Chapter XV. The effects of heating water on pH

I'm taking a break from taxonomy to provide an update on the experiment I described in Chapter XIII.  After writing "This should kill 99% of bacteria etc. but I think leave the water essentially unchanged" it occurred to me that I better look into what the likely effects of boiling water before adding it to an aquarium might be. There is very little information I could find, especially relating to fish keeping. But the always dependable Skeptical Aquarist has an article on using boiling to soften water. The linked article predicts that the water will be depleted in dissolved oxygen and may have lost some of its buffering capacity. I decided to test what the effect of heating my tap water (TW) was. I heated 15 liters of TW to 70 Celsius in a stainless steel stockpot. I measured the pH before heating and after. I then added an air stone and left it for one week, taking daily pH readings. As I described in Chapter XII, leaving my TW for a week results in a slight reduction in pH. I assume this is the result of the aerobic respiration of organisms present (see here for a great article on bio-acidification). If my heated TW had lost buffering capacity then I might expect to see a more dramatic reduction in pH than in the AW as a population of microbes developed. I went for 70C because I didn't wan't the water to bubble and drive off too much dissolved gas but I should still kill most microbes when the time came to repeat  the process for real on the AW.

TW 0h

Heated TW 70 C + 16h

Heated TW 70 C + 6 days

As you can see, either heating the TW reduced its pH or leaving it overnight has (I should have bought two stockpots, filled them both with water and only heated one of them :). There doesn't seem to have been any further drop in pH during the week so no evidence of a reduction in buffering capacity. The drop in pH is about the same as I see in the AW. Given that this heated TW must have been low in microbial life and has dropped in pH within 16 hours, it does make me wonder if bio-acidification is responsible for the reduction in pH I see in the AW. Maybe its a chemical process. To test this I measured the pH of a sample of tap water straight from the tap and filled a saucepan, left it for 30 minutes and measured the pH.

TW 0h

TW 30m

There's a clear difference and the 30 minute sample looks to be about the same pH as the heated TW after six days. I would guess the process is chemical as 30 minutes seems too fast for a biological process. My explanation would be that water for domestic use is pumped around under pressure. When I fill a container with it the water equilibrates to atmospheric pressure and gaseous exchange takes place. In my case this seems to result in a slight drop in pH (which may suggest that CO2 is being absorbed). The pH then seems to remain stable. I haven't been able to find much information on what the likely effect on pH of storing water would be but here is a link where they measure an increase in pH over 12 days. In the discussion they say "The observed increases in pH during storage could be due to the activities of the resident flora and or their death, which results in the release of inorganic substances such as ammonia". This doesn't seem to be happening with my water. My question is: what were the processes in my aquarium that had previously conspired to maintain the tank pH 7.6 or above while the water I was adding naturally drops to a pH below that after adjusting to atmospheric pressure? As I mentioned in Chapter XIII, I had never measured tank pH lower than 7.6 in the entire two years and three months of cyanobacteria. Anyway, I was satisfied that heating my water to 70C doesn't have a dramatic effect on its pH so I repeated the process on AW and used it for a water change today.

AW 6 days

AW 6days 12 hours after 70 C

It took 30 minutes to heat up the 15 litres of AW and eight hours to cool from 70 C - 20 C. To achieve this rate of cooling I left the stockpot outside on a cold day. When I got back from work I added a heater and an airstone for an hour or so to add some oxygen and warm it up to tank temperature (23 C). As you can see, heating the AW doesn't seem to have affected its pH as predicted. After the water change the fish seemed fine. Now I play the waiting game.

Chapter XIV. Cyanobacteria identification 3

Unidentified cyanobacteria 12-11-12

I noticed a new patch of cyanobacteria on the moss ball. It forms a coarser matt than my previous cyano and looks to be a different colour. The matt above took four days to form so it's quite lively. Once again it was microscopy time and, if I do say so myself, I took some great photos.

Three filamentous cyanobacteria species from a freshwater aquarium. The wide filaments may contain necridia (arrowed). Sample taken from a macroscopic matt growing on a moss ball.

