QT and Biofilm

[Humblefish]

I've been doing a lot of research lately regarding QT and biodegradation of medications, and feel I now have enough information to start a thread on the subject. In short, I believe heterotrophic bacteria can build up in anaerobic regions of a long-term QT and eventually break down the medications we dose into the water. The sole exception to this is copper, which is not a true medication and thus cannot be biodegraded.

I first read about this here, regarding the degradation of formalin in saltwater: http://www.jzar.org/jzar/article/view/131

The exact reaction that causes the degradation remains unclear, but heterotrophic bacteria, fish (Wienbeck and Koops 1990) and proteins and amino acids (Kitamoto and Maeda 1980) are possible contributors. Failure to maintain therapeutic concentration during the entire treatment cycle could lead to decreased treatment efficacy, recurrence of the pathogen, or resistance build up by the pathogen (Kuemmerer 2009).

Next up is this, Praziquantel degradation in marine aquarium water: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4824874/

Abstract
Praziquantel (PZQ) is a drug commonly utilized to treat both human schistosomiasis and some parasitic infections and infestations in animals. In the aquarium industry, PZQ can be administered in a “bath” to treat the presence of ectoparasites on both the gills and skin of fish and elasmobranchs. In order to fully treat an infestation, the bath treatment has to maintain therapeutic levels of PZQ over a period of days or weeks. It has long been assumed that, once administered, PZQ is stable in a marine environment throughout the treatment interval and must be mechanically removed, but no controlled experiments have been conducted to validate that claim. This study aimed to determine if PZQ would break down naturally within a marine aquarium below its 2 ppm therapeutic level during a typical 30-day treatment: and if so, does the presence of fish or the elimination of all living biological material impact the degradation of PZQ? Three 650 L marine aquarium systems, each containing 12 fish (French grunts: Haemulon flavolineatum), and three 650 L marine aquariums each containing no fish were treated with PZQ (2 ppm) and concentrations were measured daily for 30 days. After one round of treatment, the PZQ was no longer detectable in any system after 8 (±1) days. The subsequent two PZQ treatments yielded even faster PZQ breakdown (non-detectable after 2 days and 2 ± 1 day, respectively) with slight variations between systems. Linear mixed effects models of the data indicate that day and trial most impact PZQ degradation, while the presence of fish was not a factor in the best-fit models. In a completely sterilized marine system (0.5 L) PZQ concentration remained unchanged over 15 days, suggesting that PZQ may be stable in a marine system during this time period. The degradation observed in non-sterile marine systems in this study may be microbial in nature. This work should be taken into consideration when providing PZQ bath treatments to marine animals to ensure maximum drug administration.

The study later discusses how microbial populations (bacteria, protists, algae, phytoplankton, cyanobacteria etc.) may be able to utilize prazi as an energy source after the first exposure to the drug. It also had this to say about the "Biofilm" in a marine aquarium:

Within a marine aquarium, many microorganisms including bacteria, fungi, and protozoans inhabit a biofilm on surfaces within the aquarium system which is semi-protected from non-ideal environmental conditions with an extracellular polymeric matrix. Some biofilms have been described as so hydrophobic that they repel water and other liquids as well as Teflon (Epstein et al., 2011) making them impenetrable and unsusceptible to bleach. Though, some biofilms have been shown to be susceptible to killing by chlorine bleach at a concentration of 10 ppm after 127 min, or after only 12 min at a concentration of 90 ppm (Grobe, Zahller & Stewart, 2002). If the microorganisms in the biofilm survived the chlorine bleach treatment, they could then engage in cell dispersal after the chlorine had been removed, releasing many cells at once or few cells continuously to aid in re-colonization of the aquarium (for review on biofilm cell dispersion, see Solano, Echeverz & Lasa, 2014). In this study, the biofilm is thought to have survived 24 h at a concentration of 200 ppm Cl− and then released microorganisms back into the systems that were able to degrade PZQ in three days. The dynamics and likelihood of this hypothesis are unknown at this time, but studies investigating the rate at which a biofilm can re-colonize a system with microorganisms that can metabolize PZQ are needed to substantiate this claim.

A biofilm is any group of microorganisms which stick to each other and then adhere to a surface. Think bacteria sticking to the glass of a QT. Next up is this email exchange between @AlanM and employees of "The Seas", a Disney aquarium:

Amber,
I read the paper you wrote at Disney. Did you do further work to determine what biofilm resists chlorine to quickly grow and break down the praziquantel in tanks you tried to sterilize with bleach?

I'm treating fish with it at home and it makes a big difference in how often to dose in order to maintain therapeutic levels. I have no way of testing ppm at home, though. I'd hate to have to just burn through quarantine tanks throwing them away instead of being able to bleach them between batches.
Alan

Hi Alan,

Thanks for reaching out about the praziquantel article!

