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Old 09/01/2018, 10:58 PM   #1
tkeracer619
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Using Chloroquine Phosphate in display: Rapid Degradation

In an attempt to rid my system of Vermatid Snails and an unidentified flatworm that I was unable to eradicate through other routes I used Chloroquine Phosphate in my display. I did so during a fallow period after removing all coral and inverts. This was very successful and after a short period of time green and coralline algae returned to normal.

After moving the fish back into the display Crypto showed itself again. They were treated with Chloroquine Phosphate for 30 days in an established quarantine system prior to being re-introduced.

After much deliberation I decided to try treating the entire system with CP since it had already been run successfully without an ammonia spike. I had yet to re-introduce inverts or coral. I dosed the tank as I had before and monitored the system for two weeks prior to leaving the country for vacation. When I left there were no signs of Crypto. When I returned everything looked great and soon enough green and coralline algae returned. After a month, so did the crypto...

I decided I would dose the tank again, maybe I had gotten the dosage wrong, after all I am estimating water volume. This time I purchased a Photospectrometer in an attempt to monitor Chloroquine Phosphates levels.

Here is what I have found. On my first test, I had dosed the system to a theoretical concentration of 44mg/gal. It measured 25mg/gal. I dosed the system with a small maintenance dose (0.5 grams) and then 24 hours later took another measurement. The levels in the tank had continued to drop. I dosed a larger amount of 5.0 grams, added it directly to the display, and gave it an hour to mix before running a test. About 12 hours later I tested again, the numbers were even lower. After another 7 hours the levels continued to rapidly drop.

Here is a chart that shows the testing.


The tank has no active filtration or UV in operation other than filter bags so I can only assume this degridation is caused by one of two things (or a combination of the two).

A. It's being absorbed by the substrate.
B. Bacteria is consuming it.

I assume bacteria is the culprit here, if it were being absorbed I would think absorption would be at a constant or reduced rate. However, it seems the more I dose the faster it degrades leaving me with the thought that more bacteria have grown and are consuming the medication.

I understand this is a very small sample set over a short period of time, however, given I cannot get pukani rock anymore and the maintenance dose at the current rate would entail continuously dosing 360mg of CP per hour for a total of 260 grams over the course of 30 days (assuming consumption stays consistent) I'm throwing in the towel on this attempt. It appears that a therapeutic concentration of Chloroquine phosphate is not achievable.

I will add to this post as time goes on and I get to investigate chloroquine phosphates role in my QT procedures but I have had several failures with it over the years. Most of the time I keep my QT tanks cycled unless there is a failure in which I bleach, dry, and restart the QT. I feel like this same issue may exist in a cycled QT which may have led to some of those failures.

My next step will be to bleach some rock and repeat this test in a sterile environment to see if I see if the CP absorbs into the rock.

One thing to note is that the known concentration sample I made to calibrate the spectrophotometer on day one is still reading 40mg/Gal concentration.


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Old 09/02/2018, 11:32 AM   #2
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What exactly are you monitoring that corresponds to the chloroquine concentration?


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Old 09/02/2018, 11:57 AM   #3
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What exactly are you monitoring that corresponds to the chloroquine concentration?
Absorbance of UV light at 343nm using a Spectrophotometer. I've been told there may be more to it because Chloroquine Metabolites that may not be bio-active will also read in the 343nm range. This would however cause a false reading in the positive direction. I'm still researching what equipment is necessary to separate the two components.

Here is a good read if you are interested.
https://pdfs.semanticscholar.org/481...06677b8e44.pdf


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Old 09/02/2018, 12:03 PM   #4
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I added carbon last night. It certainly pulls a fair amount of Chloroquine from the system.




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Old 09/02/2018, 03:43 PM   #5
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You stated

“On my first test, I had dosed the system to a theoretical concentration of 44mg/gal. It measured 25mg/gal.”

Did you maybe calculate the dose based on the diphosphoric acid salt and measure the free base. That would explain 25 vs 44 mg


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Old 09/02/2018, 04:38 PM   #6
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Absorbance of UV light at 343nm using a Spectrophotometer. I've been told there may be more to it because Chloroquine Metabolites that may not be bio-active will also read in the 343nm range. This would however cause a false reading in the positive direction. I'm still researching what equipment is necessary to separate the two components.

Here is a good read if you are interested.
https://pdfs.semanticscholar.org/481...06677b8e44.pdf
Thanks!

Have some random thoughts I will pass your way when I get home.

What was the absorbance @343 nm for the initial dose? While I have only poked around down to 380 nm, organics start to absorb well down there.


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Old 09/02/2018, 05:48 PM   #7
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You stated

“On my first test, I had dosed the system to a theoretical concentration of 44mg/gal. It measured 25mg/gal.”

Did you maybe calculate the dose based on the diphosphoric acid salt and measure the free base. That would explain 25 vs 44 mg
I dosed based on the "add 40mg per gallon" directions. I didn't have the equipment until thursday which was already over a week into dosing.

Once I got it, I created a slope based off known concentration of 80mg/gal, 40mg gal, 20mg/gal, and 10mg gal.

