Rapid Degradation of Hydrogen Peroxide

[Dan_P]

Two recent posts

https://www.reef2reef.com/threads/analyzing-a-bacterial-method-for-dinoflagellates-and-cyano.635165/

https://www.reef2reef.com/threads/what-does-adding-hydrogen-peroxide-do-to-tank-chemistry-and-organizims-in-tank.640936/

contained information about the use of hydrogen peroxide that made me wonder whether hydrogen peroxide actually worked as described. Both the claim of what hydrogen peroxide does and the seemingly small effective concentration at which it achieves these things raised several questions, the first being “does it even survive in the aquarium.” This post provides data that we might use to answer the question, at least provisionally.

Hydrogen peroxide (3%) was added to a range of solutions and the resulting ~1 ppm solutions held at room temperature for twenty fours. Using the Hanna hydrogen peroxide test, the concentration of hydrogen peroxide was determined at the beginning and end of the hold period (bar chart). As expected, hydrogen peroxide was stable in RO/DI. The variation in the height of the first pair of bars gives an indication of the variation of the method in my hands.

Hydrogen peroxide mixed with freshly prepared Instant Ocean and two samples of aquarium water also showed little or no hydrogen peroxide degradation. When dinoflagellates and substrate were mixed with aquarium water, most or all of the hydrogen peroxide was consumed (last two pairs of bars). This suggests hydrogen peroxide could be totally consumed when added to an aquarium? How long would 1 ppm of hydrogen peroxide last in an aquarium?

The second graph shows the results of two consecutive doses to my fish only aquarium. 1 ppm is consumed in about one hour. Does this happen in every aquarium? Does the rate vary by time of day? What things are responsible for hydrogen peroxide consumption? Is the consumption a “useful consumption”, e.g., killing or incapacitating a microorganism, or just the conversion of H2O2 to water and O2?

I had an offline discussion with @brandon429 on the sort of experiments that might be useful concerning hydrogen peroxide dosing. Maybe this data set has raised some questions you would like answered. Let me know.

 

[brandon429]

Dan thank you tons for always bringing strong science to this topic, we've only been able to assess using rough patterns from thread activity it's great to see logged measures for it

I'm studying the information you've produced and trying to consider how orp readings posted by dosers might coincide with the direct peroxide test details. Was trying to see if their actions picked up by the meter shows any ties into this excellent initial testing. I don't think this exists anywhere else in aquarium forums! First direct peroxide measuring for aquarium science I've seen or read about

Orp peaks and troughs are how we've been guessing about degradation times

As I read the various consumption differences in your chart and look back on others orp measures, it does seem orp actions average about four hours until back to normal across tanks with orp + dosing peroxide. Orp has many other steps involved is this right vs the test above which is just that molecule bring dissolved? Something makes orp take a bit longer to rebound... residual acids from organic cleaving taking time to become bound?

 

[TARHEEL78]

4 hours is pretty much spot on for my ORP to return to normal after dosing. This thread reminded me that I was supposed to test ORP in RO water when dosing. Sorry @brandon429 I forgot all about it.

 

[brandon429]

It’s ok np! It was helpful to have updated readings for orp from the tank anyway, it continues a true pattern for us to observe in tank

First I saw the pattern/paradox of dropping ORP via peroxide was a post by boomer in reefcentral chem forum circa from about 2006 ish and it’s neat to see how consistent the effect is even through today, we expect an orp raise from dosing peroxide but instead we get an initial drop, in a reef tank. I bet it’s not dropping in Dans ro/di measure above

 

[Randy Holmes-Farley]

4 hours is pretty much spot on for my ORP to return to normal after dosing. This thread reminded me that I was supposed to test ORP in RO water when dosing. Sorry @brandon429 I forgot all about it.

Your ORP rose or fell on hydrogen peroxide dosing? Most see a fall which is not simple to understand. One possibility is cells breaking open, releasing low ORP contents.

 

[TARHEEL78]

Your ORP rose or fell on hydrogen peroxide dosing? Most see a fall which is not simple to understand. One possibility is cells breaking open, releasing low ORP contents.

It fell, takes almost exactly 4 hours to come back to normal range.

 

[Randy Holmes-Farley]

It fell, takes almost exactly 4 hours to come back to normal range.

I’d just be careful to not assume that is tracking actual presence of hydrogen peroxide, but rather some secondary effect.

 

[TARHEEL78]

I’d just be careful to not assume that is tracking actual presence of hydrogen peroxide, but rather some secondary effect.

Oh, of course. I had assumed the ORP drop was a secondary effect and I just dose 1ml/gal every 12 hours which seems to be the standard.

