Anyone want to test for phosphate binding to aragonite?

[LouH]

Lately there has been much discussion on the theories of deep sand beds vs. no sand bed or very shallow sand beds. Add to that the discussion on whether or not sand beds should be maintained or left alone so that benthic organisms can process detritus. I would like to put out an invitation to RC members to help me devise an experiment which will generate some real world data to provide to the community for consideration.

I have access to an inductively coupled plasma (ICP) machine at work, and with it I can measure elemental phosphorous. I also happen to have a reef system that has a bare bottomed display, a lighted refugium with a deep sand bed (about 4 "), and a deep sand bed (about 12") in a 20 gallon Brute garbage can that receives no direct light.

I moved into my current home in October 2012. During that move my lighted refugium was completely broken down. This included siphoning out the DSB and rinsing the aragonite before putting it back into the refugium. The Brute garbage can DSB was left intact for the move. Although I've moved my system several times over the years, I would guess that the aragonite in my system is over 8 years old if not more. During those 8 years, I've experienced a severe battle with Bryopsis, so I know that the system has been exposed to high phosphate levels. If phosphate binds to calcium carbonate (and there are plenty of studies out there that prove that it does), I should be able to measure significant levels of phosphorous bound to or released by my aragonite.

My statement that there are studies that that show evidence that phosphate binds to calcium carbonate might make one believe that I shouldn't bother with an experiment. So why would I do it? I want to do it for the following reasons:

1. I have a functioning 120 gallon artificial reef in my home;
2. I have access to both a lighted DSB, and a DSB that receives little light;
3. I have old aragonite that should be loaded with phosphate if phosphate binding occurs under the conditions found in reef aquaria;
4. I have read studies that state that phosphate binding is a very complex mechanism that is influenced by many factors including oxygen concentration in the sediment, microbe populations, and the presence of other ions that are involved in the reaction (like iron);
5. I want to see for myself what is actually happening in my system; and
6. I want to provide data to other enthusiasts so that they can make more informed decisions about how they want to design and manage their system.

I see many challenges to devising this experiment including the following:

1. There is biologically bound phosphorous in the organisms that live in the sand beds. The experiment would have to somehow strip that phosphate component away from the measurements.
2. If an acid is used to liberate bound phosphate, how do you take the data and apply it to a reef system in which the water is full of other ions and the water has a relatively high pH?
3. All samples have to be fresh water solutions, because the ICP's plasma chamber gets gunked up when attempts are made to analyze sea water (I tried it once and the lab manager was not pleased). Again, how can the fresh water solution data be extrapolated to a reef tank when the water chemistry is relatively complex (compared to distilled water).
4. If saltwater is taken from my tank and diluted so that it does not gunk up the ICP, the phosphorous concentration will likely be below the method's limit of detection.
5. I'm sure there are about 5 other challenges to overcome that I'm not identifying at this time.

So there you have it. The proposal is out there. Anyone who wants to contribute is more than welcomed to share their ideas to aid the development of the experiment. If we can get some agreement on a path forward, I'll commit to finding the time to make it happen despite the fact that my wife will hate me and I'll regret putting out the proposal.

Lou

 

[LouH]

One other thing, if this has already been done and I simply have not come across the study(ies), let me know so that I don't waste my time.

Thanks.

 

[blanden.adam]

Well, I think to move forward you need solidify in your mind exactly the question you are trying to answer.

Is the question IF phosphate binds to sand in sandbeds? Is the question does the DEPTH of the sandbed influence the amount of phosphate that binds? Is the question does the sandbed bind enough phosphate to effect the level in the water column? etc... These are all valid questions, but until you clearly define what question/questions you want to ask, it will be all but impossible to design an experiment that will answer them. Gotta know what you're looking for before you start looking if you want a reasonable chance of finding it

 

[LouH]

Well, I think to move forward you need solidify in your mind exactly the question you are trying to answer.

Is the question IF phosphate binds to sand in sandbeds? Is the question does the DEPTH of the sandbed influence the amount of phosphate that binds? Is the question does the sandbed bind enough phosphate to effect the level in the water column? etc... These are all valid questions, but until you clearly define what question/questions you want to ask, it will be all but impossible to design an experiment that will answer them. Gotta know what you're looking for before you start looking if you want a reasonable chance of finding it

Why, all of the above of course.

Likely each will have its own experiment. If I may plagiarize from your post:

1. Does phosphorous bine to aragonite under the conditions found in reef aquaria? This would be experiment 1, as all others will follow most of the same test procedure.

