Measuring Organic Matter. A Brief Study

Dan_P

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I am sharing the results of a brief study of two methods for measuring and trending dissolved organic matter: BOD5 and basic potassium permanganate consumption (COD). Study samples which served as reference standards were characterized with Triton’s NDOC test which measures the amount of organic carbon and organic nitrogen. The samples in this study were sterile filtered aquarium water and sterile filtered newly collected skimmate. BOD5 and COD tests were performed within two hours of collecting the samples. Each sample was also submitted to Oceamo for the recently introduced Organo-MS test (https://www.reef2reef.com/threads/introducing-the-oceamo-organo-ms-analysis.1112943/). Nitrate, nitrite, total ammonia and phosphate concentrations were measured for both samples (see Sample Table).

image.png


Study Samples Analysis
Water chemistry and NDOC results show that the skimmate enrichment is primarily organic carbon (1.9X) with a lower enrichment (1.3X) of dissolved organic nitrogen (DON: NDOC total nitrogen minus inorganic nitrogen species). The skimmate was colorless. The amount of organic nitrogen in the aquarium water is equivalent to 1.2 ppm nitrate.

BOD5 Results
This test measures the amount of oxygen consumed by bacteria in a water sample held in the dark at room temperature for five days. The oxygen consumption is related to the amount of digestible organic matter contained in the sample. Not all organic matter is consumed by bacteria, and therefore, the results only reflect a portion of the dissolved organic carbon present. Unless calibrated, the results from this test are used for relative comparisons.

Skimmate sample preparation involved passing the water through a 0.45 micron filter to remove particulates which included bacteria. Therefore, bacteria were added back by diluting a small amount (10%) of skimmate with sterile filtered skimmate. From previous work, the particulates added with the unfiltered skimmate increase the BOD5 by about 4%. The aquarium water sample was not filtered because previous work showed little or no difference in BOD5 results for sterile filtered aquarium water with bacteria added back. BOD5 study results are presented in the table below.

image.png


While we can’t directly compare NDOC to BOD5 results, we can note that skimmate has 1.9X more organic C than aquarium water but consumed 7.5X more oxygen (unfiltered skimmate has the highest BOD5. NDOC test not conducted). How might this be explained?

For aquarium water, 0.2 ppm O2 was consumed in the BOD5 test. If we assume all the oxygen went to making CO2, then only 0.075 ppm carbon was consumed in the process. Since the aquarium water is not nitrogen or phosphorous limited, the bulk of the organic carbon (NDOC result) is likely not readily digestible (labile) carbon. In the case of the skimmate sample, a larger BOD5 was observed, consuming up to 0.56 ppm carbon. This likely means that the 1 ppm organic carbon enrichment in the skimmate sample had a much higher proportion of labile carbon than aquarium water, and evidence that the skimmer is doing its job.

COD
This test like BOD5 measures oxygen consumption, but in this case, bacteria are replaced with a chemical oxidizer, potassium permanganate. The amount of permanganate reduced to manganese dioxide is related to the amount of organic matter in the sample. Because permanganate does not oxidize all organic matter, like BOD5, COD results reflect a portion of the total organic matter present. Study results are shown in the table below. The amount of permanganate consumed is converted to a chemical oxygen demand.

image.png


The COD results parallel the NDOC total organic results more closely then the BOD5 results. The estimated amount of organic carbon, assuming all the oxygen is converted to CO2, is about 16% of the NDOC test result for both samples. This relationship may vary as the proportion of organic matter susceptible to permanganate oxidation varies. Caution is required when comparing COD results from different samples. For this study’s samples, there is a strong correlation between COD and BOD5 results (see plot below).

image.png


Organo-MS
This test is a bit different from NDOC, BOD5 and COD. It is focused on identifying molecules of interest in the aquarium such as amino acids, vitamins, pesticides, herbicides, PFAS, pharmaceutical and plasticizers. When a molecule is detected and identified, the concentration is reported. To understand what fraction of the organic carbon is identified by this method, each sample was submitted for the Organo-MS analysis. The concentration of organic carbon and nitrogen was calculated for each chemical species that was reported with a concentration and summed (see table below).

image.png


As is clear from the results, Organo-MS is reporting on a small subset of the organic matter found in the aquarium samples. A more complete review of the data can be found in post #87 in


Conclusion
The data gives me a level of confidence to use BOD5 and COD to trend the concentration of organic matter in an aquarium water.

Any questions, comments?
 

EnterName

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Absolutely love what you are doing!

I'm not too deep into the material yet and would love to learn how you conducted the COD and BOD5 tests. I'm also wondering if other oxidants which naturally occur in seawater could skew the results in any way or make these tests even applicable, as I only know them from wastewater treatment.

