Post your test data

[threnjen]

Source: SimplyDiscus.com: Microscopes: Nitrite Toxicity in the Aquarium by Ardan Huck
"Salt added to the aquarium, usually 2 tablespoons per 10 gallons water, helps the fish because the chloride ion (cl-) from the NACL (sodium chloride, or salt) attaches to the gill cells where oxygen enters the blood. This is also the place where nitrite enters the blood, therefore the chloride blocks the nitrite from entering. This only works well if the pH is above 7.0 as in acidic conditions Nitrite (NO2) will bind with hydrogen ions, H+ (acid contains more H+) and form Nitrous acid (HNO2). Nitrous acid is not blocked by CL- ions at the gill site, and thus can pass freely into the blood. "
Salt added for fish-in helps fish live when you get to nitrites phase?

"Water changes are often used to reduce the nitrite levels in the aquarium, however, changing 90% of the water will only reduce the nitrite concentration by 30% (Kohler, 1997). "
WHAT???? I have GOT to find this study this cites!!!!!!
Edit: It was not a study but a magazine article, so I can't put enough weight on it. Also, I can't find anywhere to read the article

 

[Caliban07]

Source: SimplyDiscus.com: Microscopes: Nitrite Toxicity in the Aquarium by Ardan Huck
"Salt added to the aquarium, usually 2 tablespoons per 10 gallons water, helps the fish because the chloride ion (cl-) from the NACL (sodium chloride, or salt) attaches to the gill cells where oxygen enters the blood. This is also the place where nitrite enters the blood, therefore the chloride blocks the nitrite from entering. This only works well if the pH is above 7.0 as in acidic conditions Nitrite (NO2) will bind with hydrogen ions, H+ (acid contains more H+) and form Nitrous acid (HNO2). Nitrous acid is not blocked by CL- ions at the gill site, and thus can pass freely into the blood. "
Salt added for fish-in helps fish live when you get to nitrites phase?

"Water changes are often used to reduce the nitrite levels in the aquarium, however, changing 90% of the water will only reduce the nitrite concentration by 30% (Kohler, 1997). "
WHAT???? I have GOT to find this study this cites!!!!!!
Edit: It was not a study but a magazine article, so I can't put enough weight on it. Also, I can't find anywhere to read the article

I had read much of this about the chloride. I didn't report because our experiment and research was dedicated to fishless cycling and not exposure to fish. I have probably lead us down this route so apologies but if you are happy to go with both studies then so am I. I thought it was commonly thought that nitrite was less toxic than ammonia and it attacks the fish in a different wau although both lead to gasping. Nitrite alters the blood cells if the fish removing the oxygen. I believe

I am starting to lose track if where we are up to now. I think it would be a good idea to compile test parameters for the helpers with what we know so far as wel as compile some facts on what we have learned so far maybe on to a word document. What do you think?

 

[Mebbid]

I'm not even going to try to read all these articles but I like the stockpile that you are creating. It might be worth making a new thread of a database of all of these scientific papers and see if it can get stickied.

 

[Caliban07]

I'm not even going to try to read all these articles but I like the stockpile that you are creating. It might be worth making a new thread of a database of all of these scientific papers and see if it can get stickied.

The thing is, most of the stuff we have learned is not new knowledge. However, we have learned some things that may not have been tested during a fishless cycle.

Some interesting facts that we have uncovered (not new) is that ammonia readings of say 0.25ppm are less toxic than originally thought. This is why I suggested in the other thread that water need not be changed if ammonia stays at this level. At that pH. As you may know. Actual free ammonia toxicity increases with pH. It baffles me that the OP has even gone as far as to add prime at free ammonia toxicity levels way below the threshold harmless to fish. It appears that some advisors who understand this feel it is necessary to advocate water changes to the newbie as a precautionary measure. Whilst this is a sensible approach I feel that people have the right to expand their knowledge further if they wish and a stickies maybe the way to do this. Although I believe there are adequate materials posted on this forum already covering this particular topic. We need to do more work and research our next step is to figure out just how toxic nitrites are to fish, what levels of exposure are harmful and how much nitrite is produced from a unit of ammonia. How efficient are nitrite consuming bacteria at consuming nitrite and is it easily taken care of in low doses before it can harm fish.