(Edit 7/9/14 see Chapter22 for more recent cyanobacteria identification)

There are obviously three types of filament here. Species 1. looks a lot like the cyanobacteria in the unheated tank and that had previously dominated the tropical tank. I'll call the broad filaments species 2 and the small species 3. So back I went to phycokey where I discovered that all three are probably Oscillatoria (filaments, un-branched, un-tapered, no heterocysts, no sheath, not spiral). The two arrowed cells above are probably not heterocysts. They look like this diagram from an algae identification guide I found.

The clear cells are the separation discs (necridia). These are the site of filament fragmentation giving rise to a hormogonium, which is how some species of Oscillatoria reproduce. But not all Oscillatoria it seems as I have never seen necridia in my species 1 (note the single cell 'filament' in photo two Chapter VIII Appendix I).

Species 2 is much more interesting to look at than species 1.
Note how dark the pigment in species 3 is, almost black.

Given that the three species look so different and possibly reproduce differently, I thought maybe I could take a guess at which species I have (contradicting what I wrote in Chapter VI Appendix I) . I found a guide to identifying algae in water supplies that has a section on cyanobacteria and Oscillatoria in particular (thankyou The University of Texas). All I needed to do was measure the width of the filaments and the length of the cells that make up the filaments and It seemed I could follow the key.

Species 1. Filament diameter 2.0 micrometers (2 thousandths of a mm), average cell length 5.3 micrometers (L/W ratio 2.65). So, question one, are the cells half to equal or greater in length than the width of the filament? Answer yes. Question two, are the filaments red/purple? No. Question three, are the filaments blue/green? Yes. Question four, are the cells 2-3 times as long as the filament diameter? Yes. Question five, are the filaments tapered? At this point I am left with two choices. If I say yes I get Oscillatoria splendida, if no O. amphibia. The cell dimensions are correct for both so don't be put off O. amphibia because the filament in the linked photo looks a different colour to mine. Microscope types make a huge difference to the images. So the question is, are species 1 filaments tapered? I will make two points. Firstly I can find photos annotated as O. splendida with non-tapered filament ends (there's one in the main photo from the above link). Secondly, as I pointed out in Chapter VI Appendix I, I observe a mixture of tapered and non-tapered filaments (admittedly non-tapered dominate). I suggested then that I might have a mixture of species, and if being tapered really is being used as a species defining characteristic in the genus, then perhaps I do. However, I have made a disturbing observation.

The filament marked has a tapered and non-tapered end and wasn't the only one I observed, although they are rare. I cannot find a filament that is tapered at both ends in any of my photos so I'm not sure tapering can be used to define these filaments. In summary, my best guess at the moment is that my tank was overrun by O. splendida or O. amphibia but I've got some reading to do to check how consistent tapering is in these species. Also, these might not be the only possibilities. The University of Texas guide doesn't cover all Oscillatoria species, only those found in water supplies. If you follow the O. splendida link above you will see a related species that looks very similar but that is not mentioned in the key. Oscillatoria taxonomy is a contested area and I notice from algaebase that out of 1049 species in the genus, only 60 are accepted taxonomically. I think this will be a work in progress.

Chapter XIII. Theorise!

The results of the pH tests showed that I hadn't thought of all possible reasons why AW might reduce cyano numbers in Chapter XI. With this in mind I now think there are three possible reasons, but I would be interested in other opinions.

1. Ecological. The aging process results in the growth of a population of bacteria/unknown organisms (probably heterotrophic bacteria) in the AW who directly compete with the cyano for space and nutrients after being added to the tank.
2. Nutritional. During the aging process the AW is depleted in a factor (X) that promotes cyano growth. The mechanism could be biological (e.g. uptake by bacteria) or chemical (e.g. precipitation/chelation).
3. Chemical. The aging process results in a change in water chemistry so that it no longer favours the growth of cyano (e.g. pH).