Again, I'm not very knowledgeable regarding this topic, so maybe I misunderstood what you did but as soon as I have a reasonable understanding, I could imagine trying them for myself (as long as they are replicable in a home-lab)
 
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Dan_P

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Absolutely love what you are doing!

I'm not too deep into the material yet and would love to learn how you conducted the COD and BOD5 tests. I'm also wondering if other oxidants which naturally occur in seawater could skew the results in any way or make these tests even applicable, as I only know them from wastewater treatment.

Again, I'm not very knowledgeable regarding this topic, so maybe I misunderstood what you did but as soon as I have a reasonable understanding, I could imagine trying them for myself (as long as they are replicable in a home-lab)
Thanks for the reference. Hope you get a chance to try these methods.
 

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Thanks for sharing this.

I’m also quite curious about how you ran the COD tests. Did you use a commercial kit or make your own reagents?

SAC 254 might also be an interesting method to compare, since some companies offer it as part of their ICP services, which means there may be more data available from reefers.
 
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Thanks for sharing this.

I’m also quite curious about how you ran the COD tests. Did you use a commercial kit or make your own reagents?

SAC 254 might also be an interesting method to compare, since some companies offer it as part of their ICP services, which means there may be more data available from reefers.
The COD test is DIY alkaline permanganate, using the Hanna low range nitrate Checker to measure color intensity. Would you like me to supply details enough to run the test?

Yes, 254 nm absorbance is used as a surrogate DOC measure. I believe Oceamo offers the test.
 

EnterName

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The COD test is DIY alkaline permanganate, using the Hanna low range nitrate Checker to measure color intensity. Would you like me to supply details enough to run the test?

Yes, 254 nm absorbance is used as a surrogate DOC measure. I believe Oceamo offers the test.
I don't know if jeremie is interested, but I am so I would love to hear how you did it!
 

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There is a lot here. One thing that jumps out is the potential to further explore ratio of BOD5:NDOC which might be able to be developed into a bioavailability index (think OLI) for DOC. This could potentially lead to many insights on tank stability. Nice work!
 

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The COD test is DIY alkaline permanganate, using the Hanna low range nitrate Checker to measure color intensity. Would you like me to supply details enough to run the test?

Yes, 254 nm absorbance is used as a surrogate DOC measure. I believe Oceamo offers the test.
Great alternative to a spectrophotometer and lot less expensive! I am curious how the DIY approach would align with a HACH 1900/3900.
 
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There is a lot here. One thing that jumps out is the potential to further explore ratio of BOD5:NDOC which might be able to be developed into a bioavailability index (think OLI) for DOC. This could potentially lead to many insights on tank stability. Nice work!
Thanks for the feedback.

Exploring the “bioavailability index” idea was just placed on the “to do” list. If this turns into something really big, we’ll split the prize money!
 
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Great alternative to a spectrophotometer and lot less expensive! I am curious how the DIY approach would align with a HACH 1900/3900.
I don’t see any reason why you couldn’t use these spectrophotometers in place of the Hanna photometers. Likely to have gains in accuracy and a lower detection limit.
 

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The COD test is DIY alkaline permanganate, using the Hanna low range nitrate Checker to measure color intensity. Would you like me to supply details enough to run the test?
That would be very helpful.
I have access to a spectrophotometer that can measure absorbance at 254 nm, so I’m thinking of doing some tests to see whether the two methods correlate well.
 
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Dan_P

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That would be very helpful.
I have access to a spectrophotometer that can measure absorbance at 254 nm, so I’m thinking of doing some tests to see whether the two methods correlate well.
Here’s the procedure, obviously adjust as needed.

I use 15 mL screw cap glass test tubes
5 mL sample
1 mL 2% NaOH (2 g to make 100 mL solution in RO/DI). Seawater ecome cloudy and gelatinous precipitate can form
1 mL 390 ppm potassium permanganate (this concentration gives a good absorbance. There is room to adjust up down as needed)
Cap test tube
Mix thoroughly
Place test tube in boiling water (100 C) for ten minutes. This will generate some pressure inside test tube.
Cool to room temperature.
There is a precipitate that needs to be removed. I found 1 minute in a centrifuge ~4000 rpm packs it down enough that the supernatant can be decanted. The only step not refined yet.
Collect visible spectrum.

Let me know if anything isn’t clear.
 

EnterName

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Here’s the procedure, obviously adjust as needed.

