SAMPLE STORAGE AND ITS IMPACT ON PHOSPHATE MEASUREMENT

[Rick Mathew]

SAMPLE STORAGE AND ITS IMPACT ON PHOSPHATE MEASUREMENT​

In a discussion on R2R @BigJohnny posed a question about why his Hanna ULR Phosphorous test results were significantly higher (2-3 times) than his ICP test results…. Here is a link to the discussion

Accuracy of Phosphate ICP analysis

Curious because my Hanna ulr phosphorus shows 2-3x more on average than icp. I stopped using it though because my experience with hanna is that they are consistent but accuracy/reagents can vary so it's better for relative measurements, and that assumes perform the test perfectly and clean the...

www.reef2reef.com

One of the possible reasons that were proposed was the potential impact of sample transit time and could that have an impact on the results of the ICP test. So I set up a series of experiments to test the hypothecs. At first it was a simple one time experiment in which I took a large sample and measured the dKH, NO3, PO4 and Iodine Levels on the initial sampling. Each was measured 3 times and an average value calculated. The dKH was measured with the Hanna Checker. The Nitrate was measured using the test I developed using the Hanna HI-764 (link to details--- https://www.reef2reef.com/ams/test-meter-for-testing-nitrates.599/ )

The PO4 was tested using the Hanna HI-736 Phosphorous ULR Checker and the Iodine was tested using the method I developed ( link to details https://www.reef2reef.com/ams/using-hanna-checker-hi-707-to-test-for-iodine.743/ )

I took the rest of the remaining sample and split into two sample sets. The first one I stored inside in a controlled environment. The second one I placed in the glove compartment of my car…Uncontrolled environment. I live in Florida where we can get cool nights and hot days so there was some variability. I then tested the samples on day 5, day 10, and day 19…Below is the results. (CHARTS 1-4)

CHART 1

CHART 2

CHART 3

CHART 4
​

As you can see from the charts the PO4 and Iodine measurements changed quite significantly. The dKH and NO3 measurements stay within the expected error range over the testing period.

From these results a question arose as to whether or not the composition of the container could have an impact on the results. A second experiment was conducted using containers made of different materials…Glass, Polypropylene, High Density Polyethylene, Polyethylene, PET, Poly-carbonate and Polystyrene. I chose to test only the PO4 because it had the biggest change over time as well as this was the parameter that posed the question. In addition the measurements were taken using two different Hanna Testing Meters... the HI-736 ULR Phosphorous Meter and the HI-774 ULR Phosphate Meter. The tests were replicated at least 2 times for confirmation some were done as many as 4 times. There are a lot of charts (23) resulting from this set of experiments that I won’t include here, but if you are interested I can send you the charts.

The initial findings indicated that there were measurable differences between the different containers, with glass and PET giving the best results. Then @Dan_P raised the question could the Surface Area to Volume ratio (SA/V) be a factor given that the containers were of different sizes. Sure enough his hunch was correct. The containers with the highest SA/V ratio had the worst results. This initiated a third round of experiments using containers with equivalent SA/V ratios. I chose to use the actual sample tubes provided by the ICP vendor that are made from Polypropylene (PP) and glass sample tubes with the same volume and dimensions thus giving the same SA/V ratio. These tests were also done in duplicate. The results are below. (CHARTS 5 & 6)

CHART 5

CHART 6
​

As you can see both the PP Sample Tubes as well as the Glass Sample Tubes showed a decrease in the measured PO4 with both testers. This is a clear indicator the samples are not stable during shipment & or storage at least with respect to PO4. as tested with the Hanna Checkers

At this point a question was posed by as to whether this was due to Bio-Activity or purely a chemical reaction. I had remembered reading that one of the ICP vendors had tested the question of storage stability using a HACH standard solution and testing it over several days. They reported that the measurement was stable over the storage period. I decided to replicate this experiment.

