Organics (DOC) measurement methods for aquarium water

[Randy Holmes-Farley]

Are you sure of this in saltwater when you have a KH around 7 ?


Does not this mean that Delta pH is independed of the alkalinity

By the way - I took a pH test (Hanna) yesterday and saved 10 ml untested sample in a test tube and took a test of that after 24 hours - delta pH around 0.2. I will do some more test later on

Sincerely Lasse

Yes, the delta pH of any acid or base addition to seawater depends on alk inversely and in a more complicated way on starting pH because the buffering of seawater varies with pH and alkalinity.

 

[Randy Holmes-Farley]

For me DOC is the labile fraction of TOC. I know that yellowing substances normally are seen as a part of DOC and at least the UV method incorporate them in the result. But they are among the most hard-to-degrade substances in the organic carbon group - IMO - only strong chemical oxidisers - like O3 and in some degree H2O2 will mineralise them.

Sincerely Lasse

Thst should be called LDOC (Lasse’s DOC) so as to not confuse it with the real definition of DOC, which has nothing to do with whether organisms can metabolize it., and only has to do with it not being a particle.

 

[Lasse]

Thst should be called LDOC (Lasse’s DOC) so as to not confuse it with the real definition of DOC, which has nothing to do with whether organisms can metabolize it., and only has to do with it not being a particle.

Yes - that´s true because the yellow substances (directly translated from Swedish humus matter or maybe Humic substances) are more of an end product of the bacterial decomposition (mineralisation) of organic matter than the rocket fuel for heterotrophic bacteria activity that we maybe should be concerned of. In the Swedish language we have a word (sentence) for this part of DOC - snabb organisk kolkälla - directly translated to "fast organic carbon source" This part of the DOC has importance as an accelerator both in aerobic and anaerobic bacterial mineralisation (decomposition).

If we could find an accepted word for this part of the DOC (it is the same part that reef builders article deal with) it could help with the understanding. FDOC (Fast Dissolved Organic Carbon) or SDOC (Simple Dissolved Organic Carbon) may be a better suggestion than LDOC

Sincerely Lasse

 

[taricha]

The pH drop caused by bacteria metabolism during 1 or 2 days could be an indirect measurement of DOC......... Is there any one that know how stable is the dye in Hannas pH measurement kit for the checker and Hanna Marine Master?

By the way - I took a pH test (Hanna) yesterday and saved 10 ml untested sample in a test tube and took a test of that after 24 hours - delta pH around 0.2. I will do some more test later on

Yes, this sort of game does work. You can use a bit of pH indicator in a culture sample to track the pH while the culture is growing without having to open it. Nice way to reduce contamination risk.
Here I did a number of bacterial products vs the filtrate of 20mg/L sterilized fish flake. I was trying to grow them on low food amounts - this meant I couldn't just use turbidity to track growth because it was too small.
So I added some phenol Red to the sample media and tracked the color change over time without opening any bottles.
It gives a good picture of who ate how much and when.

(product 1 and 2 didn't activate and looked like the distilled water over 30 days. bacteria from aquarium water and sand/grunge were the most active)

 

[Lasse]

I would be very interesting if you redo the experiments sometimes in the future and beside the fish food - you ad a few drops of ethanol in some of the samples. It could be interesting to know if "turbo" mode exist in saltwater as it does in freshwater sewage plants.

Sincerely Lasse

 

[MnFish1]

I do not understand what you mean?

Sincerely Lasse

Hi Lasse, I was not referring to your experiments (nor anyone else in particular). It was meant to be a general statement. Example (made up) - I as an aquarist think that an N ratio of xxx is needed for coral to thrive and the 'ugly stage' not to occur. Depending on the design of the experiment one could easily design an experiment showing that the original theory is correct. Sorry I wasn't pointing at an experiment here again - it was a general comment

 

[Dan_P]

Yes, this sort of game does work. You can use a bit of pH indicator in a culture sample to track the pH while the culture is growing without having to open it. Nice way to reduce contamination risk.
Here I did a number of bacterial products vs the filtrate of 20mg/L sterilized fish flake. I was trying to grow them on low food amounts - this meant I couldn't just use turbidity to track growth because it was too small.
So I added some phenol Red to the sample media and tracked the color change over time without opening any bottles.
It gives a good picture of who ate how much and when.

