Clove Oil Procedure to combat cilliate protozoan infections in reef tanks

OceanPup

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Hypothesis: Metroplex-resistant cilliate protozoan can be managed by using a 1500ppm clove oil solution bath dip (e.g. 100% clove oil extract, w/o ethanol) for no longer than 10mins.

Background Information: Due to the sensitive nature of dosing medications in a reef-seahorse display tanks/reef tanks, and due to the pervasive nature of cilliate protozoan infections in LFS seahorses, treatment plans to rectify an active cilliate infection in a reef tank are limiting at best (chloroquine phosphate, formalin, etc.) due to the effects the medication can have to an established reef tank. For example, consequences the medications may be detrimental, with dire results (e.g. corals susceptible to death, invert/CUC sensitivity to the medication, loss of biological filtration, parameter fluctuations, etc.). But, now there may be a solution: Chu, T.-W.; Cheng, C.-M.; Cheng, Y.-R.; Dong, C.-D.; Chuang, C.-H.; Pan, C.-H.; Sun, W.-T.; and Ding, D.-S. (2022) focused their study of the use of clove oil to grate cilliate issues within the aquaculture industry. In short, the researchers evaluated whether Clove Extract can be used for hobbyists and reefers as an effective drug in combating Ciliate Infection in Coral (e.g. goniopora columna).

Procedure: Chu, T. W. et al. (2022) stated: “In experiment 1, it was known that the LC50 of clove extract against ciliates is 1500 ppm and that it can cause complete death within 10 min. Therefore, in the study of the ciliate disease treatment and drug tolerance of coral, 1500, 2500, 5000, 7500 and 10,000 ppm were selected as test concentrations. No medication was used on control group C. This experiment refers to Cheng et al. (2021) [10]. After the corals were infected by ciliates, they were dipped in different clove extract concentrations for 10 min, and then replaced in sterilized seawater. After 72 h, we referred to Levy et al. (2003) [38] and Chen et al. (2021) [10] for the method to judge the change in coral morphology, and the survival, chlorophyll a, zooxanthellae number, antioxidant enzymes superoxide dismutase and catalase were measured to evaluate the effect of drug treatment on the coral. After the experiment, the seawater in the beaker was centrifuged, the supernatant liquid was removed, and the ciliates in the saltwater were calculated using a hemocytometer.”

Therefore, I decided to implement a home replication of this study’s experiment due to the active cilliate infection in my seahorse display tank, I figured to give it a shot since my display tank currently contains Ricordeas, rhodactus, Yuma’s, PS and NPS gorgonians, and micro gonioporas currently affected by the metroplex-resistant cilliate infection. I took one of each coral type (soft, photosynthetic, and stony corals) and created two subsequent groups: Control Group (no dip) and Treated Group.

Control group consisted of:

• 2 ricordea frags (one bleached, one healthy)
• 1 yuma
• Red Branching Gorgonian coral
• 1 encrusting montipora frag
• Purple micro-goniopora frag

Treatment Group consisted of:

• 2 ricordea frags (one bleached, one healthy)
• 1 metallic neon green rhodactus
• Yellow Branching Gorgonian coral
• 1 encrusting montipora frag
• Peach micro-goniopora frag.

Materials:

• line, pump, and air stone
• 100% pure clove oil extract (which means no ethanol is added)
• display marine tank water (for rinsing)
• sterile saltwater (5 gallons)
• timer
• waste bucket

Procedures:

Calculated the dosage of clove oil extract to mix with the 2 gallons of display tank water to achieve a level of 1500ppm. Heavily aerated the tub for 30mins before bathing the treatment coral group. Started the 10min timer, and rinsed off each frag multiple times (3-5 rinses) with aged and clean saltwater at the end of the 10min duration.

After the bath and rinse, I took 24hr daily photos of the corals to determine if the results could be replicated (i.e. measuring polyp extensions) and measured the length of the polyps by using the iPhone measurement app.

Link to the academic study of clove oil as a potential drug option to treat cilliate infection in goniopora sp.: https://mdpi-res.com/d_attachment/biology/biology-11-00280/article_deploy/biology-11-00280.pdf

Results and Discussion:
At the end of the initial 24 hours, a significant difference was found when comparing the Control Group with the Treatment Group; polyp extensions were shorter in the Control group while the Treatment group was visually healthier. A week later (today), here are the coral’s pictures after the clove oil bath. The bleached ricordea is growing back, as shown in the attachments.

