cut it in smaller pieces and expose the established colonies. What do you think about this ?
I tried to have at least one complete unit in one treatment. A mature “unit” can release spores, but it is not realistic to wait for them to grow (time and glass). Also, this is one reason I have the control sample. It has tap water with EI. If the algae there is still alive and the one in the treatment bottle dies, then we can suggest that the treatment is responsible for the death. 

Today I exposed one sample to light after a 3 day lights-out. It is still alive. I will come back with a post with photographs showing the interim results.


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## fablau (Feb 7, 2009)

Thank you for testing this! I think your idea of using chopsticks could work. You just need some sort of "media" where BBA would attach, and then you can move it whenever you want to.


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## PAXpress (Oct 19, 2015)

g4search said:


> Mihai,
> it sounds good what you are planning. However, I just want you to know that I have tried several times to "transplant" BBA and make them grow in different containers. Unfortunately, I was never successful, even if I moved them within the same aquarium they came from. They just melted away. I have the feeling BBA has to grow from spores, and once they are in the growth-phase they can't be "uprooted" and grown elsewhere.


What if you were to shave the wood underneath it and move that?


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## g4search (Aug 10, 2014)

PAXpress said:


> What if you were to shave the wood underneath it and move that?


I don't know it may work.

Bump:

@dukydaf - it may be possible to keep BBA alive once it grows on something solid that can be moved. That way, I would do this by sacrificing a plant leaf that has BBA growth at the border. Cut the leaf in pieces so that you have some BBA on each piece. The problem may now be that you have to suspend that piece in a way so water flows around the leaf evenly.
I am curious to know whether BBA stays alive under these conditions!


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## dukydaf (Dec 27, 2004)

*Warning: Long post*



PAXpress said:


> What if you were to shave the wood underneath it and move that?


This would work. At the moment, most of the algae growth is on woodrock so I need to grow them on something else.



g4search said:


> I don't know it may work.
> That way, I would do this by sacrificing a plant leaf that has BBA growth at the border. Cut the leaf in pieces so that you have some BBA on each piece. The problem may now be that you have to suspend that piece in a way so water flows around the leaf evenly.


Thank you for your ideas @g4search. At the moment the BBA on plant leaves are very small colonies. Adding damaged leaf bits into the samples adds increased organic material :confused1:. Since we still suspect that spread of BBA is due to high organics, this approach might introduce a big confounder. I am preparing my chopsticks at the moment, waiting for the fungal bloom to pass .
Also, please note that the sample bottles hold about 30ml of water ( often I fill them with less). Stratification is not relevant in this volume. I shake the bottles once or more per day. Ideally I would put them on a shaking plate, like in the lab. But I need funding for that :grin2:

Anyway, it seems to me that the control bottle still looks good.
Here are the results for Experiment 1:
DAY4


No real difference that I can see yet:|, but it is still early. All algae have the same intense color.

Experiment 2:
DAY3

Easylife Easycarbo seems to be an effective way to get rid of this algae, algae is more light coloured. You can notice some dosage related effect. 5 times the recommended dose and 10 times the recommended dose seem to be the more effective than the recommended dose. At this point I stopped the treatment on all bottles, see if the algae dissolves or recovers. At Day 5 the effect of EasyCarbo is more obvious, all treated samples were white-isch almost transparent, unlikely that the algae recovers. Meanwhile the control is blue with a greenish tint, very dense and healthy looking.
Easylife Easycarbo is something like Seachem Excel, but more available in EU. It is produced by a company from the Netherlands. If fish are still in the aquarium it is unlikely that one can dose 10x or 5x the recommended dose. However, it was surprising for me to see that even a 1x dose is enough:surprise:. In my experience a little over 2x is tolerated by fish. *1x is 2ml of product in 50L. I started spot dosing with 2x in my aquarium and algae in the region turns red in less than 24h.

That being said, It would be interesting to see if it has an effect on the BBA spores, that is to say if you stop the treatment do spores already in the water mature and develop into visible BBA.

----------------------------------------
Description of Photography Method

Since it is easy to manipulate photographs I include here the full disclosure of what settings and changes were done. All photographs were exposed to the same treatment.

Camera settings: D90 with 18-105 Nikon lens, Exposure 1/60 at f/10, ISO 200 with external flash attached on camera (TTL), WhiteBalance Manual 6670K saved to RAW.