(product 1 and 2 didn't activate and looked like the distilled water over 30 days. bacteria from aquarium water and sand/grunge were the most active)

Have you thought about selling your aquarium water as a probiotic? I would buy a few gallons. Could help build up a couple college funds

 

[taricha]

Have you thought about selling your aquarium water as a probiotic? I would buy a few gallons. Could help build up a couple college funds

It would sell great, I have good data to support how fast and active it is.
But I'd get blasted when telegraham sent my stuff to aquabiomics and showed that my "super active, fast consumers of organics" were mostly vibrio and closely related types.

 

[taricha]

Since it's already been brought up (didn't know oceamo was doing UV254 abs) Let's talk about the optical measures: Fluorescence and Absorbance


Absorbance by organics in saltwater increases at shorter wavelength through the visible into the UV. In the visible/near UV, the trend is just a slope with increasing absorbance for shorter wavelength. In the far UV, there are actually some features where certain organics have peaks (DNA peak absorbance very near 254nm, hence UV-C sterilization). In the visible this means blue/violet is more absorbed and water goes "yellow".

Visible absorbance:

The lowest tech version of this is the common "white bucket test":
Fill a white 5gal bucket with new mix saltwater, and a second white bucket with your tank water. Some people have used the comparison that if it's noticeably different side-by-side, they change their GAC or run ozone. Both are useful for reducing the yellow compounds.

(Fun google scholar term: "marine gelbstoff". Academics like it, because if you don't know what it is you can just say "yellow stuff" but in German and it sounds like a technical description )

If you want to make it quantitative, then you can get a narrow tube - rigid PVC is fine - over a foot long, glue a glass or thin plastic viewing window on each end, and drill a fill/drain hole on one end.
Get a violet LED in the 400-420 range - connect to a 9V battery and a variable resistor, and some method of light sensor on the other end. The light sensor can be a spectrophotometer, or just a light intensity meter that'll give you raw intensity. The chips inside hanna meters simply output a voltage proportional to light intensity which is perfect - but those chips are a few bucks, no need to rip apart a checker for it. A PAR meter might also do the trick, PPFD is literally counting photons, so that'll work.
Fill the tube with distilled water, get the light intensity. Empty and fill with tank water, and get the new light intensity.
Absorbance = log10(Initial intensity/intensity through the sample)

Divide this absorbance number by the length of the tube in cm and you have Abs per cm at that wavelength, which is a pretty standard thing. The paper Lasse shared used Abs@410nm as one of the parameters for comparison.

Here’s an example of data that Dan acquired with a 56” long tube comparing the absorbance of water in the tank vs the effluent from the skimmer and GAC.

The skimmer may not be significant but the GAC (5 day old at the time) certainly was.


UV-Absorbance:
UV absorbance is a well-studied measure, because many organic compounds absorb in that region - including proteins and DNA, and because that emission wavelength is available from mercury lamps for UV-C sterilization. Now, LEDs are getting better and better making this more approachable for the hobbyist.
The open source iorodeo colorimeter now has a 255nm UV-C LED version for DNA quantification. The calibration curve for DNA has an absorbance in 1cm cuvettes of about 0.02 Abs per 1ppm DNA, this suggests to me probable sufficient sensitivity to detect the absorbance from amino acids/proteins in the tank water even in a 1cm cuvette. Additionally Oceamo’s target values of 2-8 abs/m = 0.02-0.08 abs/cm makes me think that yes, a colorimeter like iorodeo can make measurements in that range, even with the default 1cm cuvette. There’s a bunch of papers on abs at 254nm for marine waters, and how it well-correlates with DOC.

It is also possible to measure UV-C absorbance in a DIY way. Here’s a proof of concept, but not a safe enough implementation for hobby use.
I cut a window (1" square) into my UV sterilizer - a cheap 9W "green killing machine". Use a fluorescent target that is excited by the UV-C radiation, but is dim from the sterilizer light that gets through a pane of clear glass - the optical brighteners in paper or cardstock actually give me a really good result. Yellow highlighter ink works also.
I put the fluorescent target (peice of cardstock) at the bottom of a glass beaker, and placed a light meter just outside the beaker aiming at the fluorescent target. I mounted the UV-C unit just above the beaker with the window I cut over the fluorescent target.