They're all interlinked of course. A drop in pH could increase the rate X precipitates, X could be a factor which discourages the growth of bacteria Y, which compete with cyano etc.

Anyway, I thought of an experiment that might help eliminate pH as a possibility. I could add the minerals I bought before Arthurs intervention to my tap water and then age it. According to the manufacturer this would result in raising the carbonate hardness by 4 dKH, and the general hardness by 7dH. As a result the acidification of the AW should be prevented by the extra buffering compounds (bicarbonate (HCO3) mainly). I haven't tested my tap water for carbonate hardness, but I think the water here is soft. If it wasn't I wouldn't be seeing a drop in pH in the AW. Also, my unheated goldfish tank pH is 6.0 or below before a monthly water change and usually ~6.4 afterwards. This suggests that my water has very little buffering capacity. It has always surprised me that I never took a pH reading below 7.6 from my tropical tank before I started using AW. My plan was to switch to fresh tap water until the cyano came back (assuming it did), this should gradually cause a slight increase in tank pH (~7.0-7.6+) over a period of around three changes. Then if I used the modified AW, I might see a reduction in cyano with no decrease in pH. If not then it would suggest that pH was important. Unfortunately I can't risk it. A fish-keepers primary responsibility is to his or her fish. Also it would take weeks.

As an alternative I will try and eliminate 1. Ecological as a possibility. Of the three, it's the one I think most likely (I will explain why later). What I will do is age the water as usual but before using it, heat it to near boiling point for five minutes. This should kill 99% of bacteria etc. but I think leave the water essentially unchanged. If I measure no difference in pH before and after the heating I will add it to the tank. If i see an increase in cyano numbers in the tank it would suggest that the ecological composition of AW is its important quality in limiting cyano growth rather than it's chemical composition. This is a better experiment because I will hopefully be changing far fewer variables than the comprehensive change in water chemistry required for the pH experiment. One thing I will be doing different is probably adding a very large number of bacterial corpses.

Chapter XII. Safety disclaimer

I know what you're thinking, you're thinking "why don't you switch back to using fresh tap water for water changes and see if the cyano comes back?" It wouldn't be scientific proof (for that I would need eight identical cyano infected tanks in a randomised block design) but it would be reassurance that using AW was the critical factor in reducing the cyano. The reason I have been putting off doing this is because, on Saturday 6th October 2012 (week 115) one of the Cardinal Tetra died. This makes a total of six fish I have lost. The three Otocinclus from Chapter II. One of the nine Glowlight Tetra developed what looked like a tumour sometime in 2011 and died. I don't feel guilty about these deaths as they might not have been my fault. Then in week 85 one of the five Black Phantoms died. This probably was my fault. The death happened five weeks after the fish were netted for the total tank breakdown in Chapter IX. The Phantom had been visibly ill for a while and showed the classic symptoms of a bacterial infection. Swollen eyes and body, totally off his food. I think he probably picked up the infection through a wound inflicted during his netting. The Cardinals death was different, he/she developed the sunken chest and curved spine that I have only ever seen when newly added fish are failing to adjust to tank conditions. This made me suspect that a change in tank pH might be responsible. So two days after I did some pH tests.

AW pH

Tap water pH

Tank water pH

 It seems maturing my tap water for a week results in a lowering of its pH. And that by using it for water changes I have lowered the pH of my tank. I think the Cardinal Tetra that died failed to adjust to this change in tank pH. He/she died four days after the third AW change.

Chapter XI. Arthur intervenes or How I solved my Cyanobacteria problem

Edit 15-8-14
I have not been able to repeat the reduction in cyanobacteria observed in this post using aged water (see Chapter 23).

I had come to believe, that there was something about my tap water, in the context of the biochemistry of my fish tank, that inevitably led to the dominance of cyanobacteria. From my reading of the reef forums I knew that many people had found it necessary to completely control the chemistry of their tank water in order to keep sensitive corals in reef tanks. They used reverse osmosis water or distilled water, in which practically all dissolved substances have been removed. They then add added back all the necessary dissolved buffering substances and trace elements etc. at known concentrations. In this way they didn't have to worry about their tap water affecting their ecosystems. I was surprised that I would have to go to the same trouble as a reef keeper in order to keep assorted Tetra and a moss ball but that's life. I bought a 20 liter camping water container and a product called Tropic Marin re-mineral tropic. Here is the blurb.