Software: Adobe Lightroom 
Preset: Zeroed, Temp: 7100, Tint: +1, Exposure: +0.50, Clarity: +41, cropped
Joined in Photoshop and saved as TIFF, exported from Lightroom as JPEG.

If required I can release the full RAW files so they are reviewed for manipulation.


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## fablau (Feb 7, 2009)

Wow, what an excellent work you have done here! Do you know the dosage conversion of Easy Carbon to Excel?


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## kevmo911 (Sep 24, 2010)

Sewingalot liked to experiment with her tanks. Not sure if you've seen this thread:
http://www.plantedtank.net/forums/23-algae/125230-bba-loves-low-light-too.html

I figured you might find it interesting if you hadn't seen it yet. Keep up the good work!


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## Oso Polar (Apr 22, 2015)

I used 5x dose of Excel for about two months in a tank with fish (lots of young bettas) and plants trying to control BGA outbreak. Everything was fine - both fish and plants (mostly crypts) did well. BGA stopped spreading from day one of treatment and slowly started to retreat but didn't disappear completely even after about 2 months of such dosing.


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## g4search (Aug 10, 2014)

Oso Polar said:


> BGA stopped spreading from day one of treatment and slowly started to retreat but didn't disappear completely even after about 2 months of such dosing.


hey @Oso Polar, this thread is about BBA not BGA (cyanobacteria). The ONLY way to kill BGA completely is by using *Erythromycin*. Everything else works only temporarily!


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## g4search (Aug 10, 2014)

@dukydaf, Mihai, I was under the impression that you actually wanted to "grow" BBA, so you could eventually treat the growing BBA to figure out how to prevent further growth (or perhaps stop sporulation). This would be the most rational way for you to actually control BBA. 
My interpretation of your current experiments is that you are treating various trans-located BBA specimens to see which one gets more discolored? (as indication of DEAD specimen?) I'm sure that you can keep your "control" specimen for 3-4 weeks without notable changes, but the REAL question is: is it still truly alive? (look at it as cut flowers. You can keep them in a vase for one or two weeks, but eventually you have to throw them out because they are dead)

You know, I think just about everybody on this site here know how to kill BBA ( e.g. spot treatment w glut or H2O2 [or hypoclorite]). I am sure there are many more "methods" to kill BBA. It is actually not very difficult! Prevention on the other hand, is much much harder. ( Theoretically, if you could keep BBA spores out of your tank, you would NEVER have a BBA problem. That seems to be unrealistic however.)

So the only other route then is to control its growth. There are certainly many factors that have to be considered. I my opinion, LIGHT using the proper wavelength spectrum is the most important factor! At this stage, what I can tell you is that light at wavelengths around 410 nm (at what intensity?) are absolutely necessary for BBA to grow.

BTW, with respect to your pictures here, they are excellent! I am sure that nobody here would ever question whether they are doctored.


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## dukydaf (Dec 27, 2004)

First a small UPDATE:
No additional Easycarbo has been added and the treated BBA turned snow white by now. Still keeping it to be sure. The control is still looking strong. 
I started some organic rich experiments. Shrimp pellets, fish food, ground up H. pinnatifida leaves. Also found some leaves with BBA on them and used them. No growth on the chopstick, otos love to hang there.

@fablau I don't think you can calculate it because Easycarbo doesn't give any active ingredient or concentration.

Thanks for the link @kevmo911 , it was an interesting read with somewhat similar speculations.



Oso Polar said:


> I used 5x dose of Excel for about two months in a tank with fish (lots of young bettas) and plants trying to control BGA outbreak.


I think what Oso Polar said here is that a 5x dose of Excel does no harm to bettas and crypts. Thanks for the input. Keep in mind bettas are pretty hardy fish and can take oxygen from the air. I would be careful in dosing the same amount with shrimps and otos. You can see them run away from the place where you dose even 1x.

@g4search I will set up some experiments keeping your point in mind.



g4search said:


> I was under the impression that you actually wanted to "grow" BBA, so you could eventually treat the growing BBA to figure out how to prevent further growth (or perhaps stop sporulation). This would be the most rational way for you to actually control BBA


So if I understood you correctly: The aim of an the experiment should be to find relevant factors that either limit the spread of BBA or induce death of existing colonies.
These factors should not be additional chemicals and should be relevant to an aquarium with growing aquatic plants ( eg. exposing the BBA to air for 24h is not good because it would kill plants).