Then I got measurements of the light intensity from the fluorescent target (cardstock) at different depths of water as I added water in the beaker - both distilled and Tank water. To set the “zero” - I put a thin clear pane of glass in front of the sterilizer window to block all UV-C light and measure the illumination of the fluorescent target with no UV-C getting through. Below is the proof-of-concept data.

The distilled water (blue) shows no blocking of the UV-C, it gets slightly brighter as the water level rises due to refractive index/reflection effects inside the beaker. The tank water (red data) significantly absorbs the UV-C causing dimming of the fluorescent target - almost down to the “zero” level of illumination from putting a glass pane in front of the UV-C window (yellow data).
We can actually calculate approximate 254nm absorbance of tank water from this data by subtracting the value through the glass pane from the distilled and tank water data.

Abs = Log10((distilled-glass)/(Tankwater-glass))
and here’s the calculated absorbance vs depth of water in the beaker….

We get a nicely linear trend with water depth added. And we calculate a value of 0.104 Abs per cm - this would be 10.4 per m, or above the Oceamo recommendation range of 2-8 per m...

This is a reasonable result since I pulled GAC out for something a couple of months ago and forgot to put it back.

How useful is optical absorbance for predicting bacterial food content of water?
Are these optical absorbance measurements important (in the context of bacterial food sources)? In my opinion, unlikely. The visible light absorption is yellow stuff that is probably bacterial waste products (Fluorescence characteristics of humic-like fluorescent dissolved organic matter produced by various taxa of marine bacteria[pdf]) and other papers have found that a significant portion of the stuff that accumulates and sticks around in marine water is from carotenoids that have been broken down (Carotenoids are the likely precursor of a significant fraction of marine dissolved organic matter). This also aligns with yellow-er water in macroalgae heavy systems.
So if you are looking at dissolved organic matter as a source to drive bacterial growth - this yellow stuff is not growing much of anything very fast. There are certainly going to be some bacteria - maybe specialized types - that take advantage of this food source (especially when you concentrate them by binding onto GAC surface). And perhaps these groups show up in microbiome samples of tanks with yellow water - but a dilute, low nutrition source that is broken down very slowly is not a good candidate for growing pathogenic bacteria - or much bacteria at all.

So, my reasoning is that this stuff is not the DOM you are looking for - and measuring it is probably only useful to the degree that its measurements correlate with the presence of yummier, labile (easily digestible) organics.
Absorbance at 254nm is a little bit better of a marker than the visible yellow stuff, because at least at 254nm, it also can show proteins, aminos etc - which are a richer food source. But as Oceamo says “Stability measurements have been conducted, showing that the SAC254 value does not significantly change over several weeks.” - which tells us that most of it is not labile organics but instead the waste products that bacteria don’t have much use for.

Next - Fluorescence
(measuring fluorescence of tank water directly)

 

[Lasse]

The skimmer may not be significant but the GAC

I have tested (visually) GAC, oxidator and Ozone in order to get rid of the "marine gelbstoff" The experience I have indicate that ozone is most effective followed by oxydator and after that GAC. My aquarium water - changed around 60 % in 6 years is a little bit yellow and I have run oxidator all the time. It will be interesting to know what my next Oceamo test will say about it.

Sincerely Lasse

 

[Christoph]

Taricha, nice DIY measurement Just be very careful with UV-C and your eyes.
Also noteworthy thats not a monochromatic 254 nm source, which will surely affect the measurement.

 

[taricha]

Taricha, nice DIY measurement Just be very careful with UV-C and your eyes.
Also noteworthy thats not a monochromatic 254 nm source, which will surely affect the measurement.

Thanks. Yep, definitely not safe as illustrated. I also didn't like when I realized how the uv-c was reflecting off glass surfaces. Needs full shielding to be OK.

Christoph, for the 254nm abs, are you using a 1cm cuvette? seems like the absorbance is plenty high that you might not need a longer cuvette to get a good measurement.

 

[Christoph]

Thanks. Yep, definitely not safe as illustrated. I also didn't like when I realized how the uv-c was reflecting off glass surfaces. Needs full shielding to be OK.