2. Does the depth of there sand bed influence how much phosphorous binds, or put another way, is there a gradient in phosphorous binding from the surface of the sand bed through to the bottom?

3. Does bound phosphorous go back into solution?

Better?

Lou

 

[blanden.adam]

1) I think the experiment is buy a bag of new aragonite, rinse it well, set some aside, and put some in a cup anywhere in your tank. Then wait. IDK how long, but some amount of time, maybe even several time points. Then, take a small scoop out, solublize in HCl, and measure compared to the sand at the start. You could also do this with a sample of sand from the rest of the sand beds you have access to as a comparison (experiment vs real world observation). I don't think the conditions present in a reef aquarium matter for how you treat the sand to assay the phosphate because you are just trying to know the amount of phosphate at the end. HCl doesn't interfere with this and the HCl treatment will be the same for your control and test samples. Also, phosphate in biomolecules is almost universally bound via phosphate-ester bonds, which will readily hydrolyze in sufficiently concentrated acid. If you are really worried you could even heat the samples.

2) Same as experiment 1! Except have 2 or more cups with sandbeds of different depths (1" and 4" at least), and take samples from different depths. Do this with the real world sandbeds as well.

It should be noted that the first two are limited by the amount of phosphate you have in your own particular water. ie just because you don't see any phosphate bound doesn't mean phosphate doesn't bind, just not under the conditions present in YOUR aquarium. Which may be fine for you and useful to know, just something to keep in mind when interpreting and disseminating the data

3) This is a trickier one given the problem with your reaction chamber. For this one I'd prepare several samples of sand treated with varying concentrations of phosphate buffer to bind them with phosphate. Alternatively, you could just use the samples you made in the first two experiments if you want it to be more "real world," but then you are limited as in the first two. I would then treat a sample from the samples as in experiment 1 to assay the phosphate content in the sand. I would then put fresh artificial seawater over the phosphate treated sand, and wait. Again, idk how long, but some amount of time, maybe a time course. I would then take a sample of the water and treat it with lanthanum III chloride to precipitate the phosphate. Add acid to solublize, and run on the instrument.

what do ya think?

 

[LouH]

1) I think the experiment is buy a bag of new aragonite, rinse it well, set some aside, and put some in a cup anywhere in your tank. Then wait. IDK how long, but some amount of time, maybe even several time points. Then, take a small scoop out, solublize in HCl, and measure compared to the sand at the start. You could also do this with a sample of sand from the rest of the sand beds you have access to as a comparison (experiment vs real world observation). I don't think the conditions present in a reef aquarium matter for how you treat the sand to assay the phosphate because you are just trying to know the amount of phosphate at the end. HCl doesn't interfere with this and the HCl treatment will be the same for your control and test samples. Also, phosphate in biomolecules is almost universally bound via phosphate-ester bonds, which will readily hydrolyze in sufficiently concentrated acid. If you are really worried you could even heat the samples.

2) Same as experiment 1! Except have 2 or more cups with sandbeds of different depths (1" and 4" at least), and take samples from different depths. Do this with the real world sandbeds as well.

It should be noted that the first two are limited by the amount of phosphate you have in your own particular water. ie just because you don't see any phosphate bound doesn't mean phosphate doesn't bind, just not under the conditions present in YOUR aquarium. Which may be fine for you and useful to know, just something to keep in mind when interpreting and disseminating the data

3) This is a trickier one given the problem with your reaction chamber. For this one I'd prepare several samples of sand treated with varying concentrations of phosphate buffer to bind them with phosphate. Alternatively, you could just use the samples you made in the first two experiments if you want it to be more "real world," but then you are limited as in the first two. I would then treat a sample from the samples as in experiment 1 to assay the phosphate content in the sand. I would then put fresh artificial seawater over the phosphate treated sand, and wait. Again, idk how long, but some amount of time, maybe a time course. I would then take a sample of the water and treat it with lanthanum III chloride to precipitate the phosphate. Add acid to solublize, and run on the instrument.

what do ya think?

OK, here are the areas that I need clarification on/have questions about for each experiment

1.

a. I can rinse the aragonite with distilled water once I pull it out of the tank because I'm going to use HCl (at what concentration of HCl, 21%?) after the rinse to dissolve the aragonite.

b. The variable that concerns me is understanding the fraction of the result that is phosphorous that that was mineral bound vs. the dissolution of the bacterial and micro fauna life forms. The sand from my system will have issues with this more so than the cup(s) full of new aragonite.

c. Do I have to worry about the chloride ions from the acid causing buildup/scale in the plasma chamber like that observed when full strength synthetic seawater was attempted?