 

[Dan_P]

Dan thank you tons for always bringing strong science to this topic, we've only been able to assess using rough patterns from thread activity it's great to see logged measures for it

I'm studying the information you've produced and trying to consider how orp readings posted by dosers might coincide with the direct peroxide test details. Was trying to see if their actions picked up by the meter shows any ties into this excellent initial testing. I don't think this exists anywhere else in aquarium forums! First direct peroxide measuring for aquarium science I've seen or read about

Orp peaks and troughs are how we've been guessing about degradation times

As I read the various consumption differences in your chart and look back on others orp measures, it does seem orp actions average about four hours until back to normal across tanks with orp + dosing peroxide. Orp has many other steps involved is this right vs the test above which is just that molecule bring dissolved? Something makes orp take a bit longer to rebound... residual acids from organic cleaving taking time to become bound?

My pleasure. It’s fun shining a light down a rabbit hole (@taricha ) to see what’s down there , Just need to take care not to fall in.

It would be great to see those ORP curves from a dozen systems so we could overlay them and ponder their meaning.

I would expect ORP values to climb ( more positive) as the hydrogen peroxide concentration climbs (would dissolving 1 ppm of oxygen give the same effect?). The mixing time for the system and consumption time of peroxide might be such that the entire system does not see the full dose of hydrogen peroxide. This is one big black box (not for an engineer though, any takers?) that will be different for every system. So here is one inherent problem with the current approach to dosing hydrogen peroxide: large differences in dosed amounts are likely. Does this explain the variability/reliability of the approach?

The second potential issue is judging what an effective dose is for your system. What data drives the decision to dose 1 mL/10 gallons in every single aquarium?

The third issue is around the fate of hydrogen peroxide. Are we just dosing 1 ppm of oxygen, i.e., making bubbles? Do we think hydrogen peroxide is damaging microorganisms? What data is available that shows hydrogen peroxide is an effective sanitizer? Is oxygen a sanitizer? And exactly where in the aquarium is the hydogen peroxide being consumed and is it where we want it/need it to work, whatever “work” means?

The more I look down this rabbit hole the deeper it appears. Looking forwards to ideas to probe this further.

 

[taricha]

Dan, my favorite reference on peroxide vs phytoplankton is here: Applicability of Hydrogen Peroxide in Brown Tide Control – Culture and Microcosm Studies
It covers a lot of ground - A few takeaways.
Cyano gets hit hard by h2o2.
"Cyanobacteria were found more sensitive to H2O2 than eukaryotic phytoplankton. Low dose hydrogen peroxide has been shown successful in controlling a cyanobacteria bloom and microcystin toxin caused by Planktothrix agardhii. In the study of Matthhijs et al. a special dispersal device (water harrow) was used to disperse H2O2 to the lake at 2 mg L−1, it resulted in more than 99% reduction of bloom organism of Planktothrix agardhii and algal toxin microcystin within a few days, while the eukaryotic phytoplankton, zooplankton, and macrofauna remained largely unaffected."

They also subjected 12 different phyto of various classes to h2o2 - small cells fare poorly.
"Exposure of these species to H2O2 at 6.4 mg L−1 revealed cell size dependent species sensitivity (Table 2). It was found that the smaller the cell size the more sensitive it is to H2O2. For example, among the diatoms, with decreasing cell size (Tw >Sc >Tp >Mpo >Pt) the percent inhibition increased from non-inhibitive in Sc and Tw to more than 90% inhibition in Pt"

And in addition to cell size they found that a cell wall such as silica in diatoms or cellulose theca in dinoflagellates reduced the effect of h2o2.
"Other than cell size, the cellular structure, particularly the presence of rigid cell wall, may contribute to a species' H2O2 resistance. For example, the silica-mucilage cell wall of the diatoms may have given Sc and Tw their H2O2 resistance ... The rigid cell wall, if present, potentially provides a first layer of defense against H2O2 and HO·, and allowing them to be inactivated before reaching the more sensitive and biologically critical components of cell membrane and organelles."

All these photosynthesizers produce small amounts of their own reactive oxygen species in their chloroplasts when the photosystem gets too much excitation. So these organisms have means to deactivate the h2o2 - if it's not too much.
"Once H2O2 penetrates into a cell, reactive oxygen species scavenging systems would decompose and inactivate the excessive amounts of H2O2 ...This, along with the capability of the cell to repair and replace the damaged biomolecules and cellular components, permits the culture to re-grow, as was observed in both the culture and the microcosm studies at relatively low H2O2 exposure."