This post is not aimed at anyone in particular and we are still trying to do more research. Any help is appreciated

 

[Caliban07]

Interesting paper
http://repositorio-aberto.up.pt/bitstream/10216/376/2/13766.pdf

Again confirms that nitrospira thrive at lower nitrite concentrations whilst nitrobacter thrive at higher concentrations. According to this. Nitrospira did not come back when concentrations were lowered but may have been due to nitrospira's slow reproduction time and test length. It was said that the bacteria coexisted. This could mean that higher ammonia dosing and subsequent high nitrites may complete a cycle faster as nitrobacter is in fact a quicker multiplayer. Having said that are the low concentrations we use when cycling enough to spawn nitrobacter? I would say not as other studies have shown nitrospira to be the dominant strain during experiments that use considerably higher amounts of nitrite.

Even if the higher end of nitrite we get in our aquariums was enough to spawn nitrobacter this would be a gradual thing as these tests were conducted with an immediate initiation of nitrite which would not be true during a cycle as nitrite production would be reliant on ammonia consumption. This would mean nitrite would start small spawning nitrospira then the nitrobacter would start to grow and become the dominant strain suppressing nitrospira. This would results in an inefficient use of surface area and inefficient consummation of nitrite. In my opinion based on the info.

This is if course hypothetical since I believe from what I have read that the concentrations of nitrite would not be enough to encourage nitrobacter growth.

Nitrospira is a recent discovery and has yet to be sufficiently studied in order to understand its efficiency.

In relation to nitrite toxicity, I haven't found anything that suggests what levels of nitrite are lethal to fish. I read 1ppm is bad but not backed up by scientific data.

So that means if 1ppm ammonia converts to 2.7ppm nitrite an ammonia concentration of 1ppm would require a water change to dilute the nitrites or maybe this chloride buffer? Best to dilute in my opinion depending on tapwater baseline. However if ammonia was 0.25ppm that would mean we would only have 0.6ppm nitrite which is less than one (assuming 1ppm is in actual fact toxic to fish) 0.6ppm is fairly close to 1ppm which leads me to my next question.
How accurate are our test kits? Does it just turn the colour it is closest to? Could 0.25ppm actually be 0.1 ppm which would mean 0.2 ppm of nitrite. See what I'm saying?

We need to understand how toxic nitrite is to fish and at what levels? There must be a study on this?

 

[threnjen]

LOL Caliban! In one post you're trying to keep us on track, in the next one you're off on the irresistible tangent

This is all so interesting, it's hard to keep on track!

I agree with your post a few back - we need to come up with the test parameters for the volunteers. My husband advised me that first I need to make a chart of all of the "known" parameters relating to bacteria growth, using all of our sources. i.e. temperature (not just the ideal temps - how slowed is growth outside of them?), rate of doubling of colonies, proportion of colonies in fully cycled samples (I HAVE seen bits of this, somewhere, about the proportions) etc, ideal DO, etc. Then from there we form our hypotheses. He said that I have seen enough data that I should be able to easily run a simulation of what I would expect to see given different dosing scenarios. I will try to work on this today and post them.