I made up a PO4 standard at .06 ppm using salt water at 35ppt made with RODI water and reagent grade Sodium Chloride and a HACH Standard. The thinking was that this should represent a Bio-Inactive sample. The same test protocol that was used for the tank sample was used. I chose two different containers, glass and a HDPE, the results are below: (CHARTS 7 & 8)

CHART 7

CHART 8​

The test results indicated that although there was a change over the storage period the results were within testing error and did not show the significant change found in the tank samples that were Bio-Active.

After reviewing the test procedure of the HACH Standard experiment I had some question as to whether the containers were completely free of any Bio material. I did a repeat of the test after sterilizing both containers. Results are below: (CHARTS 9 & 10)

CHART 9

CHART 10
​

The above results replicate the results of the ICP vendor. Using the HACH Standard the measurements remain relatively stable over the sample storage period.

This would be an indicator that the instability of the PO4 is related to the Bio-Activity of the tank samples.

One hypothesis is that the Bio-Active samples from the tank are forming a thin bio-film on the sides of the containers that over time adsorb the organic phosphate their by reducing the amount of PO4 in the water being tested. This is only one of several possible explanations yet to be explored.

An additional experiment was set up to see it ‘Pre-Conditioning” the container might be helpful. The thought was by doing this the film would be formed and the tubes would be “conditioned” and the next sample placed in the tube would not undergo the “film forming” because the film was already there.

The PP sample tubes were filled with tank water and allowed to set for 12 days. An initial measurement was taken (Day 0) and then a second measurement on day 12. The tubes were then re-filled with a second batch of tank water without rinsing out the tubes. The second batch was then tested according to the standard test protocol. (Store samples and test on day 0, 3 & 6 and test with the 2 Hanna Checkers). The results are below: (Charts 11 & 12)

CHART 11

CHART 12
​

These test result indicate the “conditioning” under this set of parameters does not appreciably reduce the effect of loss of PO4 over time: That being said, there are additional experiments that by changing the parameters could define a set of conditions where “pre-conditioning” the container could prove to be successful. These experiments are yet to be completed.

It also could be that the hypotheses is incorrect and there is some other mechanism causing this…Bacteria interaction…some type of chelation process….and who know what else….Most of this is way above my pay grade, but many of you out there in R2R Family have the skills, knowledge and ability to contribute ideas.

SOME INITIAL CONCLUSIONS:

• Samples stored in high SA/V containers (Sample Tubes) are not stable over time with respect to PO4 measurement when tested with Hanna Checkers.
• The problem is associated with tank water (Bio-Active) and does not show up with “sterile” samples made with PO4 standards.
• The type of container does not appear to make a difference. The SA/V Ratio appears to be the controlling factor.

SOME INITIAL SPECULATIONS:

• This could be a possible explanation of why a number of Reefers get significantly different results from there testing protocol and ICP results when testing PO4.
• There could be other test parameters that are affected by this issue.
• A different sampling protocol may be necessary to insure better ICP test results related to some test perimeters.

Well that is about the long and the short of it at this point. The work is continuing and I will provide updates as they are completed. There are so many “Rabbit Holes” to this project! I have several questions yet to be explored….

• Does the initial PO4 level change the outcome?
• Are there other parameters to be concerned about? Evidence says Iodine might be an issue…
• What is the effect of temperature; would keeping the sample cold reduce the problem?
• Is there a critical Surface Area to Volume Ration where the problem is reduce or eliminated and what is it?
• Why are there some cases where the ICP results closely match the results obtained by the Hanna Checkers and yet there are other times they are vastly different (2-3 times)?

And I am sure many of you will have some great questions to be answered.


Thanks again to @Dan_P and @taricha for their help and guidance in this project...What an awesome family this R2R group is!!
One thing is for sure BRS is happy about this project. With over 200 individual tests and more to come their sales of Checker Reagent is benefiting greatly!

​

 

[Reefer5640]

Wow that’s awesome man! That’s a lot of work. I work with a lot of chemicals and we do ICP testing at work but they’re taken with Teflon bottles that are pre leeched and the samples are never left in there for more than a few hours. I’m definitely interested in following along to see what you come up with.