2. I just purchased a Hanna phosphate low range photo spectrometer, and my first couple of attempts have shown 0 ppb, which is not believable. So, I have no idea what the actual concentration of phosphate in solution is in my aquarium's water. This could prove to be problematic, but then again, it might not be. As stated earlier, this aragonite is in the range of 8 years old and has seen some nutrient laden water, so I would anticipate that bound phosphorous will be pulled out of my system's aragonite vs. fresh samples.

Just as an aside, I've read people's comments that phosphate will leach back into solution due to concentration gradients. Is this true? If it were, then by what mechanism? I've read elsewhere that calcium carbonate in the form of aragonite and live rock do nothing for buffering capacity in our tanks. If this is indeed true, then I would expect phosphorous to be the same. I can certainly understand phosphorous concentrations fluctuating over time in response to microbial and algae population crashes, but I don't get how this can happen with calcium carbonate based materials.

Lou

 

[Osteoclast]

LouH
Set up your collection cups in multiples so that you can statistically run your data and get overall means with standard deviations. If you chose cups treat each the same way and process each exactly the same. I would use 5 cups per time point and this should give you sufficient power to see some statistical difference vs old sand. You need to sample the sand at similar depths to make any conclusions compared to the test cups. Of course if your N is 5 then the tank control sand will be 5 measurements. As BA noted I would take multiple time points: 1hr, 24hr, 72hr, 7days, 14days, etc. It is imperative that you sample multiple time points or you may miss data. Of course with multiple times you will have more work and this may not be practical, nevertheless, this is how basic science data is collected and studied. Sometimes we analyze a smaller set of data to generate preliminary data that becomes the foundation for a larger more robust study. So three replicates at 24 hr, 7d, and 14 d could be experiment one followed by another more extensive study depending on where the data fall by statistical analysis. BTW you may get by with simple t-tests if the design is simple. Good luck.

 

[Osteoclast]

LouH another issue,
To work out your experimental design you may need to assay your sand first in the HCL conditions that BA suggested. You need to show that the Hanna Meter can pick up phosphate results with your protocol. So I would perfect the HCL methodology first, and then after you have developed reliable results do a small experiment with an N of 3 over a couple of time points. If this shows some promising data go for the robust study over several weeks. This final study will take careful analysis of the statistics and you may need to review your power to see real differences. In any event I hope you pull it off. Feel free to PM me for any design issues or statistical concerns.

 

[blanden.adam]

OK, here are the areas that I need clarification on/have questions about for each experiment

1.

a. I can rinse the aragonite with distilled water once I pull it out of the tank because I'm going to use HCl (at what concentration of HCl, 21%?) after the rinse to dissolve the aragonite.

b. The variable that concerns me is understanding the fraction of the result that is phosphorous that that was mineral bound vs. the dissolution of the bacterial and micro fauna life forms. The sand from my system will have issues with this more so than the cup(s) full of new aragonite.

c. Do I have to worry about the chloride ions from the acid causing buildup/scale in the plasma chamber like that observed when full strength synthetic seawater was attempted?

2. I just purchased a Hanna phosphate low range photo spectrometer, and my first couple of attempts have shown 0 ppb, which is not believable. So, I have no idea what the actual concentration of phosphate in solution is in my aquarium's water. This could prove to be problematic, but then again, it might not be. As stated earlier, this aragonite is in the range of 8 years old and has seen some nutrient laden water, so I would anticipate that bound phosphorous will be pulled out of my system's aragonite vs. fresh samples.

Just as an aside, I've read people's comments that phosphate will leach back into solution due to concentration gradients. Is this true? If it were, then by what mechanism? I've read elsewhere that calcium carbonate in the form of aragonite and live rock do nothing for buffering capacity in our tanks. If this is indeed true, then I would expect phosphorous to be the same. I can certainly understand phosphorous concentrations fluctuating over time in response to microbial and algae population crashes, but I don't get how this can happen with calcium carbonate based materials.

Lou

1) a) I don't know what the question here is . But with reguard to the HCl concentration, that's really gonna depend on the volume you add and the amount of aragonite you want to dissolve, and what your instrument can handle.

b) If you want to separate the biological fraction from the mineral bound fraction, you could try a quick treatment with bleach and a rinse to remove any biofilms, but that may or may not be significant. I have never done such an experiment, and have no knowledge to make even an order of magnitude estimation to do some quick math :/

c) I've never worked with an ICP and have no knowledge of what concentrations of different species are acceptable, so I have no idea.