As far as hypotheses, I *think*, and I can only say I think as I need to think it through more, that ultimately *my* hypothesis (I believe yours will be different) will be that:
a) The level of dosing does not influence the cycle length, but does influence the end nitrification capacity
b) Water changes can be eliminated through manual additions of phosphorus (fish food) and baking soda at cycle start
c) Cycles without water changes will complete faster than cycles with water changes
d) I feel uncertain on this one. But I'll say it. After initial dose is reached, no additional ammonia is required, and will only prolong the cycle.
e) No levels of ammonia or nitrite in our scale of application will stall the cycle

To address these in turn:
a) I'm not on board anymore that EITHER your .25 dose will end faster OR that my madcap 18ppm dose will end faster. I think it all ends up the same because I've seen so many lab graphs charting the cycle. To that end I think it will take exactly the same-ish amount of time to complete the cycle for these dosings and therefore a larger dose will prove to be more efficient
b) We only water change to stabilize pH and phosphorus, but these readily available additives make that irrelevant, and affect c
c) I think that the cycle is slowed by water changes because you are removing substrate that the nitrite oxidizing bacteria need to eat and multiply. If all is left alone, I propose the ammonia eating bacteria will produce exactly the correct amount of nitrite that the nitrite eating bacteria must consume in order to create a perfectly balanced population.
d) Why do we dose back up? Is it because we fear the ammonia eating bacteria die? Or is it because we want to be sure that the bacteria can successfully eat Xppm all at once (rather than the same individual bacteria eating some every day). If it's the latter, then a higher initial dose will do the job. aka Dose it to 8ppm and never add ammonia again. If it's the former - they don't. We have proven this.
e) I simply believe this to be true after all of our work

 

[threnjen]

We need to understand how toxic nitrite is to fish and at what levels? There must be a study on this?

There are lots! One of the papers I read on nitrite toxicity in fish mentions that there are over 40 papers about it. So the truth is out there.

 

[threnjen]

I just found a blatant error in one of our good sources. This is why only the base scientific paper is the best source.

Nitrifying Bacteria Facts
We like this paper right? I've looked at it a lot.

But it says:
"Nitrosomonas growth is inhibited at a pH of 6.5. All nitrification is inhibited if the pH drops to 6.0 or less. Care must be taken to monitor ammonia if the pH begins to drop close to 6.5. At this pH almost all of the ammonia present in the water will be in the mildly toxic, ionized NH3+ state."

Um.... no? Doesn't a low pH present the lowest amount of Free Ammonia NH3?

 

[threnjen]

Source: BMC Microbiology | Full text | Strategies of Nitrosomonas europaea 19718 to counter low dissolved oxygen and high nitrite concentrations 2010

"Cell growth [of Nitrosomonas] was not detected at an initial NO2- concentration of 560 mg-N/L and DO = 1.5 mg O2/L, even after 2 weeks of incubation (data not shown). An initial NO2- concentration of 280 mg NO2--N/L and DO = 1.5 mg O2/L, resulted in a lag phase one day longer than that in the initial absence of nitrite (Figure 4 D1-D2 and Figure 2, B1-B2, respectively). However, the overall cell yield was not impacted. The extent of NH3 oxidized to NO2- in the presence of 280 mg NO2--N/L (88 ± 5%, n = 2) was not significantly different (α = 0.05) than in the absence of nitrite (90 ± 10%, n = 2). "

Even at 280ppm, Nitrosomonas still grew, it just had slower startup.
Nitrosomonas is NOT AFFECTED by Nitrite on the scale that we use in our aquariums

 

[threnjen]

Source: JSTOR: An Error Occurred Setting Your User Cookie

Nitrobacter (not spira, possibly irrelevant) were shown to grown in culture with a doubling time of .6 days

Source: Nitrospira-Like Bacteria Associated with Nitrite Oxidation in Freshwater Aquaria
"Whether in pure culture or on biofilters, NOB are slowly growing organisms with doubling times from 12 to 32 h (3, 5, 7). Therefore, in newly set up aquaria, ammonia and nitrite can reach concentrations toxic to fish before a sufficient biomass of AOB and NOB becomes established"

Trying to find doubling times so I can chart it...

