Ammonia Testing- The Good, The Bad And The Ugly

[TwoTankAmin]

Ammonia- we talk about it a lot, we don't want it, but our bacteria do. How much is in any tank at any time?
 
Almost any fish keeper you can find probably owns an ammonia test kit. We all consider it to be an important parameter to measure in our tanks, especially during cycling. And we discuss the results, the problems with testing. There are countless discussions about things that cause false positives, about the accuracy of results or the methods for testing. But this only covers a small portion of the world of testing.
 
Laboratory and field researchers at companies and universities are also testing, and they are using completely different equipment for this than we in the hobby normally use in our tanks. the one thing almost no fish keeper will dispute is that our kits are not really accurate and the lab grade ones are. But how much do most us us really know?
 
Almost all of test ammonia using a test kit which is uses the salicylate method of testing. But do most of us know anything more than add so many drops of solution 1 and mix, then add so many drops of solution two and mix. Then we wait some number of minutes and compare the color in the little vial to the color on a card. And we all know about the issues with color interpretaion and comparison.
 
In order to come to grips with ammonia testing the first step is to know exactly what is actually going on in a salicylate test:

Low ammonium levels can be determined in FIA by using the salicylate method. The salicylate method is a variation of the Berthelot-Phenate
method but does not require the use and disposal of toxic phenol. The salicylate method involves a three-step reaction sequence. The first reaction step involves the conversion of ammonia to monochloroamine by the addition of chlorine. The monochloroamine then reacts with salicylate to form 5-aminosalicylate. Finally, the 5-aminosalicylate is oxidized in the presence of sodium nitroferricyanide to form a blue-green colored dye that absorbs light at 650nm.

From http/www.flowinjection.com/methods/ammonia.aspx
In the most expensive test kits the color is read electronically- no human color perceptions are involved. But basically the more ammonia that is present, the darker the color.
 
Now one might think that something like and API or Tetra or SeaChem kit are doing the same thing the fancy lab kits are since they both use the salicylate method. But they stop looking so similar when one examines how a lab grade kits is used. For example, Hach Company's salycilate kit requires 3 minutes of wait after the first reagent is added and then a 15 minute wait after the 2nd. The total wait time on and API ammonia test is 5 minutes.
 
The Hach kit comes with a table and some formulas and the following instructions:

Ammonia exists in water in two forms, un-ionized ammonia (NH3) and the ammonium ion (NH4+). NH3 is toxic to fish, while NH4+ is nontoxic (except at extremely high levels). At a neutral pH and ambient temperature, almost all of the ammonia exists as NH4+. As the pH and temperature increase, the proportion of NH3 increases (see Table 1). This test kit measures both ammonia forms as ammonia nitrogen (NH3–N). Use Table 1 and equations (1) and (2) to determine the amount of NH3 and NH4+ in the sample.

From http/www.hach.com/asset-get.download.jsa?id=7639982686
 
So my first question is, why do the aquarium kits need so little sitting time compared to the lab grade tests? My second question is since the even higher quality lab grade ammonia kits prefer to use an expensive photometer to read the results instead of a human visual comparison, how accurate can the colors and our interpretations of them be on the aquarium grade kits? When one gets into the really sophisticated end of testing the equipment stars to be priced in the $1,000 and up range.
 
But it only beginning to get interesting because there are a number of things that can cause inaccurate readings when using the salycilate method, even with a lab grade kit.

Sulfide will intensify the blue-green color and should be removed from the sample if possible. Hydrazine and glycine like sulfide can also intensify the color. High sample turbidity will give significant erroneous results and may need to be pre-filtered or run utilizing a different ammonia method. Iron will also interfere with the analysis, but can be eliminated by doping the calibrations samples with the same level of iron present in the sample. To a lesser extent the following ions may also interfere: sulfates (>300 ppm), phosphates (>100 ppm), nitrates  (>100 ppm), nitrites (>12 ppm), calcium (>1000 ppm) and magnesium (>6,000 ppm).

From http/www.flowinjection.com/methods/ammonia.aspx
 
Its doesn't end here. Because the first step in the test is basically to add chlorine, sites which explain how to DIY salicylate testing all explain the the test solution 1 should be refreshed daily as it degrades. Since it is basically chlorine, we know it out-gasses and loses strength. Does anybody think that the #1 bottle of solution in aquarium level kits is airtight? And even if one can get it well sealed by firmly closing the cap, how often might we not turn it quite all the way?
 