2) If you want to test the accuracy of your test kit, you can always make a known series of concentrations of sodium phosphate buffer and test them. Plot them out as a measured versus actual concentration, and find where the linear range is.

When you find the linear range, fit it with a line. The slope of the line (how close it is to 1) will tell you the accuracy of the kit in your hands, the residuals will tell you the precision (again, in your hands), and where the linear range begins will tell you the lower limit of detection.

As noted above by Osteoclast, you are always going to be battling with the compromise the amount of time/effort/cost you will put into a study versus the quality of the study. To really do the job well, a time course with multiple time points, and several preliminary experiments to optimize conditions would need to be done, but this all takes your time and money. Personally, what I'm willing to do for experiments at work and what I'm willing to do for the hobby are very different for this very reason.

3) With regard to the leaching question, there is a simple way to look at the processes involved in phosphate leaching and calcium carbonate stability, and a more involved but more correct way to look at it, but for our purposes the simple way will probably suffice.

Phosphate adsorbs onto the surface of calcium carbonate. At any given concentration of phosphate and available surface area of the calcium carbonate, an equilibrium will establish. The equilibrium will establish when the rate of phosphate binding is equivalent to the rate of phosphate unbinding. Now lets consider the situation where we have calcium carbonate bound with phosphate at equilibrium in water with phosphate present, and then immediately remove 100% of the phosphate from the water. Now, per unit time, no phosphate can bind because there is none in the water, but phosphate can still unbind at a rate proportional to the concentration of phosphate bound.

The difference between phosphate binding and calcium carbonate dissolution is explained simply, albeit incompletely, by the rate of dissolution being incredibly slow under the conditions of the reef aquarium. Calcium carbonate is always super-saturated in a reef aquarium, and the pH is almost always high, so there is almost no driving force the dissolution of the calcium carbonate. As a result, per unit time, almost no calcium carbonate dissolves, and even the small but non-zero amount that does is more than made up for by the heavily favored forward reaction (precipitation)

 

[Osteoclast]

LouH
Agree with BA that bleach should be sufficient as a batericidal agent. You will need to test this by an incubation with bleach for several minutes followed by several rinses before assaying the phosphate. You may need to treat for multiple times to achieve bacterial cell death although probably 3-5minutes is a reasonable starting point for your experiment. Check phosphate pre and post bleach to get additional data regarding your system. Again this is a preliminary trial prior to more longitudinal data. Once the bugs are worked out (literally) you can move to step two.

 

[LouH]

Gentlemen,

I appreciate everyone's input, and I can pretty much apply components of everyone's recommendations.

I like the bleach solution to the bio variable. I also agree that there is some preliminary work that will have to be done just to verify my ability to make accurate measurements.

Regarding cost, the ICP will cost nothing but some machine time, so 5 samples per variable is a non issue.

Thanks guys.

Lou

 

[LouH]

I was thinking about this experiment, and I'm wondering if the surface area of the aragonite is a significant variable. Most of my sand is very fine, but I can't say with certainty what the particle size distribution is. If needed, I can attempt to see if I can ship some samples to our R&D facility for particle size analysis with a laser scattering device. That might be tough to pull off, however. My time is free. Using someone else at another location, well that's something else entirely.

I was also considering the number of samples needed to pull this off. If I do 10 samples in cups (5 in the lighted refugium, and 5 in the unlit refugium), and I take samples at 3 depths, I'm looking at 30 samples right there. I can double this number for the samples collected in my established refugia, so I'm at 60 samples. Add to that the blanks, and I'm at 65. While I'm doing all of this, I may as well digest and measure several samples of an equivalent mass of fish food fed to the tank daily. Yikes!

Lou

 

[LouH]

I forgot about repeating the tests at 3 time intervals. That just trippled the sample number.

 

[Osteoclast]

LouH

The particle size of the sand is certainly a confounding variable but your number of replicates takes that in to account to give a range of measurements that are then expressed as a mean +/- the SD. The surface area, influenced by the grain size, for binding needs to be controlled by using the same type and grade of sand and even lot (if possible) in all conditions. Further, your individual samples need to be standardized by weight to even out the distribution. Dry weight is best to discount any water weight. So removing samples of sand followed by a drying procedure (best a dessicator) weighing each carefully followed by a bleach treatment, followed by an acid incubation would be a possible protocol. Usually multiple rinses are done between the steps times 3 is typical using phosphate free water. The volumes of bleach, HCl, water, and concentrations where appropriate need to be the same. Calibrated pipettes are best for this problem.