 

[threnjen]

Going back to the paper about Nitrospira from Dr. Tim. I like this paper because it has a nice graph about the entire nitrogen cycle length, and also made a point to detect when certain bacterias appeared. This appears to be one of the few papers out there that basically just studies "cycling" (although its primary purpose was to prove Nitrospira, its secondary effect was a study on the cycle)
Nitrospira-Like Bacteria Associated with Nitrite Oxidation in Freshwater Aquaria

"Time of Nitrospira-like bacterial appearance.
The daily concentrations of ammonia, nitrite, and nitrate over the first 33 days after setup of a new aquarium are presented in Fig. ​Fig.6.6. The trends were as expected, with ammonia peaking about day 12. Nitrite values increased starting at day 12, peaked at day 21, and decreased to below detection limits by day 26. Nitrate values steadily increased from about day 15 onwards. DGGE showed that the band corresponding to clone 710-9, the putative NOB, first appeared on day 12, with the relative intensity of the 710-9 band increasing daily based on relative fluorescence units of rDNA amplicons"

One important thing to note, as you look at the graphs, is that all of the additions of ammonia are in mM. So that means when it says 4mM ammonia it means 68ppm ammonia. NitrIte in this study reached 11mM, or 506ppm, before dropping.
Again: lab work is so far outside of our scope and does not stall. Our cycles will not stall due to levels of ammonia or nitrite that we encounter. They may stall due to OTHER THINGS but not for that SPECIFIC reason.

"Time series.
Three aquaria were set up as previously described with 4.53 kg of gravel and were filled with 30 liters of city water which had been passed through activated carbon. The test was run for 138 days, during which the aquaria were individually dosed with 8.9 mmol of filter-sterilized (0.2 μm) ammonia (as ammonium chloride) on the first and second days of the test. From days 12 to 78 of the test, further additions of 8.9 mmol of ammonia were done on average every 3 days. A total of 246 mmol of ammonia was added to each tank during the test. The water was sampled three times a week for chemical analysis. The aquaria were run for 80 days with freshwater, at which time the water was switched to seawater (32 ppt) by draining and refilling with water mixed with artificial sea salts (Marineland Commercial Aquariums, Moorpark, Calif.). After the switch, the testing continued for an additional 57 days."
Total of 246 mmol ammonia added. 4189ppm ammonia. lol!

"Time of NOB appearance.

Three all-glass aquaria were established as described above. A 34-liter sample of city tap water, which was passed through activated carbon, was added to each aquarium, which contained 4.53 kg of gravel. Initially, 0.71 mmol of filter-sterilized (0.2-μm-pore-size filter) ammonium chloride was added to each tank, followed by an additional dosing of 5.0 mmol of NH4Cl on the fourth day. On days 10, 15, 18, 23, and 30, further ammonia additions of 8.9 mmol were made to each aquarium. During the test, a total of 50.4 mmol of ammonia was added to each aquarium. Water samples were collected daily.
Two 10-g samples of gravel were collected from each aquarium daily for 33 days."
This one had an initial dosing of 12ppm ammonia with 4th day dose of 85ppm and maintenance doses of 151.6ppm.

 

[threnjen]

So mM MEANs moles per liter.
So mMol or mM conversion to ppm is exceptionally simply. In practice it is nothing more than the concentration of the solution * the molecular weight of the solution.
So if they say "50 mM of Ammonia" you just multiply 50 * 17.031 (molecular weight of ammonia) to get 851.55 mg/liter which is 851.55ppm

Source: http://www.ehow.com/how_8412601_convert-millimoles-ppm.html

ppm = A x mmol/l
mmol/l = ppm/A
Where A = atomic mass of the ion

mmol/L is millimoles per litre
mM is "milliMolarity" which is millimoles per litre
both are millimoles of solute per litre of solution
So we may see it written either way but it means the same thing.

 

[Caliban07]

LOL Caliban! In one post you're trying to keep us on track, in the next one you're off on the irresistible tangent

This is all so interesting, it's hard to keep on track!