Christoph, for the 254nm abs, are you using a 1cm cuvette? seems like the absorbance is plenty high that you might not need a longer cuvette to get a good measurement.

yes, we use 1 cm optical path quartz cuvettes.

All the best,
Christoph

 

[Malcontent]

Adding tannins is the trendy thing in freshwater now. I find it amusing that you can get the same result by just not changing your water and letting the humic substances accumulate...

 

[Lasse]

Adding tannins is the trendy thing in freshwater now. I find it amusing that you can get the same result by just not changing your water and letting the humic substances accumulate...

Its bumping back - back in the 50-60 ties old yellow water was pure gold for aquarists - the only that quarantine that some fishes did not die. And if someone dare to test these hypotheses with a WC - all fish die. What few people known at that time was that the pH in the old water was around 4, total ammonium 200 - 400 mg/L because all nitrification was stopped as alkalinity was around zero too. WC => toxic NH3 was formed - all fish die. The solution was WC from the beginning, nitrification filter in order to reduce both NH3/NH4 and NO2 - NH3/NH4 could be toxic and NO2 is toxic in chloride concentrations below 100 mg/L.

Sincerely Lasse

 

[Malcontent]

This is one of the earliest papers on DOC.

DOC inhibits fish growth at lower levels than nitrate.

DOC appears to be controlled by filter cleaning while nitrate is controlled by changing water.

The trendy thing in the freshwater hobby is to obsess over nitrate but not to clean filters which seems backwards to me since DOC is more dangerous than nitrate.

 

[HomebroodExotics]

This is one of the earliest papers on DOC.

DOC inhibits fish growth at lower levels than nitrate.

DOC appears to be controlled by filter cleaning while nitrate is controlled by changing water.

The trendy thing in the freshwater hobby is to obsess over nitrate but not to clean filters which seems backwards to me since DOC is more dangerous than nitrate.

I disagree that it’s doc that is the danger. Bacteria and pathogens will consume doc and proliferate. These will kill the fish and also starve the fish for oxygen in the process. It’s what the doc is growing that matters.

 

[taricha]

This is one of the earliest papers on DOC.

DOC inhibits fish growth at lower levels than nitrate.

DOC appears to be controlled by filter cleaning while nitrate is controlled by changing water.

The trendy thing in the freshwater hobby is to obsess over nitrate but not to clean filters which seems backwards to me since DOC is more dangerous than nitrate.

Nice paper.
I don't know what this means in terms of what it says about the origin material or processes that create this DOC....
"Ultrafiltration of the culture water revealed that more than a half of
the total organic substances existed as organic carbon with a molecular
weight of the order of 10^4....
The fractionation of dissolved organic matter in
coastal seawater by ultrafiltration has shown that the fraction with a
molecular weight of an order of 10^4 usually accounts for the largest proportion of the total DOC (Ogura, 1974). "

What sorts of things are aquarium food inputs that are organics greater or equal to 10^4 molecular weight?
Or are breakdown processes making larger weight organic waste out of smaller stuff?

 

[taricha]

What sorts of things are aquarium food inputs that are organics greater or equal to 10^4 molecular weight?
Or are breakdown processes making larger weight organic waste out of smaller stuff?

One answer to my question - protein-like stuff is this size. So we put in animal and plant proteins through foods that would be this size, and also aquarium organisms make proteins this size out of smaller simpler things.

but proteins are a bacterial-digestible source that would be broken down over time in some sense, so my picture of what's going on here is still fuzzy about what processes make stuff that's a) really big, b) full of organic carbon and c) mostly not digestible.

 

[BeanAnimal]

The other reason is that the consume of Oxygen from DOC is fast but the POC consumption take a little bit longer. You get more of the POC demand if the test period is longer. For aquarium purpose - BOD3 (as suggested from @taricha ) may be the best because its mostly DOC that use oxygen during that period. However - there is DOC that is very slow in their oxygen demand and its only a COD (Chemical Oxygen Demand) analyse that can give you these figures. These include (among others) - the yellow colouring substances

It would appear to me then that meaningful information could be obtained by comparing the BOD3, BOD5 and BOD7 ratios of different samples? As in DOC : POC based on the measurement trend? Allowing some better estimate as to what was POC vs DOC?