I'm actually going to pass your question on to Dr. Stamper (co-author on the paper, head of the research department at The Seas, and also cc'ed on this email). Hopefully he can help you with your sterilization concerns.
Amber

Thanks, Amber.

I've been corresponding with someone from Georgia Aquarium who presented a poster at MACNA 2016 on their new technique for cheaply and quickly (relatively speaking) quantifying PZQ in saltwater. She says that the concentration can drop very quickly in tanks that have seen it before, that the speed of the drop is a bit unpredictable, and sometimes is entirely gone within "hours" which is consistent with your paper. If they are treating with it, they always test repeatedly since it can be used up so quickly. They haven't looked in to sterilizing tanks with Cl to try to keep the concentration high, but from your paper it sounds like maybe that isn't a practical method anyway since the bacteria that uses it is so resilient.

Her poster is here: http://masna.org/wp-content/uploads/2016/11/P005-S_MASNA2016Poster_Kailen_Gilde.pdf

Dr. Stamper, I'd be interested in whether or not you have succeeded in killing off whatever biofilm is using the PZQ to allow it to stay at high concentration for a number of days to treat fish or if you just estimate and re-dose frequently to maintain treatment levels over a long period since it's hard to do damage to the fish by overdosing it slightly. Or maybe you just use copper or chloroquine phosphate or other things instead of or in addition to PZQ?
Thanks again,
Alan

From our research the only way to tackle the biofilm is detergent, alcohol, and possibly H2O2. Ideally you would find a fish-safe detergent followed by 20 min alcohol application. H2O2 would be a second choice but don't know concentration or contact time. Bleach is no good in this situation. Every system is different. Good luck!

Conclusions: In light of this information, I feel it is unwise to maintain a long-term QT. Especially if you are using Prazipro, CP, metronidazole, antibiotics, etc. The concern here is that after awhile heterotrophic bacteria (and other biodegraders) will form a biofilm in the QT which will render the aforementioned treatments ineffective. This also explains why the use of medications in a DT environment (full of microbes) often fails, and why it is so important to only use meds in a sterile QT.

A practical solution to this problem is to periodically break down your QT, clean everything using vinegar and then allow to air dry thoroughly before reusing in order to sterilize.

Dosing chlorine, or one of the other chemicals mentioned, may eliminate some of the bacteria but NOTHING kills bacteria 100%. Unless you drain and allow to air dry thoroughly. That will sterilize only because you are (essentially) eliminating their environment.

For those who wish to disinfect using chlorine, here is a good dosing chart: http://dec.vermont.gov/sites/dec/files/dwgwp/DW/chlorinedosageemergencydisinfection.pdf

IME; it takes about a week to fully evaporate 10 ppm chlorine in a well circulated QT.

 

[melypr1985]

Well this doesn't bode well for my fish system at work. It suggests that my running prazi every week will eventually not be effective at all without breaking down the entire 900 gallon system. It's been almost a year since I last broke down the system to sterilize. It's a huge several day affair and not easy on the fish either. Would it be possible to break down a system that large only twice a year and be effective? It doesn't sound like it, but it may be the best I can do. What are your thoughts humble?

 

[Humblefish]

Would it be possible to break down a system that large only twice a year and be effective? It doesn't sound like it, but it may be the best I can do. What are your thoughts humble?

Can you dose chlorine every 2-3 months to at least knock some of the bacteria back? I'm still doing research into this, but what I'm seeing is that biodegradation doesn't become a serious problem until bacteria levels are high. Dosing chlorine should at least somewhat keep their numbers in check.

 

[melypr1985]

Can you dose chlorine every 2-3 months to at least knock some of the bacteria back? I'm still doing research into this, but what I'm seeing is that biodegradation doesn't become a serious problem until bacteria levels are high. Dosing chlorine should at least somewhat keep their numbers in check.

I may be able to do that. It would still involve removing all the fish and housing them elsewhere for about a week according to your experience with it. It would be quicker to spend three days sterilizing the whole system with bleach or vinegar ( I did bleach last time). The whole system has started becoming cloudy for a few days every time I dose the prazi and it didn't before. I'm thinking that it's in dire need of a "re-start". I guess I'm needing to decide on which method would be best and how often. It wouldn't hurt to try the chlorine method at least once and see if it's easier. Another problem is convincing management that it's a good idea to keep the tanks empty for a whole week. Hmm.. *puts on thinking hat*

 

[Humblefish]

@melypr1985 The cloudy water could be from bacteria in the water interacting with the praziquantel.

 

[Humblefish]

I really want a spectrophotometer now.

In addition to being able to measure CP in the water; if one were to see a rapid decrease after dosing fresh CP, one could assume biodegradation was occurring and it was high time to sterilize one's QT.

 

[CarrieB]

TTM instructions indicate that 24 hours "drying time" between tank uses is enough to kill off any remaining parasites. Would that hold for biofilm as well?