Another experiment is to find relevant factors that promote the germination of BBA from spores.

(These need to be separated. It may be the same factor it may be different. For example, understanding the factors that contributed to one getting the flu does not mean that by eliminating those factors the person would be healed.)



g4search said:


> You know, I think just about everybody on this site here know how to kill BBA


To my knowledge there is little information regarding the use of Easylife EasyCarbo on the forum. I suspected it did the same thing as Excel to BBA, but wanted to try it out and see for how long and how much. It came as a surprise that just 1x was enough to kill BBA in 3 days. I am redoing this experiment to confirm my result.



g4search said:


> I'm sure that you can keep your "control" specimen for 3-4 weeks without notable changes, but the REAL question is: is it still truly alive? (look at it as cut flowers. You can keep them in a vase for one or two weeks, but eventually you have to throw them out because they are dead)


Well, we need laboratory equipment to determine if they are alive or not for sure. Another way to verify if they are alive is to propagate them (NOT multiplying does NOT mean they are dead). I think there is little reason to assume the algae died at the moment it was removed from the rock. I think your comparison to the flowering meristems of a terrestrial macrophyte is not the most adequate, it is to specialised. Would say that a leaf of H. corymbosa is dead or dying? You can actually grow a full plant from it. However, it is still possible that the spore of BBA gets buried deep inside the rock and cannot be removed and is essential to the regeneration and propagation of the BBA colony.


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## Oso Polar (Apr 22, 2015)

dukydaf said:


> I think what Oso Polar said here is that a 5x dose of Excel does no harm to bettas and crypts.


Yeah, exactly, I just wanted to say that at least some fish and plants can handle 5x prolonged Excel overdosing without any harm to them. Chocolate gouramis don't mind permanent 2x Excel dose (I just didn't try higher) - and they are pretty delicate.


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## dukydaf (Dec 27, 2004)

Hello fellow algae growers, 

Though it was time for a weekly update. In short: easy carbo 1x kills BBA detached or on leaf. Differences in water type is inconclusive. Putting BBA in the dark for one week does nothing. Experiments with BBA and organic matter were inconclusive (yet).

Experiment 1: Day 14

No difference in the way algae look. But there is some string algae developing in the RO water and 1/2 tap.


Experiment 2: Day 14 - only control and 1x shown



8 days after stopping the experiment, the 1x sample is still dead white. Control still going strong. All other samples looked like 1x.


Experiment 3: Day 14


Nothing different in the algae held in the darkness. 

Experiment 4: Day 7

Experiment 4: Description 
Taking into consideration the feedback received I tried to experiment on algae on leaves. I tried to expose the algae to organic material. 

Leaf extract = 5 leaves of H. pinnatifida grinded in 10ml water.(not a scientific way to measure but should still release a lot of what is in the leaf)
Crushed solution= 2ml of leaf extract in 8ml water (w/ macros, see first post)
crushed 1/2 = 1ml of leaf extract+9ml water

I also tried the CO2 experiment once again (1x), in water with no organics and in water with organics.

Experiment 4: Results

It is a little difficult to see in the photos from Experiment 4. In the Easycarbo 1x treated bottles (marked with CO2), both the freestanding algae and the algae on the leaves was dead. In crushed 1/2 and crushed samples with BBA on Althernathera, the algae is still there, not necessarily growing but healthy. In the "crushed" sample with no leaf, the detached colony of BBA is dead and white. 

In sample(not shown) with leaf extract and BBA on H. pinnatifida leaf, the BBA turned white and now only low patches of BBA are visible (looks like GDA but with the color of BBA).

Maybe H. pinnatifida has some algae killing toxin in the cells that was in high enough concentration. I will try with snail poop and fish food next, see if I manage to get some rapid propagation. The problem that I see is that green algae also develop in these conditions and might outcompete the BBA.


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## fablau (Feb 7, 2009)

Very interesting results! Pretty much what we could expect with Easycarbo. About your mentioned sample with leaf extract and BBA on H, what does the "H" mean? 

Thank you for doing this!


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## Solcielo lawrencia (Dec 30, 2013)

Hygrophila


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## fablau (Feb 7, 2009)

Oh... Thanks!