I agree with your post a few back - we need to come up with the test parameters for the volunteers. My husband advised me that first I need to make a chart of all of the "known" parameters relating to bacteria growth, using all of our sources. i.e. temperature (not just the ideal temps - how slowed is growth outside of them?), rate of doubling of colonies, proportion of colonies in fully cycled samples (I HAVE seen bits of this, somewhere, about the proportions) etc, ideal DO, etc. Then from there we form our hypotheses. He said that I have seen enough data that I should be able to easily run a simulation of what I would expect to see given different dosing scenarios. I will try to work on this today and post them.

As far as hypotheses, I *think*, and I can only say I think as I need to think it through more, that ultimately *my* hypothesis (I believe yours will be different) will be that:
a) The level of dosing does not influence the cycle length, but does influence the end nitrification capacity
b) Water changes can be eliminated through manual additions of phosphorus (fish food) and baking soda at cycle start
c) Cycles without water changes will complete faster than cycles with water changes
d) I feel uncertain on this one. But I'll say it. After initial dose is reached, no additional ammonia is required, and will only prolong the cycle.
e) No levels of ammonia or nitrite in our scale of application will stall the cycle

To address these in turn:
a) I'm not on board anymore that EITHER your .25 dose will end faster OR that my madcap 18ppm dose will end faster. I think it all ends up the same because I've seen so many lab graphs charting the cycle. To that end I think it will take exactly the same-ish amount of time to complete the cycle for these dosings and therefore a larger dose will prove to be more efficient
b) We only water change to stabilize pH and phosphorus, but these readily available additives make that irrelevant, and affect c
c) I think that the cycle is slowed by water changes because you are removing substrate that the nitrite oxidizing bacteria need to eat and multiply. If all is left alone, I propose the ammonia eating bacteria will produce exactly the correct amount of nitrite that the nitrite eating bacteria must consume in order to create a perfectly balanced population.
d) Why do we dose back up? Is it because we fear the ammonia eating bacteria die? Or is it because we want to be sure that the bacteria can successfully eat Xppm all at once (rather than the same individual bacteria eating some every day). If it's the latter, then a higher initial dose will do the job. aka Dose it to 8ppm and never add ammonia again. If it's the former - they don't. We have proven this.
e) I simply believe this to be true after all of our work

Ok i agree with some of this.
a) i have to admit im leaning more towards the conclusion that it takes x amount of time to colonise bacteria so deal with it.
b) no need to change water agreed.
c) Agreed
d) i dont really know. Fear? fear that bacteria will die out one dosing of 4ppm would be enough IMO. remember bacteria was still able to consume lab levels of ammonia months later.

We can however not assume that low ammonia dosing would be faster or slower. It hasnt been donr this way in labs?

Do we even have to bother with conditions? afterall these lab tests would have been carried out in optimum conditions anyway. I can say that your goal has been fulfilled. You do not need to change water during a cycle. Patience is key.

we have established useful things that help. Maybe we could suggest that fishless cycles require a water temp of 30 degrees with a close to neutral pH in a dark room. one test with 4ppm ammonia and the next starting at .25

 

[Delapool]

Can you try it with a case already worked out? I just had a quick look to what it had and it looked a nice find.

 

[Caliban07]

So mM MEANs moles per liter.
So mMol or mM conversion to ppm is exceptionally simply. In practice it is nothing more than the concentration of the solution * the molecular weight of the solution.
So if they say "50 mM of Ammonia" you just multiply 50 * 17.031 (molecular weight of ammonia) to get 851.55 mg/liter which is 851.55ppm

Source: How to Convert Millimoles to PPM | eHow

ppm = A x mmol/l
mmol/l = ppm/A
Where A = atomic mass of the ion

mmol/L is millimoles per litre
mM is "milliMolarity" which is millimoles per litre
both are millimoles of solute per litre of solution
So we may see it written either way but it means the same thing.

yes i see this math. so 851.55ppm of ammonia would = to about 2299.185 nitrite! lol

so one dosing of 4ppm ammonia would equal a total of 10.8 nitites throughout the whole of he cycle? since we read that even a 90% water change only removes 30% nitrite this could be a reason we nitrite readings remain 'off the charts' after 4 50% water changes that are often encouraged. even after that nitrite would be dark purple.