Each time I take a measurement I use new sw with matching salinity to zero it out. I had saved some water from the tank prior to dosing to compare with the new sw and they came back identical.


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Old 09/02/2018, 05:54 PM   #8
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Thanks!

Have some random thoughts I will pass your way when I get home.

What was the absorbance @343 nm for the initial dose? While I have only poked around down to 380 nm, organics start to absorb well down there.
Were you using quartz or glass cuvvetes? If organics were at play I would expect the reading to be higher than with new saltwater. It wasn't. My tank is pretty clean though, not a ton of docs.


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Old 09/02/2018, 07:59 PM   #9
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I dosed based on the "add 40mg per gallon" directions. I didn't have the equipment until thursday which was already over a week into dosing.

Once I got it, I created a slope based off known concentration of 80mg/gal, 40mg gal, 20mg/gal, and 10mg gal.

Each time I take a measurement I use new sw with matching salinity to zero it out. I had saved some water from the tank prior to dosing to compare with the new sw and they came back identical.
The actual amount of active ingredient of chloroquine in 40 mg chloroquine phosphate is 320/516 * 40 mg. This does not explain the falling concentration, just the discrepancy between dosed vs measured amount of chloroquine.

I follow your approach for zeroing the instrument.

With regard to the absorbance of chloroquine phosphate, you might not be getting the same value when working in seawater at a pH near 8 when the work in the paper was performed in water with a pH likely below pH 6 (chloroquine diphosphate is a diphosphoric acid salt). This would contribute to deviations between calculated and measured chloroquine levels. I owe you thoughts on this.


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Old 09/02/2018, 08:06 PM   #10
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Were you using quartz or glass cuvvetes? If organics were at play I would expect the reading to be higher than with new saltwater. It wasn't. My tank is pretty clean though, not a ton of docs.
I was using glass.

I never saw it discussed, but how do we know that new salt water is DOC free? The comparison of new versus tank salt water may not be valid for judging whether organics are in play, but I agree, organics may not be relevant to answering your question.

How do you know the level of DOC in your system?


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Old 09/02/2018, 08:31 PM   #11
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OK, here are the random thoughts on the concentration decay.

The phosphate of chloroquine is reported stable under acid conditions. Not clear to me whether it remains stable in seawater at pH 8 near 80 F. Ditto solubility. Once the phosphates are neutralized, would the free base or at least partially deprotonated chloroquine crystallize from solution. Add to this its sensitivity to light, not just UV light, and decomposition and precipitation are at least plausible explanations for its loss.

At the higher pH of tank water, the free base being less soluble in water and nearly neutral than the phosphate salt would be more likely to adsorb to hydrophobic surfaces. This would include water-air interfaces of the tank and in the skimmer. Certain plastic surfaces like PVC might provide adsorptive sites.

Comparing the stability (concentration over time) of a chloroquine solution in new saltwater against that in tank water in a glass container might be instructive.

Well, that’s the random thoughts for tonight.

Good luck!

Dan


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Old 09/02/2018, 09:04 PM   #12
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The actual amount of active ingredient of chloroquine in 40 mg chloroquine phosphate is 320/516 * 40 mg. This does not explain the falling concentration, just the discrepancy between dosed vs measured amount of chloroquine.
As I understand it since my calibration was done in saltwater with measured amounts there shouldn't be a discrepancy since the ratio is built in. From what I understand the numbers I came up with in my calibration were just the chloroquine component so while there may not be an actual 40mg/gal of chloroquine there is 40mg/gal of powder since what I based my calibration on was weight of powder added.

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Comparing the stability (concentration over time) of a chloroquine solution in new saltwater against that in tank water in a glass container might be instructive.
This one should be pretty easy to accomplish and I think should be done.

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I was using glass.
I forget the cut off where you have to go to quartz but I believe at 343nm the glass has started to absorb on its own.

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I never saw it discussed, but how do we know that new salt water is DOC free? The comparison of new versus tank salt water may not be valid for judging whether organics are in play, but I agree, organics may not be relevant to answering your question.

How do you know the level of DOC in your system?
I don't necessarily know the level of DOCs just that the water quality is fairly high, at least there isn't significant color change when viewing through the 8ft dimension. That and Zeroing the spectrophotometer with new SW vs tank SW without CP showed no significant difference in absorption at 343nm so I have little concern regarding interference between the zero reference and the tank water with CP.

Is there an abbreviation for Spectrophotometer and if there isn't can we make one up? It's a keyboard full

Thanks for your input, I'm not a chemist, i'm just winging this but love CP when it works and don't when it fails. At this point I haven't been able to correlate a reason why QT with it has been a failure or success. I think the more we wrap our heads around its behavior in our tanks the more successful we will be with it.


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Old 09/03/2018, 01:21 AM   #13
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Looks like carbon and whatever was degrading the CP made short work of it. Bottomed out. The spectrophotometer reads an absorbance of .005 which since we are calibrating zero with new saltwater I feel that is within a reasonable error to call it zero. I'll turn the UV back on tomorrow and will change out carbon next weekend.