85625FB5-2C38-4BD3-8AFE-438100AA4C16.jpeg CC32534C-43E6-451A-8F3B-16E3A8DF46A4.jpeg 3DB841EA-EF8B-4093-A436-01F88ED4A696.jpeg 55A2205F-8EE5-4600-91EA-736FF71E720D.jpeg 10460A3B-86A2-4176-88B8-174C1365B6F3.jpeg C9588664-9528-4630-AAA5-AC63C873074D.jpeg D255AD59-4B05-41EB-97DA-376D3EAF18C0.jpeg F192E5D8-5A3A-43AA-ABF7-3AD017D9516B.jpeg F2DC5EFA-2503-4B67-8E5B-A63A6AB98922.jpeg
 

MnFish1

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Hypothesis: Metroplex-resistant cilliate protozoan can be managed by using a 1500ppm clove oil solution bath dip (e.g. 100% clove oil extract, w/o ethanol) for no longer than 10mins.

Background Information: Due to the sensitive nature of dosing medications in a reef-seahorse display tanks/reef tanks, and due to the pervasive nature of cilliate protozoan infections in LFS seahorses, treatment plans to rectify an active cilliate infection in a reef tank are limiting at best (chloroquine phosphate, formalin, etc.) due to the effects the medication can have to an established reef tank. For example, consequences the medications may be detrimental, with dire results (e.g. corals susceptible to death, invert/CUC sensitivity to the medication, loss of biological filtration, parameter fluctuations, etc.). But, now there may be a solution: Chu, T.-W.; Cheng, C.-M.; Cheng, Y.-R.; Dong, C.-D.; Chuang, C.-H.; Pan, C.-H.; Sun, W.-T.; and Ding, D.-S. (2022) focused their study of the use of clove oil to grate cilliate issues within the aquaculture industry. In short, the researchers evaluated whether Clove Extract can be used for hobbyists and reefers as an effective drug in combating Ciliate Infection in Coral (e.g. goniopora columna).

Procedure: Chu, T. W. et al. (2022) stated: “In experiment 1, it was known that the LC50 of clove extract against ciliates is 1500 ppm and that it can cause complete death within 10 min. Therefore, in the study of the ciliate disease treatment and drug tolerance of coral, 1500, 2500, 5000, 7500 and 10,000 ppm were selected as test concentrations. No medication was used on control group C. This experiment refers to Cheng et al. (2021) [10]. After the corals were infected by ciliates, they were dipped in different clove extract concentrations for 10 min, and then replaced in sterilized seawater. After 72 h, we referred to Levy et al. (2003) [38] and Chen et al. (2021) [10] for the method to judge the change in coral morphology, and the survival, chlorophyll a, zooxanthellae number, antioxidant enzymes superoxide dismutase and catalase were measured to evaluate the effect of drug treatment on the coral. After the experiment, the seawater in the beaker was centrifuged, the supernatant liquid was removed, and the ciliates in the saltwater were calculated using a hemocytometer.”

Therefore, I decided to implement a home replication of this study’s experiment due to the active cilliate infection in my seahorse display tank, I figured to give it a shot since my display tank currently contains Ricordeas, rhodactus, Yuma’s, PS and NPS gorgonians, and micro gonioporas currently affected by the metroplex-resistant cilliate infection. I took one of each coral type (soft, photosynthetic, and stony corals) and created two subsequent groups: Control Group (no dip) and Treated Group.

Control group consisted of:

• 2 ricordea frags (one bleached, one healthy)
• 1 yuma
• Red Branching Gorgonian coral
• 1 encrusting montipora frag
• Purple micro-goniopora frag

Treatment Group consisted of:

• 2 ricordea frags (one bleached, one healthy)
• 1 metallic neon green rhodactus
• Yellow Branching Gorgonian coral
• 1 encrusting montipora frag
• Peach micro-goniopora frag.

Materials:

• line, pump, and air stone
• 100% pure clove oil extract (which means no ethanol is added)
• display marine tank water (for rinsing)
• sterile saltwater (5 gallons)
• timer
• waste bucket

Procedures:

Calculated the dosage of clove oil extract to mix with the 2 gallons of display tank water to achieve a level of 1500ppm. Heavily aerated the tub for 30mins before bathing the treatment coral group. Started the 10min timer, and rinsed off each frag multiple times (3-5 rinses) with aged and clean saltwater at the end of the 10min duration.