 

[Rick Mathew]

Wow that’s awesome man! That’s a lot of work. I work with a lot of chemicals and we do ICP testing at work but they’re taken with Teflon bottles that are pre leeched and the samples are never left in there for more than a few hours. I’m definitely interested in following along to see what you come up with.

Thank you...It has been quite a reveling project... It for sure changed the way I handle my tank samples for testing....No more over night sitting before testing....I am aware of the strict sampling protocols used in the water testing industry and the special containers required most likely for the very reasons we are seeing these results

rick

 

[Dan_P]

SAMPLE STORAGE AND ITS IMPACT ON PHOSPHATE MEASUREMENT​

In a discussion on R2R @BigJohnny posed a question about why his Hanna ULR Phosphorous test results were significantly lower (2-3 times) than his ICP test results…. Here is a link to the discussion https://www.reef2reef.com/threads/accuracy-of-phosphate-icp-analysis.381738/page-3

One of the possible reasons that were proposed was the potential impact of sample transit time and could that have an impact on the results of the ICP test. So I set up a series of experiments to test the hypothecs. At first it was a simple one time experiment in which I took a large sample and measured the dKH, NO3, PO4 and Iodine Levels on the initial sampling. Each was measured 3 times and an average value calculated. The dKH was measured with the Hanna Checker. The Nitrate was measured using the test I developed using the Hanna HI-764 (link to details--- https://www.reef2reef.com/ams/test-meter-for-testing-nitrates.599/ )

The PO4 was tested using the Hanna HI-736 Phosphorous ULR Checker and the Iodine was tested using the method I developed ( link to details https://www.reef2reef.com/ams/using-hanna-checker-hi-707-to-test-for-iodine.743/ )

I took the rest of the remaining sample and split into two sample sets. The first one I stored inside in a controlled environment. The second one I placed in the glove compartment of my car…Uncontrolled environment. I live in Florida where we can get cool nights and hot days so there was some variability. I then tested the samples on day 5, day 10, and day 19…Below is the results. (CHARTS 1-4)

CHART 1

CHART 2

CHART 3

CHART 4
​

As you can see from the charts the PO4 and Iodine measurements changed quite significantly. The dKH and NO3 measurements stay within the expected error range over the testing period.

From these results a question arose as to whether or not the composition of the container could have an impact on the results. A second experiment was conducted using containers made of different materials…Glass, Polypropylene, High Density Polyethylene, Polyethylene, PET, Poly-carbonate and Polystyrene. I chose to test only the PO4 because it had the biggest change over time as well as this was the parameter that posed the question. In addition the measurements were taken using two different Hanna Testing Meters... the HI-736 ULR Phosphorous Meter and the HI-774 ULR Phosphate Meter. The tests were replicated at least 2 times for confirmation some were done as many as 4 times. There are a lot of charts (23) resulting from this set of experiments that I won’t include here, but if you are interested I can send you the charts.

The initial findings indicated that there were measurable differences between the different containers, with glass and PET giving the best results. Then @Dan_P raised the question could the Surface Area to Volume ratio (SA/V) be a factor given that the containers were of different sizes. Sure enough his hunch was correct. The containers with the highest SA/V ratio had the worst results. This initiated a third round of experiments using containers with equivalent SA/V ratios. I chose to use the actual sample tubes provided by the ICP vendor that are made from Polypropylene (PP) and glass sample tubes with the same volume and dimensions thus giving the same SA/V ratio. These tests were also done in duplicate. The results are below. (CHARTS 5 & 6)

CHART 5

CHART 6
​

As you can see both the PP Sample Tubes as well as the Glass Sample Tubes showed a decrease in the measured PO4 with both testers. This is a clear indicator the samples are not stable during shipment & or storage at least with respect to PO4. as tested with the Hanna Checkers

At this point a question was posed by as to whether this was due to Bio-Activity or purely a chemical reaction. I had remembered reading that one of the ICP vendors had tested the question of storage stability using a HACH standard solution and testing it over several days. They reported that the measurement was stable over the storage period. I decided to replicate this experiment.