I've started doing an automatic water change to bring salinity down to hypo levels... I don't anticipate running any more tests on CP for the next couple months but will pick back up as soon as my QT systems are ready for fish.

Dan, do you know of the proper procedure to use one of these for measuring copper concentration? My spectrophotometer has a range of 200-1000nm. I'm terrible at color comparison and have been using the Hanna Copper checker but since they don't recommend it in saltwater...


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Old 09/03/2018, 05:10 AM   #14
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Dan, do you know of the proper procedure to use one of these for measuring copper concentration? My spectrophotometer has a range of 200-1000nm. I'm terrible at color comparison and have been using the Hanna Copper checker but since they don't recommend it in saltwater...
I have no experience with copper testing but do have experience using Hanna Checkers for unintended purposes.

The Hanna Checker might not be recommended for saltwater not because the chemistry fails but because the instrument is calibrated with freshwater solutions. You seem to be comfortable with making standard curves. Make a standard curve in saltwater with a copper (II) salt using both the Hanna Checker and the spectrophotometer set at the wave length of the light source of the Hanna Checker (see the instruction card for this spec). That would quickly define what’s possible. If you need more random ideas, shoot me a PM.

Dan


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Old 09/03/2018, 08:10 AM   #15
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While this is WAY above me I still like the read.
How about "Specc" for the abbreviation?


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Old 09/13/2018, 09:28 AM   #16
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Sorry you are having tank troubles. I am fascinated by the work you are doing to figure this out. I presume the crypto is a resistant form and is protected during a prolonged tomont ( is that the right name?) stage. Do you have any pharmacist friends? I think one could offer a different perspective. As a nurse I have a different idea to throw out there. Calcium causes malabsorption of many antibiotics by binding/ blocking with the medications active ingredients. an example would be bactrim. You should not eat or drink anything with a lot of calcium for up to two hours before or after taking the medication. lots and lots of calcium in a salt water tank. lol. or perhaps the calcium in LR interacts differently than the calcium in solution. Although no longer allowed to be prescribed for muscle cramps, CP is effective for muscle cramps too. muscles use calcium and other electrolytes to communicate to cause muscle contraction. I keep thinking there is something in this relationship, but I am not sure. I think Randy might also have some insight here.


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Old 09/19/2018, 12:36 AM   #17
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Sorry you are having tank troubles. I am fascinated by the work you are doing to figure this out. I presume the crypto is a resistant form and is protected during a prolonged tomont ( is that the right name?) stage. Do you have any pharmacist friends? I think one could offer a different perspective. As a nurse I have a different idea to throw out there. Calcium causes malabsorption of many antibiotics by binding/ blocking with the medications active ingredients. an example would be bactrim. You should not eat or drink anything with a lot of calcium for up to two hours before or after taking the medication. lots and lots of calcium in a salt water tank. lol. or perhaps the calcium in LR interacts differently than the calcium in solution. Although no longer allowed to be prescribed for muscle cramps, CP is effective for muscle cramps too. muscles use calcium and other electrolytes to communicate to cause muscle contraction. I keep thinking there is something in this relationship, but I am not sure. I think Randy might also have some insight here.
Thanks for your input! I do plan on running a series of tests in the future to determine if calcium does exactly that.

I have a good friend who owns a compounding pharmacy but it would be unethical to ask him to slip me some of this in an unmarked envelope. I won't even approach the subject with him. I'm headed to the vet tomorrow with both of my pups and will talk to the vet at length about this.

I did purchase a Kilogram of CP from Fishman. I will say they are great to work with. I tested it against my Ebay supply which was dwindling. The Fishman tested out at 4.6% stronger than the Ebay supply. The Fishman CP did have a finer texture, was more hygroscopic, and was easier to become airborn than the Ebay version. From what I remember, the last time I purchased 50grams of CP from a compounding pharmacy, it behaved in a similar manner as the Fishman does.

If I could get a known pure sample I would be able to go further but until then...


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Old 09/19/2018, 12:17 PM   #18
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You can have your vet precribe it and get it from the local pharmacy but it is more expensive than the stuff marketed for fish. My vet writes the script for Nemo Music. And a pharmacist I work with has said I can come pick her brain about this.


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Old 09/19/2018, 01:57 PM   #19
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Cool, see what she says!

I think with the resurgence of this medication and the lack of testing ability for 99.99 percent of hobbyists it's important to figure out its relationship in saltwater. Like I said, I've had success and I've had failure with it, I'd like to know why.


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Old 09/19/2018, 08:25 PM   #20
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Yeah, no go on the script. Not my normal vet, she wouldn't even pick up the documentation.


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Old 10/06/2018, 06:33 PM   #21
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Yeah, no go on the script. Not my normal vet, she wouldn't even pick up the documentation.
That is pretty crappy. My pharmacy said it is not all that uncommon, and they suggested I ask my vet actually.


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Old 10/09/2018, 12:17 AM   #22
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Yeah, common issue with vets not jumping to help write a script on something they know nothing about. Kinda surprises me when they say "I only do cats and dogs", figure they would take 3 minutes to listen and understand what you are trying to describe.


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