What I am seeing in all this is that testing for ammonia (and likely most other things) with the more commonly used aquarium kits is pretty much a coin toss as to how meaningful any result may be. How can a $6 vs $85 ammonia test kit be measuring with the same degree of accuracy or reliability? And there is certainly no comparison to those that can run into the many hundreds or even thousands of dollars.
 
While looking for information I ran across a site that gave directions for making ammonia test solutions to check how accurate a given kit/tester may be. I forgot to bookmark it (DOH). But I do remember it saying in a pinch one can use the same ammonia this site recommends for fishless cycling in the UK. So if folks wanted to they could use a clean bucket of water (a gallon or two), add the amount of ammonia based on the site calculator here to dose to 2 ppm, stir it up and then test with your kit. You can dilute the solution to test at lower levels. Even better would be to use deionized water. I will try to track down the site with the directions.
 
I just poked around the API site looking to see what is in their two bottles for testing ammonia, One bottle basically contains sodium hydroxide (lye) and sodium hypochlorite (chlorine) while the other basically contains sodium salicylate and polyethylene glycol. The curious can find the information here http/cms.marsfishcare.com/files/msds/ammonia_test_122309.pdf

 

[daizeUK]

While looking for information I ran across a site that gave directions for making ammonia test solutions to check how accurate a given kit/tester may be. I forgot to bookmark it (DOH). But I do remember it saying in a pinch one can use the same ammonia this site recommends for fishless cycling in the UK. So if folks wanted to they could use a clean bucket of water (a gallon or two), add the amount of ammonia based on the site calculator here to dose to 2 ppm, stir it up and then test with your kit. You can dilute the solution to test at lower levels. Even better would be to use deionized water. I will try to track down the site with the directions.

 
The problem with that is that most people are using cleaning fluids as a source of ammonia, the exact concentration is not reliable and the exact ingredients are unknown since the manufacturers are not required to list them all.  Also ammonia also gasses off so the concentrations inside these bottles will decrease depending on the age of the bottle.
 

I just poked around the API site looking to see what is in their two bottles for testing ammonia, One bottle basically contains sodium hydroxide (lye) and sodium hypochlorite (chlorine) while the other basically contains sodium salicylate and polyethylene glycol. The curious can find the information here http/cms.marsfishcare.com/files/msds/ammonia_test_122309.pdf

 
Just to add further information about the API test, this is also from Mars Fishcare:
 

The first reaction is the ammonia contained in the water sample combines with chlorine to form monochloramine.

Monochloramine reacts with salicylate to form 5-aminosalicylate.
Then the 5-aminosalicylate is oxidized in the presence of sodium nitroprusside working as a catalyst to form a blue colour which is masked by the yellow colour from any excess reagent present which produces a green-coloured solution in the test tube.

 
On the subject of Salicylate versus Nessler tests, you pointed me in the direction of Seachem ammonia test kit which says this:
 

Product Description
This kit measures total (NH3 and NH4+) and free ammonia (NH3 only) down to less than 0.05 mg/L and is virtually interference free in marine and fresh water. Free ammonia is the toxic form of ammonia (vs. ionized Ammonia NH4+ which is non-toxic) and thus it is much more important to keep an eye on the level of free ammonia in your system. This kit is based on the same gas exchange technology that is used in the Ammonia Alert™ and thus is the only kit on the market that can read levels of free ammonia while using ammonia removal products such as Prime®, Safe™, AmGuard™ and any similar competing products. The other kits (salicylate or Nessler based) determine the total ammonia by raising the pH of the test solution to 12 or greater. At this high pH all ammonia removal products will breakdown and rerelease the ammonia, thus giving you a false ammonia reading.

 
So Seachem Multitest works on a system that is neither salicylate nor Nessler?  How then does this compare in terms of accuracy?

 

[TwoTankAmin]

Well for one thing SeaChem provides an ammonia reference solution with the kit that allows you to to test it.