I made up a PO4 standard at .06 ppm using salt water at 35ppt made with RODI water and reagent grade Sodium Chloride and a HACH Standard. The thinking was that this should represent a Bio-Inactive sample. The same test protocol that was used for the tank sample was used. I chose two different containers, glass and a HDPE, the results are below: (CHARTS 7 & 8)

CHART 7

CHART 8​

The test results indicated that although there was a change over the storage period the results were within testing error and did not show the significant change found in the tank samples that were Bio-Active.

After reviewing the test procedure of the HACH Standard experiment I had some question as to whether the containers were completely free of any Bio material. I did a repeat of the test after sterilizing both containers. Results are below: (CHARTS 9 & 10)

CHART 9

CHART 10
​

The above results replicate the results of the ICP vendor. Using the HACH Standard the measurements remain relatively stable over the sample storage period.

This would be an indicator that the instability of the PO4 is related to the Bio-Activity of the tank samples.

One hypothesis is that the Bio-Active samples from the tank are forming a thin bio-film on the sides of the containers that over time adsorb the organic phosphate their by reducing the amount of PO4 in the water being tested. This is only one of several possible explanations yet to be explored.

An additional experiment was set up to see it ‘Pre-Conditioning” the container might be helpful. The thought was by doing this the film would be formed and the tubes would be “conditioned” and the next sample placed in the tube would not undergo the “film forming” because the film was already there.

The PP sample tubes were filled with tank water and allowed to set for 12 days. An initial measurement was taken (Day 0) and then a second measurement on day 12. The tubes were then re-filled with a second batch of tank water without rinsing out the tubes. The second batch was then tested according to the standard test protocol. (Store samples and test on day 0, 3 & 6 and test with the 2 Hanna Checkers). The results are below: (Charts 11 & 12)

CHART 11

CHART 12
​

These test result indicate the “conditioning” under this set of parameters does not appreciably reduce the effect of loss of PO4 over time: That being said, there are additional experiments that by changing the parameters could define a set of conditions where “pre-conditioning” the container could prove to be successful. These experiments are yet to be completed.

It also could be that the hypotheses is incorrect and there is some other mechanism causing this…Bacteria interaction…some type of chelation process….and who know what else….Most of this is way above my pay grade, but many of you out there in R2R Family have the skills, knowledge and ability to contribute ideas.

SOME INITIAL CONCLUSIONS:

• Samples stored in high SA/V containers (Sample Tubes) are not stable over time with respect to PO4 measurement when tested with Hanna Checkers.
• The problem is associated with tank water (Bio-Active) and does not show up with “sterile” samples made with PO4 standards.
• The type of container does not appear to make a difference. The SA/V Ratio appears to be the controlling factor.

SOME INITIAL SPECULATIONS:

• This could be a possible explanation of why a number of Reefers get significantly different results from there testing protocol and ICP results when testing PO4.
• There could be other test parameters that are affected by this issue.
• A different sampling protocol may be necessary to insure better ICP test results related to some test perimeters.

Well that is about the long and the short of it at this point. The work is continuing and I will provide updates as they are completed. There are so many “Rabbit Holes” to this project! I have several questions yet to be explored….

• Does the initial PO4 level change the outcome?
• Are there other parameters to be concerned about? Evidence says Iodine might be an issue…
• What is the effect of temperature; would keeping the sample cold reduce the problem?
• Is there a critical Surface Area to Volume Ration where the problem is reduce or eliminated and what is it?
• Why are there some cases where the ICP results closely match the results obtained by the Hanna Checkers and yet there are other times they are vastly different (2-3 times)?

And I am sure many of you will have some great questions to be answered.


Thanks again to @Dan_P and @taricha for their help and guidance in this project...What an awesome family this R2R group is!!
One thing is for sure BRS is happy about this project. With over 200 individual tests and more to come their sales of Checker Reagent is benefiting greatly!