Also here's a post I made about an old beaker test I did on a troublesome dinoflagellate vs h2o2. Ostreopsis is large (~50 microns) and has a cellulose theca (armor). I found it took enough h2o2 - 2ml/L that I figured it was probably not practical for tank treatment. And worse, if I put a sliver of chaeto or caulerpa in the beaker, then the effect on the dinos went down - meaning that the presence of other targets increases the amount needed to inactivate the nuisance.

BTW 1mL/ 10Gal of 3% h2o2 comes out to ~0.8 mg/L (I think) and cyano killing doses reported in this paper and elsewhere range generally from a couple of tenths of a mg/L to about 2 mg/L. So the standard aquarium recommended dose has some backing. Also these levels in the published lit are found to harm very little else.

My Qs on this issue are - does peroxide kill stuff other than photosynthetic organisms in a tank context? I only ever run across discussion of it targeting those.

And secondly on the ORP, the mechanisms of peroxide effect seem to involve h2o2 diffusing through the cells and when I've examined killed dino or diatom cells they seemed entirely intact. If it's not lysing these cells, what is it busting up to release a bunch or reducing agents to drop ORP?

 

[Rick Mathew]

Two recent posts

https://www.reef2reef.com/threads/analyzing-a-bacterial-method-for-dinoflagellates-and-cyano.635165/

https://www.reef2reef.com/threads/what-does-adding-hydrogen-peroxide-do-to-tank-chemistry-and-organizims-in-tank.640936/

contained information about the use of hydrogen peroxide that made me wonder whether hydrogen peroxide actually worked as described. Both the claim of what hydrogen peroxide does and the seemingly small effective concentration at which it achieves these things raised several questions, the first being “does it even survive in the aquarium.” This post provides data that we might use to answer the question, at least provisionally.

Hydrogen peroxide (3%) was added to a range of solutions and the resulting ~1 ppm solutions held at room temperature for twenty fours. Using the Hanna hydrogen peroxide test, the concentration of hydrogen peroxide was determined at the beginning and end of the hold period (bar chart). As expected, hydrogen peroxide was stable in RO/DI. The variation in the height of the first pair of bars gives an indication of the variation of the method in my hands.

Hydrogen peroxide mixed with freshly prepared Instant Ocean and two samples of aquarium water also showed little or no hydrogen peroxide degradation. When dinoflagellates and substrate were mixed with aquarium water, most or all of the hydrogen peroxide was consumed (last two pairs of bars). This suggests hydrogen peroxide could be totally consumed when added to an aquarium? How long would 1 ppm of hydrogen peroxide last in an aquarium?

The second graph shows the results of two consecutive doses to my fish only aquarium. 1 ppm is consumed in about one hour. Does this happen in every aquarium? Does the rate vary by time of day? What things are responsible for hydrogen peroxide consumption? Is the consumption a “useful consumption”, e.g., killing or incapacitating a microorganism, or just the conversion of H2O2 to water and O2?

I had an offline discussion with @brandon429 on the sort of experiments that might be useful concerning hydrogen peroxide dosing. Maybe this data set has raised some questions you would like answered. Let me know.

Excellent!...As always well done...lots to explore here...Pretty deep rabbit hole...will follow this one

 

[Dan_P]

Dan, my favorite reference on peroxide vs phytoplankton is here: Applicability of Hydrogen Peroxide in Brown Tide Control – Culture and Microcosm Studies
It covers a lot of ground - A few takeaways.
Cyano gets hit hard by h2o2.
"Cyanobacteria were found more sensitive to H2O2 than eukaryotic phytoplankton. Low dose hydrogen peroxide has been shown successful in controlling a cyanobacteria bloom and microcystin toxin caused by Planktothrix agardhii. In the study of Matthhijs et al. a special dispersal device (water harrow) was used to disperse H2O2 to the lake at 2 mg L−1, it resulted in more than 99% reduction of bloom organism of Planktothrix agardhii and algal toxin microcystin within a few days, while the eukaryotic phytoplankton, zooplankton, and macrofauna remained largely unaffected."

They also subjected 12 different phyto of various classes to h2o2 - small cells fare poorly.
"Exposure of these species to H2O2 at 6.4 mg L−1 revealed cell size dependent species sensitivity (Table 2). It was found that the smaller the cell size the more sensitive it is to H2O2. For example, among the diatoms, with decreasing cell size (Tw >Sc >Tp >Mpo >Pt) the percent inhibition increased from non-inhibitive in Sc and Tw to more than 90% inhibition in Pt"

And in addition to cell size they found that a cell wall such as silica in diatoms or cellulose theca in dinoflagellates reduced the effect of h2o2.
"Other than cell size, the cellular structure, particularly the presence of rigid cell wall, may contribute to a species' H2O2 resistance. For example, the silica-mucilage cell wall of the diatoms may have given Sc and Tw their H2O2 resistance ... The rigid cell wall, if present, potentially provides a first layer of defense against H2O2 and HO·, and allowing them to be inactivated before reaching the more sensitive and biologically critical components of cell membrane and organelles."