Reference test
The proper performance of this kit may be validated by running a total ammonia testin the normal manner except that the reference sample is used in place of a aquarium sample. It is not necessary to run a reference test to use this kit. The only time you might choose to run a reference test is if you have cause to believe the test is giving in-correct results. If you run a test using the reference sample and obtain the correct result (based on the known ref-erence value provided) then you know the test is giving correct results. To run a reference test use the Total Am-monia Referenceas the sample in a total ammonia test. Reference value is 1.0 mg/L.

From http/www.seachem.com/Library/Instructions/0950-AmmoniaWE-4.0.pdf
 
I am having a problem discovering what "ingredients" are being used in the SeaChem test. You should not that the instructions tell you to wait 15 minutes for lower levels before reading and to wait 30 minutes for high ammonia levels before reading. Again we are seeing a big difference between the API etc. 5 minute wait time and the more sophisticated/accurate salicylate tests at 15 or the SeaChem wait time.
 
If you want to create your own reference solution:

7.11 Stock solution: Dissolve 3.819 g of anhydrous ammonium chloride, NH4Cl  (CASRN 12125-02-9), dried at 105°C, in reagent water, and dilute to 1 L. 1.0 mL = 1.0 mg NH3-N.
 
7.12 Standard Solution A: Dilute 10.0 mL of stock solution (Section 7.11) to 1 L with reagent water. 1.0 mL = 0.01 mg NH3 -N.
 
7.13 Standard Solution B: Dilute 10.0 mL of standard solution A (Section 7.12) to 100.0 mL with reagent water. 1.0 mL = 0.001 mg NH3-N.
Reagent water - Ammonia free: Such water is best prepared by passage through an ion exchange column containing a strongly acidic cation exchange resin mixed with a strongly basic anion exchange resin.

Fromhttp/www.caslab.com/EPA-Methods/PDF/EPA-Method-3501.pdf
 
What you should find as you start to surf for information on ammonia testing is there are a number of things that can cause the results to be inaccurate in many of the test methods. Some of the more common ones that will or may be present in tanks are: iron, sodium ions (as caused by the addition of baking soda/sodium bicarbonate).
 
One reason aquarium caliber ammonia tests are so cheap, is they are so inaccurate. Lets start with reading the results. I can not think of anything more inaccurate than the white card with the colored bars for making a comparison for a liquid sample. Even in the less expensive end of the ammonia test spectrum the more accurate method is using liquid references solutions. If you have a swimming pool and use test kits, you would be familiar with these.
 
Yes I know that is a chlorine and pH one, but its there as an example. The point is one compares liquid to liquid. Lamotte has this sort of system but unfortunately for fw it is a Nessler based kit.
 
However, what I did find is an adjunct for getting around some of the shortcomings of the API ammonia kit. It is a calibration product and a colorimeter and software. Basically, you use the supplied materials- 9 capped vials which each contain a series of 5 ml reference solutions from 0 to 8 ppm of ammonia. You run the API test on each of the 9 vials. You then run each solution through the colorimeter and the results go into the software program. Now when you are ready to test your tank water, you run the normal API test and put the sample into the colorimeter and it tells you the exact ammonia level.
 
Of course this adjunct to make your $6.50 API test kit give more accurate readings will cost you about $125-$150. You can see it all here-
The calibration kit- http/www.iorodeo.com/content/api-ammonia
The Colorimeter etc.- http/www.iorodeo.com/content/educational-colorimeter-kit
 

 
Of course none of this will help much with issues such as iron in the water, too much nitrate (100ppm or more) too much sodium ion etc.  I am wondering if what you are interpreting as something in the tank that may be stalling your cycle may actually be something that is throwing off your test results.
 
What I have come to believe in all of this is that an ammonia test kit that costs a few dollars is not going to provide reliable or accurate readings. What would not surprise me is when one calls APO to ask questions about the ammonia kit is that the person you are talking is likely a trained chemist who knows what the facts really are. An lets just suppose for a minute that A company's test kit is not real reliable, that certain factors throw it off. Do you really think that the person one the other end of your phone call would even come close to admitting this? I would bet it costs a company less than $1.00 to manufacture an aquarium grade ammonia test kit. That API ammonia kit I can buy online for about $6.50 is likely sold to the retailer for about $3.00. 
 
So exactly how accurate do we think an ammonia test kit that cost around a dollar (about 70p) to make really is?
 