​

This is a top notch investigation. The Pharmaceutical Industry pays many tens of millions of dollars a year on this type of study. A big thank you for your gift.

I wish we knew how all the other analytes are behaving. Many people worry about the concentration of the micro nutrients in their systems, and maybe now they should take ICP result with a grain of salt.

 

[Rick Mathew]

This is a top notch investigation. The Pharmaceutical Industry pays many tens of millions of dollars a year on this type of study. A big thank you for your gift.

I wish we knew how all the other analytes are behaving. Many people worry about the concentration of the micro nutrients in their systems, and maybe now they should take ICP result with a grain of salt.

Thank you very much...It is totally my pleasure to explore these kind of questions..you are correct about other micro ingredients...I am for sure going to look at Iodine base on my initial testing...

Thanks again Dan for all your help and encouragement

 

[taricha]

I'm late to this, but seriously great work.

• Samples stored in high SA/V containers (Sample Tubes) are not stable over time with respect to PO4 measurement when tested with Hanna Checkers.
• The problem is associated with tank water (Bio-Active) and does not show up with “sterile” samples made with PO4 standards.
• The type of container does not appear to make a difference. The SA/V Ratio appears to be the controlling factor.

SOME INITIAL SPECULATIONS:

• This could be a possible explanation of why a number of Reefers get significantly different results from there testing protocol and ICP results when testing PO4.
• There could be other test parameters that are affected by this issue.
• A different sampling protocol may be necessary to insure better ICP test results related to some test perimeters.

Seeing people order hach PO4 standards and confirming hanna readings are exactly as expected, then turning around and shipping a sample to ICP only to find sizable disagreement between hanna checker and the ICP test was very suggestive.
You nailing this down is very satisfying.
This issue doesn't seem un-fixable. If I wanted my water to stay PO4-stable in shipment, I could add a few pebbles of aragonite from my sand surface.
But that only works if I know the procedures the ICP vendor will use. If they filter it, great! If they acidify, then that's bad - might pull way too much off the aragonite. Or they might decide I'm shipping them sand for mischievous purposes

 

[Dan_P]

I'm late to this, but seriously great work.



Seeing people order hach PO4 standards and confirming hanna readings are exactly as expected, then turning around and shipping a sample to ICP only to find sizable disagreement between hanna checker and the ICP test was very suggestive.
You nailing this down is very satisfying.
This issue doesn't seem un-fixable. If I wanted my water to stay PO4-stable in shipment, I could add a few pebbles of aragonite from my sand surface.
But that only works if I know the procedures the ICP vendor will use. If they filter it, great! If they acidify, then that's bad - might pull way too much off the aragonite. Or they might decide I'm shipping them sand for mischievous purposes

Do you wonder why details of their procedures are not readily available?

 

[taricha]

Do you wonder why details of their procedures are not readily available?

I took at face value the argument that they have to do something fancy to make their expensive laser blaster work with saltwater that it isn't supposed to, and that [competition secrets] is why they don't detail what they do to our samples.

It could be true, it could also just be convenient if you don't want knowledgeable people pointing out where your methods are prone to error.

 

[Lasse]

• Samples stored in high SA/V containers (Sample Tubes) are not stable over time with respect to PO4 measurement when tested with Hanna Checkers.
• The problem is associated with tank water (Bio-Active) and does not show up with “sterile” samples made with PO4 standards.
• The type of container does not appear to make a difference. The SA/V Ratio appears to be the controlling factor.

This is no news - is well known from sampling of wastewater and later analyses - totally independent of type of jar - its the same with glass. In order to analyse PO4 remotely - you need to acidify the sample. But ICP does not analyse PO4 - it analyse P and after that available P is recalculated into PO4. Assuming that all P is in the form of PO4 in aquarium water. If anything - ICP should overestimate the PO4 - IMO.