Tropic Marin's Re-Mineral Tropic adds all natural minerals and substances which are vital for the well-being of the fish and lush vegetation and the reverse osmosis water gets back all its beneficial natural minerals and substances. At the same time the carbonate hardness is raised and stabilized at its optimal value. Re-Mineral Tropic is also excellent for aquarium conditioning of soft tap water, rain water and other soft water. Pure natural composition. No by-products. no nitrates. no phosphates.

Carbonate hardness determines waters ability to buffer against changes in pH that would occur as a result of the many biochemical reactions that generate Hydrogen (H+) ions. 

I planned to use distilled water from work, heat it to tank temperature with a small 25W heater, add the minerals and use it for water changes. At this point Arthur intervened.

Over the years I have asked for advice from local fish-keepers about my cyano problems. The guy at my local fish shop explained to me that excess nitrates and phosphates were probably the cause and advised me to buy nitrate and phosphate adsorbing media for my filter. A couple of fish-keepers at work explained that direct sunlight was probably the problem or that sometimes you are just unlucky and get a cyano infection. But Arthur said something different, something new. Arthur has kept tropical fish for 40 years and in that time has seen it all in fish-keeping. He made two points. Firstly, that weekly 37% water changes was too often and secondly, not to use fresh tap water for a water change. Instead use water that had been allowed to stand for at least a week. I thought it was an interesting idea because at the time, I had been reading about bacterial blooms in fish-tanks. Anyone who has set a a new tank will tell you that in the first week you can get milky water and a film of bacteria develops on the tank surfaces. Both usually go away naturally as the tank matures. From what I read the blooms were heterotrophic bacteria (bacteria that use organic compounds as food rather than autotrophic bacteria like cyano). It seemed logical to me that adding water that already had an established bacterial population to the tank might change the ecology of the tank and tip the balance away from the cyano. Or that the heterotrophs would deplete the water of mystery substance X and limit cyano growth as a result. In order to encourage bacteria to grow I added an air stone and the heater to 20 liters of water and left it for a week.

The water carrier needs to be supported or spillages can occur, here I'm using a washing-up basin.

Then in week 112 I did a 37% water change as usual but used what I'll call 'aged water' (AW). I stuck with weekly changes because it felt right, my tank needs a clean after a week. I wish I had photos of the tank before I started, but here are a series of photos from three videos shot immediately before AW changes. I posted two of them on youtube.

Week 114. After two AW changes. 50% gravel coverage, cyano on tank walls, bog-wood and plants.

Week 115. 5% gravel coverage, tank walls largely clear, some cyano on the bog-wood , plants heavily coated.

Week 116. Gravel clear, tank walls and bog-wood mainly clear, biofilm on
the plants showing signs of breaking down

As I sit here, the day before a water change in week 120, I can stop writing (variably) in the past tense. Its time this blog went live. Here is a photo I just took.

Until at last I threw my enemy down, and smote his ruin on the mountainside.

It seems that using AW dramatically reduced my cyanobacteria problem within five weeks. It is possible that this was a coincidence and switching to using AW was not the reason for the gradual reduction in cyanobacteria I observed. But it seems to me that it might be the reason and I want to understand why it works. This blog will now be where I explore my ideas about why using aged water reduced the cyanobacteria in my freshwater aquarium.

Chapter X. Measuring relative levels of DOCs

Cyanobacteria always returned to my tank in the same way. First I would see it on plants, then the bog-wood, then the gravel, then the sides of the tank and equipment. The first sign of it on this occasion was on green hair algae growing on some Java Moss. For a long time I thought it would be a milder infestation than I had seen before. I believed that my now scrupulous gravel cleaning and carbon filtration was bound to reduce it, but history repeated itself. It got worse and worse until it was as bad as it had ever been. To be clear what I mean by that. No matter how much cyano was removed at a water change, within seven days it would grow back to cover all tank surfaces. The only place it didn't grow was on the fish.