All these photosynthesizers produce small amounts of their own reactive oxygen species in their chloroplasts when the photosystem gets too much excitation. So these organisms have means to deactivate the h2o2 - if it's not too much.
"Once H2O2 penetrates into a cell, reactive oxygen species scavenging systems would decompose and inactivate the excessive amounts of H2O2 ...This, along with the capability of the cell to repair and replace the damaged biomolecules and cellular components, permits the culture to re-grow, as was observed in both the culture and the microcosm studies at relatively low H2O2 exposure."

Also here's a post I made about an old beaker test I did on a troublesome dinoflagellate vs h2o2. Ostreopsis is large (~50 microns) and has a cellulose theca (armor). I found it took enough h2o2 - 2ml/L that I figured it was probably not practical for tank treatment. And worse, if I put a sliver of chaeto or caulerpa in the beaker, then the effect on the dinos went down - meaning that the presence of other targets increases the amount needed to inactivate the nuisance.

BTW 1mL/ 10Gal of 3% h2o2 comes out to ~0.8 mg/L (I think) and cyano killing doses reported in this paper and elsewhere range generally from a couple of tenths of a mg/L to about 2 mg/L. So the standard aquarium recommended dose has some backing. Also these levels in the published lit are found to harm very little else.

My Qs on this issue are - does peroxide kill stuff other than photosynthetic organisms in a tank context? I only ever run across discussion of it targeting those.

And secondly on the ORP, the mechanisms of peroxide effect seem to involve h2o2 diffusing through the cells and when I've examined killed dino or diatom cells they seemed entirely intact. If it's not lysing these cells, what is it busting up to release a bunch or reducing agents to drop ORP?

Thanks for the reference. It addressed my questions. We need a library on R2R and a aggregator to pull together the “science behind the methods”.

You and Google are among the best innovations in the past several decades!

I recently posted an example of dosing H2O2 to a saline solution, where it produced the same large drop in ORP and slow recovery. I think we can close the books on the notion that the negative drop means anything and that it is a useful diagnostic tool. Colorimetric tests for H2O2 are the way to go.

I was disappointed that the authors of the paper you referenced did not follow the time course of H2O2 concentration with growth inhibition. They did reference other work that supported the notion of significant H2O2 stability, but I wonder whether that was after a referee called them on it. I have an infestation of big dinoflagellates, maybe the ones you said were resistant to H2O2, and will use them to study H2O2 stability. It might be possible to use KMnO4 to monitor COD and look for cell exudate or lysis products. At this point my studies would be more academic then an attempt to validate the H2O2 method against dinoflagellates. BUT I am still curious about its dose dependent effectiveness against macro algae and the filamentous cyanobacteria I am culturing.

 

[Randy Holmes-Farley]

And secondly on the ORP, the mechanisms of peroxide effect seem to involve h2o2 diffusing through the cells and when I've examined killed dino or diatom cells they seemed entirely intact. If it's not lysing these cells, what is it busting up to release a bunch or reducing agents to drop ORP?

Were those free floating cells, or cells in a biofilm? Were you looking for broken cells in the bulk water?The cells right where the H2O2 hits the water in high concentration will be hit hardest and may be breaking while cells elsewhere take longer to break apart (dead cells will always break open eventually). That said, this is just a hypothesis and other explanations may be the real reason for the ORP drop.

 

[brandon429]

I'm 100% sure the one mil per ten came about this way:

someone made it up not sure who lol it wasn't based on scientific testing because there isn't any aquarium specific studies for in tank peroxide use I know of, never found any on scholar.


It was a random guess threshold where targets die and nontargets live, consistently as well. Repeated by thousands not from reading peer reviewed publishings, but from friends reviewing peer tanks before and after testimonies using that guesstimate dilution.

*strength of solution differs home to home due to new bottle vs half flat, that accounts for some range in thread outcomes.

the demand for working algae control outpaced what legit slow accurate lab reviewed science could provide, the current advice of the 90s worked great for reef sages, Bob Ross naturals, but not for average folks as we followed the rules but still got algae

So a swarm of hobbyists who could no longer afford to lose aquariums awaiting the next algae control published offer had to speed things up, and they did.