 

[melypr1985]

@melypr1985 The cloudy water could be from bacteria in the water interacting with the praziquantel.

Right.. That's what I'm thinking as well.

 

[Sandroni]

Great thread well done, this makes me think of my DT... I have this film of my glass that builds up, my magfloat doesn't do anything to it so it makes my glass and tank look cloudy. Could this be a reason too much bacteria???

 

[Humblefish]

TTM instructions indicate that 24 hours "drying time" between tank uses is enough to kill off any remaining parasites. Would that hold for biofilm as well?

Yes

Great thread well done, this makes me think of my DT... I have this film of my glass that builds up, my magfloat doesn't do anything to it so it makes my glass and tank look cloudy. Could this be a reason too much bacteria???

You sure it isn't coralline algae? It often is white before it turns red.

 

[Sandroni]

Yes

You sure it isn't coralline algae? It often is white before it turns red.

Hmmm, I didn't know that. I will post a pic of what it looks like when I get home later.

 

[Sandroni]

Could it also be a calcium build up? It seems more like a calcium build up then coraline

 

[Humblefish]

Could it also be a calcium build up? It seems more like a calcium build up then coraline

Coralline will often form on top of calcium build ups.

 

[revhtree]

This is pretty big news!

 

[leahfiish]

Well this doesn't bode well for my fish system at work. It suggests that my running prazi every week will eventually not be effective at all without breaking down the entire 900 gallon system. It's been almost a year since I last broke down the system to sterilize. It's a huge several day affair and not easy on the fish either. Would it be possible to break down a system that large only twice a year and be effective? It doesn't sound like it, but it may be the best I can do. What are your thoughts humble?

I was just thinking the same thing... Depending on the plumbing for your system, would it be possible to just turn off a few tanks at a time (like close the ball valves?)

 

[melypr1985]

I was just thinking the same thing... Depending on the plumbing for your system, would it be possible to just turn off a few tanks at a time (like close the ball valves?)

It would be. I talked to the store manager today and he vetoed the whole idea - for now. He knows we need to do it, but we are hoping to get fish on monday (dumb time to get fish) and we need a place to put them. We aren't having big losses, so I can't show him an immediate reason to do it.

 

[gig 'em]

Yes

You sure it isn't coralline algae? It often is white before it turns red.

I don't want to reign too much on your statement here, but I want to raise a point just so some people don't become too confident in a drying method to eliminate biofilms. In college I worked in a USDA pathogenic microbiology lab studying biofilms. It was understood that when bacteria form biofilms they become remarkably resistant to environmental dangers and antibiotics making them more difficult to eradicate. Unfortunately biofilms are designed to survive harsh environments including periods of being dried out. Of course every strain is different and what we were studying could be very different from the bacteria in question here, but I would warn against simply relying on drying out a QT tank to rid of the antibiotic resistant bacteria or microbes that are consuming medications. In place I would recommend physically washing with a mild detergent and spraying with Lysol and/or a high % rubbing alcohol. You can also subject the QT tank to high pressure and heat in an autoclave to be certain you have killed all bacteria [emoji23] (sarcasm on that last option as your tank may be useless without silicone).

 

[Humblefish]

I don't want to reign too much on your statement here, but I want to raise a point just so some people don't become too confident in a drying method to eliminate biofilms. In college I worked in a USDA pathogenic microbiology lab studying biofilms. It was understood that when bacteria form biofilms they become remarkably resistant to environmental dangers and antibiotics making them more difficult to eradicate. Unfortunately biofilms are designed to survive harsh environments including periods of being dried out. Of course every strain is different and what we were studying could be very different from the bacteria in question here, but I would warn against simply relying on drying out a QT tank to rid of the antibiotic resistant bacteria or microbes that are consuming medications. In place I would recommend physically washing with a mild detergent and spraying with Lysol and/or a high % rubbing alcohol. You can also subject the QT tank to high pressure and heat in an autoclave to be certain you have killed all bacteria [emoji23] (sarcasm on that last option as your tank may be useless without silicone).

I was hoping to encounter someone like you when I started this thread.

Would wiping the entire QT down with a vinegar or bleached soaked paper towel suffice? Rinse and then let it completely air dry? I just think more people would be comfortable using one of those instead of detergent, Lysol, rubbing alcohol, etc. But if that's how it's gotta be...

 

[ascheff]

@melypr1985 The cloudy water could be from bacteria in the water interacting with the praziquantel.

As I understand it, the alcohol in commercial Praziquantel products like PraziPro, added to make in soluble, is a carbon source and can fuel a bacterial bloom in the water column, not dissimilar to other forms of carbon dosing for nutrient control. That is one reason why it is important to keep the water well aerated, as the extra bacteria depletes oxygen. I have however not personally experienced this.

 

[Humblefish]

I have however not personally experienced this.

I have when combining Prazipro with other meds, especially Chloroquine phosphate. I think oxybispropanol found in Prazipro is the culprit.