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## g4search (Aug 10, 2014)

Mihai,

I wonder what you can do to show me that the BBA in your "control" experiment is actually still alive? 

(Just because the color didn't change, does not mean the organism is still viable)



Looking forward to your next set of experiments!


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## fablau (Feb 7, 2009)

Mihai, another interesting experiment to try is to see resistance of BBA to toxicity levels of CSM+B. Maybe at different concentrations? We are discussing about that on this thread:

http://www.ukaps.org/forum/index.ph...uses-BBA?-Part-2---Bacterial-imbalance.38375/


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## dukydaf (Dec 27, 2004)

Some short news first:

The chopstick in the aquarium is beginning to be colonized by BBA, many small colonies at the moment. Took about 2 weeks. I will keep the stick in there so that they continue to grow.

The control sample for fish food was severely contaminated and I need to restart the experiment.

The sample I kept in the dark for 23 days looks fine, like nothing happened...



g4search said:


> I wonder what you can do to show me that the BBA in your "control" experiment is actually still alive?
> (Just because the color didn't change, does not mean the organism is still viable)
> Looking forward to your next set of experiments!


Thank you @g4search for closely following these experiments and being skeptical about the results. What do you mean by viable ? If anybody sees any problems with my reasoning or methods please let me know. Better to have doubts in the right places than be ignorant. Ah, what is alive and what is dead ? Very hard to define life if you think about it, but I will try to do so with BBA. 

As previously stated, to undoubtedly prove that something is alive we need establish that it is capable of metabolic activity and multiplication. For algae that would mean photosynthesis during light hours, maybe measured as oxygen production, and spore production. I don't have the equipment necessary at home to measure that, so I use surrogate endpoints. Would be nice to have a lab though . Multiplication was not yet observed and may come from contamination with spores already available. 

Example of surrogate endpoint: long term integrity of the cell and cell organelles under normal conditions as a good surrogate for a live cell. For me this is a good surrogate. Very few organisms manage to maintain their structural integrity for a long time after death (again, normal conditions). Additionally, pigments like chlorophyll would be one of the first to degrade if the organism has died so if we observe no chlorosis (transparent/white leaves) we can assume there is still metabolic activity.

Another way of thinking, less scientific but practical nevertheless is .... does it look like a dead BBA ? Does it act like a dead BBA ? Do you have any reason to assume it is a dead BBA ? If no, assume it is not dead.

I didn't specialize in algae, so maybe somebody with more information on algae physiology can comment. Do we have any reason to think that the moment a BBA colony is detached it dies ?

Now your turn  Can you prove this BBA colony is dead ?




fablau said:


> another interesting experiment to try is to see resistance of BBA to toxicity levels of CSM+B. Maybe at different concentrations?


Thank you for the link to thread, didn't have time to read all of it. I like the idea behind the bacterial population as an intermediary, but of little relevance to our aquariums. It is an interesting theory, though I highly doubt BGA would be affected by CSM levels that are not toxic to plants and invertebrates. I don't have access to CSM+B in the EU but can test it with another trace mix such as JBL Ferropol.

More coming soon.


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## Solcielo lawrencia (Dec 30, 2013)

I just want to throw in an observation about BBA. BBA has always stayed short in my tank; it's never grown tall. However, after reducing/ceasing CSM+B dosage, the BBA grew twice as tall! Thus, micronutrient toxicity does negatively affect BBA growth patterns.


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## fablau (Feb 7, 2009)

Solcielo lawrencia said:


> I just want to throw in an observation about BBA. BBA has always stayed short in my tank; it's never grown tall. However, after reducing/ceasing CSM+B dosage, the BBA grew twice as tall! Thus, micronutrient toxicity does negatively affect BBA growth patterns.



Solcielo, did the BBA eventually disappear later on? Could that me an effect of toxicity levels in some way?


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## Solcielo lawrencia (Dec 30, 2013)

fablau said:


> Solcielo, did the BBA eventually disappear later on? Could that me an effect of toxicity levels in some way?


It'll take a couple of weeks to know for sure. However, the growth that is there is much slower and fewer. CO2 levels are under 25ppm. CO2 turns on with the lights so there is a gradual concentration increase over the photoperiod. So the correlation between fluctuating CO2 levels and BBA? This is strong evidence against it. Speculation: it's possible that a micronutrient toxicity is what induces BBA growth.