 

[threnjen]

Can you try it with a case already worked out? I just had a quick look to what it had and it looked a nice find.

I don't even think we could do it on such a large scale. I am just not sure how we could even measure the PPM required to match this. The API test isn't made for such a large test!
I think at *LEAST* we can have one of the test parameters be what anyone in aquaria would consider an "outrageous" dose of ammonia, like 20ppm, which can't even be measured directly but can be added by finding the correct amount for 1ppm and doing 20x

OH! Caliban - The tests from the Dr Tim paper also used aquariums, even with these OUTRAGEOUS doses, implying to me that there is PLENTY OF PHYSICAL SPACE to colonize. Agree?

Also - I bet it can be mathematically estimated the amount of ammonia to add at the beginning if you want your end nitrification capacity to be x. Although I have failed to find the EXACT rate of conversion of ammonia->nitrite->nitrate expressed in time, we might just have to assume it takes the same amount of "time" for a single bacteria to eat and excrete, and from there just use the doubling time of the bacteria to determine this. So like if nitrosomonas doubles on avg every 10 hours and nitrospira every 22, can we use this information to estimate the amount of initial dosing of ammonia that will ultimately grow NOB capable of converting 4ppm ammonia in 24 hours.

 

[threnjen]

I found this interesting thread on another forum from 2008 where this person "proves" that waterchanges improve the cycle.
However I hypothesize that what the water changes actually did was replenish phosphorus, dissolved oxygen and pH just enough to help the cycle complete ONE DAY faster.
Overall I find his conclusions statistically insignificant. To get a true matching conclusion, he would have needed to consider these other variables and "dose" them to his no-water-change tank.

Why Water Changes During Cycling Are Good - Tropical Chit Chat - Tropical Fish Forums

Edit: ultimately I guess he was just graphing some function he wrote?
How does that function take all the other things that the bacteria "eat" into account? I guess it just doesn't make sense to me.

 

[aqua_chem]

How is it that these conversations always break out when I'm on vacation out in the woods somewhere without internet? There's so much stuff that I would have liked to comment on, but now we're 200 odd posts in, it's hard to really comment on anything specific.


So right now, exactly what are you trying to answer? That is to say, what it your current line of inquiry?

 

[threnjen]

WHY HELLO. Your forum handle and cute picture of Bill Nye tells me you will be a delightful addition to our thread!

I just tried to consolidate our purpose to my father-in-law, so let me post what I told him and perhaps that will tell you our "question"
"Even though the process of artificial colony building (called "fishless cycling") is quite popular, there are heavily varied recommendations for how to correctly perform this task, and no standard practices. There are also a lot of perpetuated myths/inaccuracies (which I can see without even being a chemist/biologist), and even worse people "stall" all the time in their cycling attempts which leads to a lot of frustration. So on a forum, some other other people and I have been poring over scientific journals in an effort to better understand the cycling process (the name for the establishment of this complete bacterial colony) and eventually have enough scientifically validated data to present our closest estimate of a "standard practice" for fishless cycling which will yield success in 99.99% of cases."

What we want is to:
* invalidate perpetuated myths about fishless cycling
* produce cycling instructions that will actually work in most cases
* produce the fastest possible cycling instructions
* as a side-project, improve information about fish-in cycling

Some of the major hypotheses just one page back on page 23.

If you really have a good grasp for science, I'm sure you could seriously help us. I think the heavy stuff starts around page 12?

Also, we're not claiming we're discovering a bunch of new things, but we ARE finding actual scientific studies and citations to back up some of things we read about cycling.

Also please do comment if you see something interesting, we can pick it back up.

 

[aqua_chem]

* invalidate perpetuated myths about fishless cycling

What particular myths are in your crosshairs? It seems like this thread has a lot of good information, but lacks goal-oriented research. I think you would benefit from selective only a few (for now) to use as a jumping off point for your research, adding new questions as they become relevant or as you become satisfied with your previous ones.