What we really need is somebody who is a trained chemist to weigh in on this subject.

 

[daizeUK]

A comparison of my API kit and Salifert kit using Dr Tim's ammonium chloride solution.
 
The ammonium chloride states that 1 drop per (US) gallon will provide an ammonia-nitrogen concentration of 2mg/L.  I found that results from my API ammonia test were reading about half the expected concentration, so I bought a Salifert test for a second opinion.
 
I filled a bucket with 1 gallon of untreated tap water.  The bucket is probably marked in imperial gallons, so that's 1.2 US gallons.  Then I added 2 drops of ammonium chloride solution.  This should give a concentration of about 4mg/L-N.  So adjusting for the extra dilution and the conversion of nitrogen to ion scale (1 to 1.2 ratio), that's (4 * 1.2) / 1.2.  What happy coincidence!  So I should expect a reading of exactly 4ppm on the ion test.
 
Here's the API test, which seemed to read around 1ppm:
 

 
And the Salifert test, do not adjust your eyes I took it upside down.  At first I thought the colour matched 0.25, certainly in intensity of colour, but the tone was slightly more orangey.  I'm tempted to say the colour might possibly match the >2.0 band better, only it's far too pale to read.

 
So... is the ammonium chloride giving the wrong concentration?  Is my API kit just unable to read ammonium chloride properly (at least one website states that this is the case).  Is my Salifert test completely broken?
 
I tried again with another 2 drops of ammonium chloride in the bucket.  A total of 4 drops in 1.2 gallons should give an total ammonia-ion concentration of 8ppm.
 
The result is a very pale solution with specks of orange precipitate (which stuck to and discoloured the sides of the tube).  I really don't think this is what is supposed to happen in a Salifert test, would you agree this is broken?
 

 

[TwoTankAmin]

I hate looking at pics of results. The API reading to my eyes can be anything from 1-4ppm from that pic. My best guess is 2 ppm. i wished you had shown the API test for the 2nd level of ammonia test.
 
The Salifert must be defective. I can tell that sample one and two are different, but I wont even try to match them to their chart, which goes up to 2 ppm max.
 
I am beginning to believe there is something in your water that affects the ammonia test results more than it might be retarding a cycle. Given your readings for GH and KH it could be any number of the potential things known to interfere with salicylate tests. Iron is a major one, but even nitrate or nitrite will.
 
And yes, I assume it is possible for the strength of the ammonium chloride to degrade over time.

 

[snazy]

It's a pity because that Salifert test is supposed to be more accurate but until one produces a test that has a proper colour chart with easily distinguished colours and is accurate enough to at least detect ammonia present, then I am going to rely on my fish to show me a problem. I've seen my fish many times scrape themselves after I do a thorough filter maintenance. I'd take the tests out and test the water, 0 ammonia, 0 nitrItes. But I've noticed the fish getting irritated so many times over the same thing that I am certain that either the ammonia tests aren't accurate at all at measuring harmful ammonia levels or the fish get annoyed from non-detectable ammonia levels that no test can show with the current colour chart.

 

[daizeUK]

I am beginning to believe there is something in your water that affects the ammonia test results more than it might be retarding a cycle. Given your readings for GH and KH it could be any number of the potential things known to interfere with salicylate tests. Iron is a major one, but even nitrate or nitrite will. 

 
I'm inclined to believe you may be right.  I got a replacement Salifert test kit, different batch number, different expiry date, and it gives exactly the same results.  Looks like Salifert kits just don't work for me.
 
I thought the dechlorinator was supposed to remove heavy metals, shouldn't that prevent any iron from interfering with the kit?  (in the tank, I didn't dechlorinate for the bucket tests)

 

[TwoTankAmin]

I am not sure re the dechlor and iron. But I would think maybe it doesn't remove iron. I base this on planted tanks. Many folks dose iron. Some also use dechlor. If dechlor that removed heavy metals was removing iron, the plants would not be doing well. This would be even more apparent in the low tech tanks where folks are not adding anything for the plants.
 
Researching heavy metals one will discover that it is not a real scientific term. It is most often used to refer to the metals that are toxic to humans and other life, mercury, lead, plutonium etc. But it can also include metals which are essential for human and other life such as iron, copper and zinc. The sort of things lifeforms need in small quantities. But here is how metals are rendered harmless in most cases via dechlor products.
 