I use 15 mL screw cap glass test tubes
5 mL sample
1 mL 2% NaOH (2 g to make 100 mL solution in RO/DI). Seawater ecome cloudy and gelatinous precipitate can form
1 mL 390 ppm potassium permanganate (this concentration gives a good absorbance. There is room to adjust up down as needed)
Cap test tube
Mix thoroughly
Place test tube in boiling water (100 C) for ten minutes. This will generate some pressure inside test tube.
Cool to room temperature.
There is a precipitate that needs to be removed. I found 1 minute in a centrifuge ~4000 rpm packs it down enough that the supernatant can be decanted. The only step not refined yet.
Collect visible spectrum.

Let me know if anything isn’t clear.
Okay so I could get a 0.01mol KMnO₄ solution and dilute it down to 0.0025mol KMnO₄ (not easy to get KMnO₄ here unfortunately). But you will need to provide some explanation on how to use the HannaChecker to make it work, as I don't have spectrophotometer at hand.
 
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Okay so I could get a 0.01mol KMnO₄ solution and dilute it down to 0.0025mol KMnO₄ (not easy to get KMnO₄ here unfortunately). But you will need to provide some explanation on how to use the HannaChecker to make it work, as I don't have spectrophotometer at hand.
I can help. What you need is a calibration curve of at least three concentrations of permanganate in saltwater. The Hanna low range marine nitrate Checker gives a reading around 2.2 when 1 mL of 390 ppm permanganate is mixed with 6 mL of saltwater. Make two more dilutions and obtain the Checker reading for each. This line tells you how much permanganate is left after you run a test. This is an approximate calibration curve because it is not obtained in exactly the same matrix as the test is run. You can worry about that later if you need to increase the accuracy of the test. You might also want to actually titrate the permanganate at the end of the test to double check its concentration independent of the Checker. That can wait.

1 ppm organic carbon in my system is barely but reliably detectable by this method. If your system is very depleted of carbon, you might not see permanganate consumed. If you want to make sure the method is working, test skimmate diluted with aquarium water or new saltwater.

Let me know if this explanation makes sense.
 

EnterName

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I can help. What you need is a calibration curve of at least three concentrations of permanganate in saltwater. The Hanna low range marine nitrate Checker gives a reading around 2.2 when 1 mL of 390 ppm permanganate is mixed with 6 mL of saltwater. Make two more dilutions and obtain the Checker reading for each. This line tells you how much permanganate is left after you run a test. This is an approximate calibration curve because it is not obtained in exactly the same matrix as the test is run. You can worry about that later if you need to increase the accuracy of the test. You might also want to actually titrate the permanganate at the end of the test to double check its concentration independent of the Checker. That can wait.

1 ppm organic carbon in my system is barely but reliably detectable by this method. If your system is very depleted of carbon, you might not see permanganate consumed. If you want to make sure the method is working, test skimmate diluted with aquarium water or new saltwater.

Let me know if this explanation makes sense.
Ordered the 0.01mol permanganate solution, I'll see how it works out and come back to you if you don't mind :)
 

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Ordered the 0.01mol permanganate solution, I'll see how it works out and come back to you if you don't mind :)
Looking forwards to your results!
Hey Dan,

I have prepared freshly mixed 35ppt water, the 390ppm KMnO4 solution, and the 2% NaOH solution. I can also sterile filter tank water and skimmate of course.

What other dilutions for the KMnO4 solution do you recommend I should try?

Your instructions say to use 5mL sample, 1mL NaOH solution and 1mL KMnO4 solution. The sample vials require 10mL but I assume 7mL will be enough to reach the height of the photosensor in the HannaChecker.

I assume you are zeroing the HannaChecker with the same water used for the sample?
 
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Hey Dan,

I have prepared freshly mixed 35ppt water, the 390ppm KMnO4 solution, and the 2% NaOH solution. I can also sterile filter tank water and skimmate of course.

What other dilutions for the KMnO4 solution do you recommend I should try?

Your instructions say to use 5mL sample, 1mL NaOH solution and 1mL KMnO4 solution. The sample vials require 10mL but I assume 7mL will be enough to reach the height of the photosensor in the HannaChecker.

I assume you are zeroing the HannaChecker with the same water used for the sample?
Oh sorry, one important detail I left out for using the Checkers for measuring a 5 mL (or 7 mL) solutions. You need to put spacer under the vial to raise it up to the light path. Since the lid won’t close you will need to cover the protruding vial. I use an opaque pill bottle but aluminum foil will work or anything that will prevent stray from light entering the Checker.

As for the spacer, i think it’s 1.7 cm but I will confirm that tomorrow. I cut the rounded end of a cylindrical pipette bulb to size. Luckily this was the correct diameter to easily fit into the Check and securely support the vial. There must be hundreds of things that can serve as a spacer.
 

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