It may be indicative of my feelings towards my fish tank during this period, that I can find no photos or videos of the tank. From week 86 (when I first noted that cyano had returned) until week 110, I did weekly 37% water changes with gravel cleaning and watched the inevitable march of the cyano. The only explanation I could think of at the time, was that there was something  in my tap water that encouraged the dominance of cyano, I'll call it X. I believed this because, one of the most depressing aspects of my cyano problem was that I always observed the most vigorous growth within the first 12-24 hours after a water change. It seemed to me that this indicated that the fresh water itself stimulated a surge in cyano growth which then declined, perhaps as a result of the cyano depleting the water of X. This is heresy in terms of aquarium fish-lore. The idea that water changes might be the cause of a cyano problem went against everything I had read on the forums. There was another problem with the theory, the cyanobacteria had first appeared during a period where I was doing bi-weekly water changes, which at the time had seemed insufficient as it had caused a green water algal bloom (see Chapter II). But that was over two years ago. Perhaps my tap water had changed and the initial causes of cyano dominance were no longer the cause. I didn't believe X was phosphate because I had never detected phosphate in my tap water. I didn't think it was nitrate as my tap water had never tested >1ppm. But maybe my tap water was high in DOCs.

I needed a test for DOCs and, although I couldn't find a commercial kit, I did find a method that I think answered my question. I got the idea from this site which describes the various uses potassium permanganate is put to in the aquarium hobby. One of these uses is as a crude test for the relative levels of DOCs. The method is based on the fact that potassium permanganate (KMnO₄) solutions change colour as they oxidise DOCs. A good example of the colour changes observed can be found here. So I took a 10 ml sample of my tank/tap water and added five drops of a KMnO₄ stock solution.

Both samples were the same colour to begin with.

Here is a quote from the skeptical aquarist link above.

You can see the reaction happening, as the magenta pink color of unreacted KMnO₄ oxidizes first to a rosy tea color, then to amber and brown; the time it takes to spend itself depends on the concentration of dissolved organic matter. In fact a rough-and-ready field test for dissolved organics measures the time it takes for KMnO₄ to completely oxidize in a water sample.

4 Hours

As you can see, the tank water sample had changed to a rosy tea colour by 21 hours.

21 hours

The tap water sample had remained unchanged, which told me that my tap water was lower in DOCs than my tank. So a water change would be expected to reduce the level of DOCs in my tank, contradicting the idea that X = DOCs. This situation reminded me of Chapter IV when I didn't know if my tap water was high in nitrates and phosphates. Back then, even though I had measured that nitrates and phosphates were low, I went ahead and spent weeks lowering them further. This time I decided a complete change in tank water chemistry was the only way I would be rid of cyano.

Out of Interest

I think the unidentified animal in the final photo from Chaper VIII Apendix I is a Rotifer.

One of over 300 species of bdelloid rotifers

Here is the wikipedia page on rotifers. The rotifer above looks most like the photos of bdelloid rotifers.

Chapter VIII-Appendix I. Cyanobacteria identification 2

Here are the photos of the cyano from the unheated goldfish tank mentioned in Chapter VIII.

Filamentous, un-branched

Un-tapered, no heterocysts, no visible sheath, not spiral, Oscillatoria

I got worms

I got ?

I think it's the same genus as the cyano in the tropical tank, but it might not be the same species. It does make the point that you can't avoid having cyano in your tank. The question is: what are the factors that control how much? In the case of my tropical tank it doesn't seem to be nitrate, phosphate, circulation, direct sunlight or DOCs. I would be very interested to know why cyano doesn't dominate in my unheated tank.