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## fablau (Feb 7, 2009)

Ok, please, keep us posted. I am doing pretty much the same experiment and I will post my own results. I also have noticed taller growth of BBA after reducing CSM. Some common grounds here!


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## mistergreen (Dec 9, 2006)

Can you experiment on pH?
My hypothesis is that BBA has a harder time in low pH.

Tanks with CO2 is usually low in pH.


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## dukydaf (Dec 27, 2004)

So my BBA ridden chopstick is almost ready. There is some algae (other than BBA) contamination on it that worries me. 
It is interesting to see that this algae grows at a gradient toward the light. The stick is at ~ 45° with soil/glass. Almost no BBA grew on the underside. More BBA grew at the top of the stick than towards the bottom, speaking in terms of visible colonies.


Solcielo lawrencia said:


> However, the growth that is there is much slower and fewer. CO2 turns on with the lights so there is a gradual concentration increase over the photoperiod. So the correlation between fluctuating CO2 levels and BBA? This is strong evidence against it. Speculation: it's possible that a micronutrient toxicity is what induces BBA growth.


Strong evidence it is not. If this is the same as the CSM+B thread, there are only confounded and very subjective observations. Many things, other than CO2 may have affected the growth rate of the BBA in your aquarium. Good to hear it is slowly going away.
It may be possible that high CO2 is one factor that suppresses spore germination. I myself do not think the variation of CO2 levels is correct, but that is just a personal belief.
A toxicity would damage the leaves of plants, which is a great spot to germinate as a spore. So there may be an indirect relation.


mistergreen said:


> Can you experiment on pH?
> My hypothesis is that BBA has a harder time in low pH.
> Tanks with CO2 is usually low in pH.


But BBA also grows in aquariums with CO2/ low pH. Nevertheless, it is a good starting point. Yes, I can experiment with pH levels but they will be lowered by some other acid , not CO2. I think I will be able to start at the end of December. 

Thank you for all the great ideas and feedback guys. If you keep this up I will have to fill my aquarium with chopsticks


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## Audionut (Apr 24, 2015)

Other acids will require the reduction of carbonate concentration to reach a lower pH endpoint. CO2 will affect H+ concentration without (significantly) affecting CO3 concentration.

With these samples it should become clear whether it's H+ concentration, CO2 concentration, CO3 concentration or some combination that has the greatest effect on BBA.



High H+, low CO3
Neutral H+/OH-, low CO3
High OH-, high CO3
High H+ (high CO2), low CO3
High H+ (very high CO2), high CO3
Neutral H+/OH- (high CO2), high CO3
High OH- (no CO2), high CO3

The first three samples would have CO2 concentration at equilibrium with the atmosphere. Lots of surface agitation will help to ensure consistent CO2 concentration, and minimize any effects on CO2 concentration from the transformation of CO3 to HCO3 to H2CO3 <> CO2 + H2O. Ideally the acid injection would be maintained with CO3 injection to maintain a steady concentration of both, however, given the (overall) sample size, a reasonable conclusion can probably be obtained even with samples varying to some degree around a base concentration. If you're after ideas, I would setup an acid solution and bicarbonate solution with some form of consistent (think minutes, not days) dosing. A balance of dosing should be found to target pH endpoint, where monitoring of KH and pH will inform of correction needed.

If KH low and pH drops > increase HCO3 injection.
If KH correct and pH drops > reduce acid injection.


The next three samples would be significantly easier to control since CO2 injection is easy to maintain consistently, and doesn't affect CO3.

The last sample would be extremely difficult to control (read: I don't know how), since I know of no way to remove CO2 from water with stability over time. A large plant mass could probably easily reduce CO2 concentration, however the concentration would be variable with variable light. edit: Actually this variability in concentration probably isn't such a bad thing since it will more accurately represent real life conditions.


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## mistergreen (Dec 9, 2006)

You can add peat moss into the container to get steady acid, H+.


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## Audionut (Apr 24, 2015)

A form of peat extract would be better imo.

There is some consensus that it is the variability of things that promotes algae, and hence any experiments to determine the effects of a thing need to control variability. Otherwise we don't know if it was the concentration of the thing that had the effect, or the variability of the thing, or some combination.


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## mistergreen (Dec 9, 2006)

Yeah, maybe vinegar might be better since tannins will block out some light.


Sent from my iPad using Tapatalk HD


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