Chelated is referred to in metals. For example chelated iron. This word means a chemical compound in the form of a heterocyclic ring, containing a metal ion attached by bonds to at least two nonmetal ions.
 
This chemical process renders the metal absorbent and non-toxic to plants and most life on the planet.
The chelating agents vary - a common synthetic chelator is EDTA.

• 
  
• Quite often water conditioners contain a chelation agent to render any potential toxic metals in the tap water harmless and prevent metal poison disease.

• Plant fertilisers like PMDD or commercial products contain chelated metals for good plant growth.

From http/www.theaquariumwiki.com/Chelated
 
I am not so sure this process means the chelation will also prevent the metals, like iron, from still interfering with the testing. I have been using hobby test kits for years now. I have never ever seen a single maker of salicylate ammonia test kits say a single word about what might throw off the results with the exception of discussing the effects of their product on Nessler test kits or false positives related to the break down of chloramines. None of them discuss the very things which interfer with test results and are specifically mentioned when one reads in connection with the salicylate lab grade test methods. Those makers specifically mention such potential agents,  And for iron suggest how to compensate to make the test accurate. They also suggest in some cases one needs to filter out the solids in the water as it will throw off the color appearance.
 
daize one question. I noticed that the salifert kits use a bit of a different method than the API, are you 100% certain you are doing the testing correctly? Do the instructions look like this?

 

 

[daizeUK]

Yes those are the exact instructions, I must have read them at least five times to make sure I'm doing it right.
 
I followed Snazy's suggestion to use distilled water to try to rule out any undesirables in the tap water.  I used household de-ionised water, I hope this was suitable.
 
Tests
A) Test a 1ppm solution of Dr Tim's ammonium chloride in de-ionised water
B) Test a 20ppm solution of Dr Tim's ammonium chloride in de-ionised water
C) Test a 1.5ppm solution of BioMature ammonium in de-ionised water
D) Test a 0.5ppm solution of BioMature ammonium in de-ionised water
 
Results
A) Salifert clear, API not tested
B) Salifert a bit above 1.5ppm, API around 4ppm.
C) Salifert 1.5ppm, API 1.5ppm
D) Salifert clear, API 0.5ppm
 

 
Methods
A) Made a stock solution with 454g (one tenth of a gallon) de-ionised water measured on electronic kitchen scales.  Added 1 drop Dr Tim's ammonium chloride to make a 20ppm solution (as per instructions on Dr Tim's bottle).  Made Salifert test solution with 0.1ml of stock solution and 1.9ml de-ionised water to make a 1ppm solution.
B) Test solutions for both Salifert and API were the stock solution made for {A}.
C) Made a rough stock solution with 2 drops of BioMature and about 200ml of de-ionised water.  I estimated this would give about 10ppm ammonia solution based on past experience with BioMature and API readings.  Salifert test solution made with 0.2ml of stock solution and 1.8ml de-ionised water.  API test solution made with 0.5ml stock solution and 4.5ml de-ionised water to achieve what I guessed would be around 1ppm ammonia solution.
D) Added another 400ml de-ionised water to the stock solution used in {C} to dilute to one-third concentration.  Methods for making API & Salifert test solutions were identical to those described in {C}.  Since {C} resulted in 1.5ppm for both tests I expected this to result in a 0.5ppm solution.
 
Discussion
Wow, my own little scientific paper!  haha
A) The Salifert test had no discernible colour, I didn't test API but if I had I would have expected it to show around 0.25ppm in line with the usual results that I get.  Neither result agrees with the Dr Tim instructions to achieve a given concentration but if the Salifert test was working it should be showing a little colour but it isn't.
B) A stronger reader for Salifert but not reaching the >2ppm colour mark, therefore still not in agreement with API.  The colour was evenly distributed and a little stronger than previous tests.
C) Salifert and API both agreed that the BioMature solution was 1.5ppm, which was pretty close to what I'd expected.  The Salifert result was rather transparent, I'm not exactly sure if it's supposed to be like that.
D) API gave the expected result of 0.5ppm.  I'm not sure if there was some faint hint of colour on the Salifert test but it's not readable.
 