You need to design and test this extraction methodology first and analyze the variability among the samples to see if more refinement or alternatively more samples will be needed to look for differences among your control and test groups. I would suspect that weight itself of the sand sample would be a variable that leads to changes in levels of phosphate. You may need to play with the method on different weights initially to see what will produce data that is reproducible and even measurable.

The extraction protocol is the first thing and is basically your preliminary experiment. Once you have worked out the kinks then you can expand it out to begin you comparisons. And as a last point (you may have checked already) but there are likely phosphate extraction protocols already in use that can be modified for this experiment.

 

[LouH]

Osteoclast,

"Your individual samples need to be standardized by weight to even out the distribution. Dry weight is best to discount any water weight. So removing samples of sand followed by a drying procedure (best a dessicator) weighing each carefully followed by a bleach treatment, followed by an acid incubation would be a possible protocol. Usually multiple rinses are done between the steps times 3 is typical using phosphate free water. The volumes of bleach, HCl, water, and concentrations where appropriate need to be the same."

I am in agreement with the exception that I would likely want to do the bleach rinse first to remove biomass from the aragonite.
"You need to design and test this extraction methodology first and analyze the variability among the samples to see if more refinement or alternatively more samples will be needed to look for differences among your control and test groups. I would suspect that weight itself of the sand sample would be a variable that leads to changes in levels of phosphate. You may need to play with the method on different weights initially to see what will produce data that is reproducible and even measurable."

I agree 100%. This may take as more time than running the actual tests once methods have been established.

"As a last point (you may have checked already) but there are likely phosphate extraction protocols already in use that can be modified for this experiment."

I have not looked yet Osteoclast, but I will. Thank you for the advice. Feedback like yours will help me save time. This will likely be a very long process. I'm guessing on the order on 6 months if I'm steadily making progress.

Lou

 

[LouH]

To be honest, this is going to be a daunting challenge. Six months may be an ambitious timeline.

 

[Reefin' Dude]

here is some info on Phosphates.

Calcium Carbonate Phosphate reaction.

Absorption of Phosphate on Calcium Carbonate.

Authigenic Sediments

Variable rates of phosphate uptake by shallow marine . carbonate sediments:
Mechanisms and ecological significance

The other way.

Phosphate solubilizing bacteria

The Marine Phosphate Cycle

if phosphate did not bind and were able to be released from inorganic sources. life on earth would not exist. it is all about the bacteria.

G~

 

[LouH]

Looks like I have some reading to do. :thumbsup:

 

[LouH]

I take away the following from these articles, threads and studies:

1. Phosphate in my system will bind to the surface of calicium carbonate which includes aragonite and live rock.
2. The mechanism is absorption, and it is finite (can hit saturation).
3. Phoshpate attached to suspended calcium carbonate solids will become coated with a biofilm and these particles are likely removed by skimming.
4. Running high pH additives like soda ash and calcium hydroxide likely facilitate phosphate precipitation.
5. Lower pH systems will have higher phosphate concentration in the water column than high pH systems (all other things being equal).
6. Biological activity facilitates the release of phosphate bound to calcium carbonate thereby creating a cycle of absorption and release, which resultes in a phosphate equilibrium in the system.
7. Phosphate interferes with biological calcium carbonate crystal formation by binding to the surface of the coral's skeletal structure.

My takeaway is that you can approach the problem of phosphate accumulation in reef aquaria through several methods including:

1. Minimal live rock and aragonite systems that employ other means of phosphate removal such as bacterial consumption and heavy skimming (carbon dosing methods).
2. Significant quantities of live rock and aragonite to provide lots of area for phosphate binding combined with phosphate removal mechanisms like macroalgae cultivation and GFO.
3. Hybrids of methods 1&2.

And so we come full circle back to debating one method's superiority over the others.

 

[LouH]

Update: 3 weeks have passed and I haven't gotten anything done.

I have, however, been thinking about this. If aragonite truly does bind phosphate, then isn't it simply functioning like a big pile of GFO? If that were the case, then wouldn't you just remove it and replace it after it has become saturated?

I'm sure that I read this somewhere at some point and my brain now thinks that it has had an original thought.

Lou