The only way to know if there is any significant P losses in transport (storage) is to take one larger sample and do a direct ICP test on one part of the sample. Next step storage the other parts for 7 and 14 days and after that analyse the samples exactly the same way as the first. This is basic in all test comparisons. First be sure you test the same parametre - second treat the samples exactly the same way.

At least one of the vendors - that I know of - doing saltwater ICP tests have done just this and have seen no significant losses.

All other way of testing this is to compare apples and pears and not even give any indications in one or another way. You can´t compare PO4 testing with one device with total P testing with another device.

Sincerely Lasse

 

[Rick Mathew]

This is no news - is well known from sampling of wastewater and later analyses - totally independent of type of jar - its the same with glass. In order to analyse PO4 remotely - you need to acidify the sample. But ICP does not analyse PO4 - it analyse P and after that available P is recalculated into PO4. Assuming that all P is in the form of PO4 in aquarium water. If anything - ICP should overestimate the PO4 - IMO.

The only way to know if there is any significant P losses in transport (storage) is to take one larger sample and do a direct ICP test on one part of the sample. Next step storage the other parts for 7 and 14 days and after that analyse the samples exactly the same way as the first. This is basic in all test comparisons. First be sure you test the same parametre - second treat the samples exactly the same way.

At least one of the vendors - that I know of - doing saltwater ICP tests have done just this and have seen no significant losses.

All other way of testing this is to compare apples and pears and not even give any indications in one or another way. You can´t compare PO4 testing with one device with total P testing with another device.

Sincerely Lasse

Lasse thank you for the reply...Good information

2 questions

1) Do you know if the test you described that the vendor did was with active tank water or a lab prepared saltwater spiked with a standard solution of PO4?

2) Being that I essentially did the experiment that you described ...Taking a large sample of tank water doing an initial P measurement using the Hanna Phosphorous tester HI-736) (then converting to PO4). Then storing the samples and tested them over a series of days using the same test...Would you expect my results testing for P would parallel that of the ICP test...Not that it would be any where as precise or accurate in measuring total P but would our values track


Thanks for your insights

rick

 

[Lasse]

1) I can´t answer the question because I do not know. But if they centrifugate the sample (as at least some says they do) - it does not matter because the film (if any) will be homogated with the water. However - I do not believe its preliminary a film on the tube that is biological active. All living water contains many, many small particles of different origin in the water - perfect for bacteria growth (fast if there is oxygen, slow if it is not). I have noted very fast down going of PO4 in samples stored in glass containers with 200 ml sample - only a matter of hours and the result is significant lower. IMO - there is a probably - if it really is a biological process - high risk that bacteria on particles takes up PO4. But after homogenisation - the ICP will pick up all P

2) - We have to sort out some definitions. When we analyse PO4 with photospectral methods - like the Hanna checkers we can express the result as PO4 - it means that it is the concentration of the whole molecule - including the O4 - we express the concentration as. This is common in the hobby and farming - to express it as PO4. But in the environmental way of expressing - it is normally expressed as PO4-P and it is only the concentration of this P (that is in the PO4 molecule) that will be expressed as concentration. Therefore you can say that 1 mg/l PO4-P is the same as 3.06*1 mg/l PO4.

HI-736 and HI-774 is basically the same meter - both analyse UL PO4 but HI-736 give the result in PO4-P and HI-774 in PO4.

Hanna does not say that it is PO4-P - they only use P as parameter and that has give a huge confusion for most hobbyists.

However - P can exist in many other forms that can´t be analysed with this type of methods - and in science you talk about total P. Total P include all forms of P in a sample. In the old days - the only way to analyse total P was to digest the sample in high temperatures, pressure and strong acids. This process converted all forms of P into PO4 and you could analyse it with methods similar to Hanna Checker. This process is both difficult and dangerous to do. In my old lab - there was a brown stain in the ceiling after a test tube where the cork was not properly tightened before digesting!