I just don't know what to make of any of this.  Test C is the only one that makes any sense to me but even then the colour is very faint.  At first I thought maybe the problem is only with Dr Tim's but then test D didn't show any colour either.  I guess I should just give up with Salifert.  Anyone got any ideas what it all means?

 

[snazy]

Looks like for C) and D) and respectively BioMature the API is giving ok results. I would have loved to see the 1ppm test with the ammonium chloride as well on the API test. I wouldn't pay much attention to the 20ppm one unless you tried a 2nd time diluting the 20ppm to a mix of 50%(20ppm solutions) and 50% de-ionised water, which should show half the ammonia on the test. That way you would be sure for example on the API test, that what you think is 4ppm, is not actually 8ppm and above, or confirm the result, which would give you exactly the same colour regardless of the dilution because it will still be above 8ppm.

There's a possibility that Dr. Tim's ammonium chloride isn't very concentrated. It does say how many drops to put to achieve a certain ppm result, but does it say what the concentration of ammonium in it is? Maybe there's cheating there to make you think the next day the ammonia has gone down
Also, the Salifert test may just be overhyped and turn out to be less accurate than API.  After all it's all about how one advertises a product and then a bit of a push on this and that forum, and people get easily convinced one way or the other without using their own brain too much. It may have more reliable tests from the entire range, but ammonia may not be their strong point.

 

[daizeUK]

The BioMature doesn't state what concentration you get though, it only instructs you to add 1ml per 30 litres of water and keep testing until you get the desired concentration of ammonia.  So I can't say if the API test is accurate.
 
Having said that, the API test has been very reliable for me.  It may or may not be accurate but it always registers a fall or rise in ammonia, it's very easy to tell the difference between zero ammonia versus some level of ammonia and the results are predictable insofar that if I dilute a solution by third then it will give me a third of the reading. 
 
I've still got that 20ppm stock solution so I could dilute it by half and test to see if I get 2ppm with the API test.  I'm actually pretty confident that I would though.  I'm also conscious of conserving my only test kit that seems to work lol.  I think these things are more expensive in the UK than in the US.
 
I think it's possible that there's something odd about the Dr Tim's ammonium chloride.  They don't say what the concentration of ammonia in the bottle is, only that it's formulated to produce a 2ppm solution with 1 drop per gallon.  I guess it's possible to work out the concentration from that.  I suppose a concern is that if the bottle isn't as concentrated as it claims to be then it might not build up enough bacteria to support an initial stocking of fish... but Dr Tim covers himself here by recommending a 'few hardy fish' for initial stocking.
 
I can't believe that these kits are typical of Salifert, loads of people use them with no problems.  They're hard to get hold of for customer support though.

 

[snazy]

Since you stated the solution in exact ppm, I presumed you were actually able to measure how much ammonia you are putting in and are expecing a certain result. If not, then there's no way you can make consclusions out of that since all you've tested is whether the test detects present ammonia.

 

[daizeUK]

Nope exactly, if you look at my Methods for test C you can see I estimated the amount of BioMature that would give me roughly around 1ppm solution, but this was based on previous API readings combined with the BioMature product.  So it's no indication of accuracy of the API kit unfortunately.  The purpose of the test was only to see if I could get the Salifert and API readings to agree with each other.

 

[snazy]

Ok. I didn't realize you wanted to see whether they'd give you the same result. I thought you had issues with the test showing the wrong ammonia levels so I presumed that's what you were testing for.
In this case, it's obvious they don't give the same result. Which one is wrong and which one right is yet unknown.

 

[daizeUK]

At the moment I have no way of knowing whether the API kit is telling me the wrong levels or if the Dr Tim's ammonium chloride is the wrong concentration.  They can't both be right but neither supplier will admit that their product is wrong!  That was originally why I got the Salifert kit, hoping that it would agree with one or the other and confirm which was right.  Instead I'm now vainly trying to get the Salifert kit to work, but it doesn't...
 
I'm thinking of trying the Seachem ammonia test next, but it's a bit more expensive and I didn't want to waste money if it's going to be another failure like Salifert.  At least your idea of using deionised water has helped to rule out something in my tap water corrupting the tests.  It must be the test at fault, not my tap water.