Chapter IX-Cyanobacteria control by reducing dissolved organic compounds

From week 62 on I just did 37% water changes/cyano siphoning/gravel siphoning every two or three days and nothing else. I no longer had any hope that the cyano would be affected, it was just so I could bare to look at the tank (and to reduce the smell). I remember a brief period when I did water changes but didn't remove any cyano with the water (except to scrape it off the tank walls). I think the idea was that the cyano population would grow so large that it would use up whatever it was feeding on and 'crash'. I lasted about 10 days but the tank got so disgusting I couldn't stand it. I also worried that if the cyano did crash and start rotting, it would use up oxygen in the water and harm the fish. I also carried on reading.

I can't remember where I first read about dissolved organic compounds (DOCs), I think it was on one of the reef forums. Reef tanks, and salt water tanks in general, also get cyano problems. Usually the cyano is red so it's often called red slime algae. Once I started searching for DOCs and cyano I discovered a whole new world where  it was well known that DOCs had to be removed or algae and cyano problems would occur. That's what protein skimmers were for, and from what I read they were pretty much universally used in the reef world. Another technique recommended for reducing DOCs was the use of activated carbon.

So what are DOCs? Basically they are a pool of organic matter at various stages of decomposition. Everything that had ever died in my fish tank (bacteria, fungi, algae, plants) and all the fs would have contributed to them as they decayed. Some DOCs are short lived and are broken down to compounds that can be used by plants (e.g. nitrates) but apparantly some degrade slowly and can persist (e.g. tannins) 

It made perfect sense to me, the food source for my cyano was DOCs. Maybe, I thought, cyano could use some of the slowly degrading complex DOCs as a food source but algae couldn't, and that was why cyano always seemed to dominate my tank. I guessed the DOCs were mainly coming from the dirty gravel. I had done a pretty good job of cleaning it but it had apparently not been enough. I also started reading criticisms of under gravel filters (UGFs). The criticism was that UGFs make it harder to keep a tank clean. Because any waste is drawn down into the gravel it is harder to siphon off and, even if you keep the gravel clean, you can get a build up of decaying matter under the filter plate. In week 73 I installed an internal canister filter and left it for a month. The filter had two carbon/wool filter pads, two sponge pads and a compartment for 'biomax' pellets (pellets that encourage the growth of nitrifying bacteria).

Week 74. Still from a video one day after a water change. The canister filter is
installed and the gravel looks clean-ish. I notice there's hardly any Ellodea left. 

Then in week 77 I turned off the UGF. I removed the riser tube, blocked up the hole in the filter plate and carried on with the water changes as before. I hoped that because the tank water was not being drawn through the gravel bed, that less DOCs would build up in the water. Or that maybe the different conditions in the gravel would result in a change in tank chemistry, perhaps due to different types of bacteria living there. I also hoped that the carbon in the filter and mechanical filtration would reduce DOCs to a point where the cyano at least slowed down in it's growth. It didn't. I thought OK, if the gravel is dirty enough then turning off a UGF won't do. Maybe you reach a point where there's so much organic matter in the gravel that you always get sufficient DOCs for a cyano bloom. I decided to start again, but this time to use a fine grained gravel so any fs would just lie on the surface and be easy to siphon off.

In week 80 I siphoned off 20 liters of water and put the canister filter and the fish in the 20 liters. I then removed the remaining plants and treated them with antibiotics before completely breaking the tank down. I removed the gravel and UGF. I should say that the gravel and filter plate were absolutely filthy despite all the siphoning etc. I scrubbed  the tank and fittings (no chemicals) and put in new fine grain black gravel, 34 liters of water then the canister filter and fish with the 20 liters of original tank water. The fish seemed fine after they calmed down. A couple of days later, I washed the plants and put them back in. Weekly water changes as before but I used a new gravel siphon, one of the ones with the wide riser tube that the gravel goes up a few inches while you're siphoning. I found this was the best product I had ever used for gravel cleaning, you could see it siphoning off everything. You may be wondering if cyanobacteria returned to my fish tank and if so how long it took? The answer is it returned in six weeks.