ICP give this type of total P - it means all forms of P in the sample. If there is biological bound P, chemical bound P, inorganic bond P (in PO4) or other type of P in the sample - it does not matter. The ICP will give you the total P. The homegation of the sample (the centrifugation) will give an equal contribution of all compounds for the picolitre that is needed for the test. Including film an particles in the sample. this is the reason why I state that if there will be a fault in the PO4 calculation - it will be to high - not to low. When all P is analysed (you see that in the P column) - the assumption that all analysed P is in the form of PO4 is done and the total P result is multiplied with 3.06

Once again - the P that you get from the Hanna HI-736 give you the PO4-P of the sample (it means all P included in PO4 in the water. THe ICP give you total P - it means all P (every form of P) in the water sample.

Your investigation is well done and but - IMO - it is only showing that you should not analyse PO4 or PO4-P in stored sample. It does not say anything about what happens with stored samples that will be analysed for total atom content of more than 30 elements there total P is on of them

Sincerely Lasse

 

[Randy Holmes-Farley]

Lasse thank you for the reply...Good information

2 questions

1) Do you know if the test you described that the vendor did was with active tank water or a lab prepared saltwater spiked with a standard solution of PO4?

2) Being that I essentially did the experiment that you described ...Taking a large sample of tank water doing an initial P measurement using the Hanna Phosphorous tester HI-736) (then converting to PO4). Then storing the samples and tested them over a series of days using the same test...Would you expect my results testing for P would parallel that of the ICP test...Not that it would be any where as precise or accurate in measuring total P but would our values track


Thanks for your insights

rick

Inorganic phosphate that gets incorporated into organic forms like DNA or phospholipids will be removed from detection by a typical phosphate kit (including the Hanna), but not by ICP, unless the molecules (or the whole organism they are in) is removed from the water that is tested somehow. That "removal" might be from bacteria growing attached to the container walls, or perhaps if they centrifuge it before analysis.

 

[Rick Mathew]

1) I can´t answer the question because I do not know. But if they centrifugate the sample (as at least some says they do) - it does not matter because the film (if any) will be homogated with the water. However - I do not believe its preliminary a film on the tube that is biological active. All living water contains many, many small particles of different origin in the water - perfect for bacteria growth (fast if there is oxygen, slow if it is not). I have noted very fast down going of PO4 in samples stored in glass containers with 200 ml sample - only a matter of hours and the result is significant lower. IMO - there is a probably - if it really is a biological process - high risk that bacteria on particles takes up PO4. But after homogenisation - the ICP will pick up all P

2) - We have to sort out some definitions. When we analyse PO4 with photospectral methods - like the Hanna checkers we can express the result as PO4 - it means that it is the concentration of the whole molecule - including the O4 - we express the concentration as. This is common in the hobby and farming - to express it as PO4. But in the environmental way of expressing - it is normally expressed as PO4-P and it is only the concentration of this P (that is in the PO4 molecule) that will be expressed as concentration. Therefore you can say that 1 mg/l PO4-P is the same as 3.06*1 mg/l PO4.

HI-736 and HI-774 is basically the same meter - both analyse UL PO4 but HI-736 give the result in PO4-P and HI-774 in PO4.

Hanna does not say that it is PO4-P - they only use P as parameter and that has give a huge confusion for most hobbyists.

However - P can exist in many other forms that can´t be analysed with this type of methods - and in science you talk about total P. Total P include all forms of P in a sample. In the old days - the only way to analyse total P was to digest the sample in high temperatures, pressure and strong acids. This process converted all forms of P into PO4 and you could analyse it with methods similar to Hanna Checker. This process is both difficult and dangerous to do. In my old lab - there was a brown stain in the ceiling after a test tube where the cork was not properly tightened before digesting!