The list of sensitive animals are those who can die within that 1:10 range and no fish we keep are on it, amazing imo spread, and no corals other than Xenia are found in pattern analysis to be harmed, it covers nearly everything we keep in the 1:10 dosage.
Outliers can happen with corals, a guy lost some zoanthids dosing kalk once too.

Most sensitive is lysmata, somehow their metabolic machinery cannot take peroxide that too is amazing. Ld50 lysmata = any drop you add to the water can kill them...amazing sensitivity among organisms who've adapted alongside with amazing resistance. Any lysmata that survives peroxide is the outlier.

We have cured to a very high degree of repeatability using daily 1:10 dosing:
Gha tanks although dosing water is old school there's a better way
Invasive macro caulerpa tanks
Cyano
Dinos, Lees thread gen forum one of the longest running peroxide thread maybe the longest
Greenwater
Bac blooms although when not carbon dosing I'm skeptical of bandwagon hazy water = bac bloom. We find sandbed non rinsing to play a role at times such that 20 + page threads exist solely to remedy that and hazy water stops for many.

in general peroxide helps clear water column this is commonly reported.

 

[brandon429]

Taricha amazing post really, reading up. Thank you for the highlights

 

[Dan_P]

Were those free floating cells, or cells in a biofilm? Were you looking for broken cells in the bulk water?The cells right where the H2O2 hits the water in high concentration will be hit hardest and may be breaking while cells elsewhere take longer to break apart (dead cells will always break open eventually). That said, this is just a hypothesis and other explanations may be the real reason for the ORP drop.

These are really great questions. They will guide my future experiments.

I need to also tap into @taricha experience

 

[taricha]

Were those free floating cells, or cells in a biofilm? Were you looking for broken cells in the bulk water?The cells right where the H2O2 hits the water in high concentration will be hit hardest and may be breaking while cells elsewhere take longer to break apart (dead cells will always break open eventually). That said, this is just a hypothesis and other explanations may be the real reason for the ORP drop.

Two circumstances. One was dinos in a beaker killed with 2mg/L h2o2 examined under microscope.
Another was a dish with a couple mL of tank water under scope. Dropped h2o2 directly in water on dino and diatom cells.
Both cases these were harvested from a filter floss that was hung in the water to catch whatever. Mostly dinos and a few diatoms attached.
In neither case did cells appear lysed. Whereas freshwater disrupts dino cells (sheds armor) in seconds.

Actually, I have an idea to check...
Red cyano turns water pink when killed (with h2o2 or any other reason). If the pigment goes into the water quickly after h2o2, then the cells have been compromised. How fast is the ORP drop that we measure? Like 10 min or 1 min?

 

[taricha]

I see Dan found the root answer to the ORP drop question, that it's chemical and not biological. See post quoted below.

This drop seems normal. It is a pH effect, otherwise, it should go up.

https://aquariustech.com.au/wp-content/uploads/bsk-pdf-manager/ORP_Chlorine_peroxyacetic_10.pdf

“Note that the dissociation constant (pKa) of hydrogen peroxide is 11.62, and hypochlorous acid (HOCL) is 7.4, expressed in terms of pH. It then becomes obvious that the peak activity of the Eo for hypochlorous acid is very near the pH of the water being treated, and the H2O2 is considerably higher. Thus, H2O2 would express a much lower ORP value at pH 7.4 than would hypochlorous due to the vast difference in the pKa constant.”

From Researchgate

“Simon - did you find the answer to this question? I have experienced the same result but I can't find a good explanation for the cause. My test conditions are 0.5M NaCl at 65°C. I injected H2O2 to produce 3 ppm H2O2 in solution and found that the potential dropped by about 150 mV”

But I had done this test anyway....

Actually, I have an idea to check...
Red cyano turns water pink when killed (with h2o2 or any other reason). If the pigment goes into the water quickly after h2o2, then the cells have been compromised

Took 100mL tank water and some sand/debris with a little cyano in it. Mixed thoroughly and split in two, Added h2o2 (1mL) to one beaker to simulate the high concentration that might occur near the point of h2o2 addition.
about 15 min later, ran water from control and h2o2 addition through .44 micron filter to remove whole cells/particles and see if any pigment leaked out of broken cells and went into water.
Fluorescence showed the red cyano pigment phycoerythrin in the water that was hit with h2o2 but not the control.

Conclusion, at least cyano cells (maybe other cells too) are in fact compromised by peroxide within minutes. I don't know if heterotrophic bacteria would also meet this fate from h2o2 or not. They aren't as easy to track.