ICP give this type of total P - it means all forms of P in the sample. If there is biological bound P, chemical bound P, inorganic bond P (in PO4) or other type of P in the sample - it does not matter. The ICP will give you the total P. The homegation of the sample (the centrifugation) will give an equal contribution of all compounds for the picolitre that is needed for the test. Including film an particles in the sample. this is the reason why I state that if there will be a fault in the PO4 calculation - it will be to high - not to low. When all P is analysed (you see that in the P column) - the assumption that all analysed P is in the form of PO4 is done and the total P result is multiplied with 3.06

Once again - the P that you get from the Hanna HI-736 give you the PO4-P of the sample (it means all P included in PO4 in the water. THe ICP give you total P - it means all P (every form of P) in the water sample.

Your investigation is well done and but - IMO - it is only showing that you should not analyse PO4 or PO4-P in stored sample. It does not say anything about what happens with stored samples that will be analysed for total atom content of more than 30 elements there total P is on of them

Sincerely Lasse

Thank you for such a detailed response...Very kind of you to take the time to help me understand...I was not aware of the HI-736 measuring PO4-P ...You are correct that is a clear point of confusion....

So just so I am clear. The ICP folks measure Phosphorous in all forms and that is the value they report as in micrograms /L of P and then they convert that to PO4 via a calculation that they report as PO4 level... Or am I still confused?

My last analysis was 13 micrograms / L of P and reported value of .04 mg/L PO4---

Thanks for your help

 

[Randy Holmes-Farley]

So just so I am clear. The ICP folks measure Phosphorous in all forms and that is the value they report as in micrograms /L of P and then they convert that to PO4 via a calculation that they report as PO4 level... Or am I still confused?

That is correct. Many report both values, but it is one measurement.

 

[Rick Mathew]

Inorganic phosphate that gets incorporated into organic forms like DNA or phospholipids will be removed from detection by a typical phosphate kit (including the Hanna), but not by ICP, unless the molecules (or the whole organism they are in) is removed from the water that is tested somehow. That "removal" might be from bacteria growing attached to the container walls, or perhaps if they centrifuge it before analysis.

Thank you for your reply...much appreciated,,,,Does this mean that I would in general measure a lower PO4 level than the ICP measurement unless as you mentioned it is being removed from the water by some other means?

 

[Rick Mathew]

That is correct. Many report both values, but it is one measurement.

Thank you sir!

 

[Randy Holmes-Farley]

Thank you for your reply...much appreciated,,,,Does this mean that I would in general measure a lower PO4 level than the ICP measurement unless as you mentioned it is being removed from the water by some other means?

It means that you should measure a lower value or the same value, if the organic phosphate is much lower than the inorganic phosphate.

I've not seen much data comparing different forms in reef aquaria.

This paper suggests both soluble organic and inorganic forms are significant on coral reefs:

https://www.int-res.com/articles/meps/14/m014p159.pdf

 

[Julian@Triton]

Here is some information that we posted recently on our forum (with focus on the section pertaining to Phosphate):

What an ICP-OES machine CANNOT test

What an ICP-OES machine CANNOT test As the company that developed the method for sea water analysis and making broadband testing available to aquarists, we at TRITON often get asked what an ICP-OES machine CAN test. These days it is getting more and more confusing with the proliferation of...

www.reef2reef.com

 

[Dan_P]

Here is some information that we posted recently on our forum (with focus on the section pertaining to Phosphate):

What an ICP-OES machine CANNOT test

What an ICP-OES machine CANNOT test As the company that developed the method for sea water analysis and making broadband testing available to aquarists, we at TRITON often get asked what an ICP-OES machine CAN test. These days it is getting more and more confusing with the proliferation of...

www.reef2reef.com

Great explanation, but if the sample requires several days of transport before it is analyzed, the phosphorous, orthophosphate or organophosphate, can be assimilated by microorganisms. If those microorganisms are attached to the surface of the sample container, will the assimilated phosphorous be detected?

 

[Randy Holmes-Farley]

Great explanation, but if the sample requires several days of transport before it is analyzed, the phosphorous, orthophosphate or organophosphate, can be assimilated by microorganisms. If those microorganisms are attached to the surface of the sample container, will the assimilated phosphorous be detected?

Ive had that same concern about trace elements since these businesses started.