After the bath and rinse, I took 24hr daily photos of the corals to determine if the results could be replicated (i.e. measuring polyp extensions) and measured the length of the polyps by using the iPhone measurement app.

Link to the academic study of clove oil as a potential drug option to treat cilliate infection in goniopora sp.: https://mdpi-res.com/d_attachment/biology/biology-11-00280/article_deploy/biology-11-00280.pdf

Results and Discussion:
At the end of the initial 24 hours, a significant difference was found when comparing the Control Group with the Treatment Group; polyp extensions were shorter in the Control group while the Treatment group was visually healthier. A week later (today), here are the coral’s pictures after the clove oil bath. The bleached ricordea is growing back, as shown in the attachments.

85625FB5-2C38-4BD3-8AFE-438100AA4C16.jpeg CC32534C-43E6-451A-8F3B-16E3A8DF46A4.jpeg 3DB841EA-EF8B-4093-A436-01F88ED4A696.jpeg 55A2205F-8EE5-4600-91EA-736FF71E720D.jpeg 10460A3B-86A2-4176-88B8-174C1365B6F3.jpeg C9588664-9528-4630-AAA5-AC63C873074D.jpeg D255AD59-4B05-41EB-97DA-376D3EAF18C0.jpeg F192E5D8-5A3A-43AA-ABF7-3AD017D9516B.jpeg F2DC5EFA-2503-4B67-8E5B-A63A6AB98922.jpeg
Very nice... Excellent experiment and references. Did you know what ciliate you were dealing with?
 
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OceanPup

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Very nice... Excellent experiment and references. Did you know what ciliate you were dealing with?
Hi and thank you!

The fish vet was able identify 3 separate ciliate protozoan species; two of the cilliate species died from the metroplex medication while the third continued to prosper. Although the vet was only able to confidently say the resistant strain was not urenoma or vibrio, he was not able to identify the resistant strain.
However, the clove oil bath seemed to work well, and I will be repeating the dips weekly to see how the display tank is done. At the end of the month, I’m planning on bringing several samples to the fish vet to see if he can confirm if the clove oil is affecting the cilliate population on the corals.
 
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OceanPup

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Nice work and an interesting read.
Thank you! It’s supposed to intrigue the audience, considering the flack I received from FB reef groups for even considering this experiment So hopefully, constructive criticism will be delivered by members when this experiment ends!
 

Muffin87

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Calculated the dosage of clove oil extract to mix with the 2 gallons of display tank water to achieve a level of 1500ppm.
How did you calculate that?
What information did you have about the clove oil exctract?
 

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Great job! I’m wondering if there’s a clove oil dose that kills uronema but is safe for fish?
 
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Great job! I’m wondering if there’s a clove oil dose that kills uronema but is safe for fish?
Thank You!

In response to your question, I found a related, academic article investigating the sedative effects of various clove oil dosages in clownfish (click here for the academic article).

Based on the results of the article, hypothetically, if the clove oil dosage found in the Clove Oil and Cilliate infection is also within the parameters of the related, clove oil and clownfish study, then I believe clove oil as a homeopathic drug may have an effect on urenoma as a potential treatment for fish. So let us check the math down below:

This is my very basic calculations for the clove oil PPM, so future members can follow along/replicate the experiment with a bit more ease:

In order to calculate the PPM (parts per million) of clove oil in sterile 1.020 saltwater, I used the following formula (shared with me by Dan U.),

PPM = [Mass of Solute divided by Mass of Solvent] * 10^6th power

Or: Concentration in PPM = Concentration as a percentage × 10^4

Per Sciencing.com, they stated, "a basic PPM calculation by volume or by mass will help to cement the concepts you need to understand. For mass, imagine you have 0.98 liters of water with 0.2 g of salt dissolved in it, noting that 1 L of water = 1 kg of mass, so the total mass in the example is 1 kg."

Since I used 1 gallon of SW as the treatment volume, I converted it 1 gallon of SW to 3.8 KG as the total mass of the solvent. The mass of the solute (clove oil) is considered as the unknown variable. Since the target clove oil PPM to treat ciliate protozoan infections within acceptable safety margins was 1500ppm, I replaced the missing clove oil dosage as "x". Therefore, I replaced the formula parts with their respective data:

1500ppm = [x/3.8kg)*10^6th power

1500ppm *3,800 g = x * 10^6

5,700,000g =1,000,000*x

5.7g = "x," the required weight of 100% clove oil extract.

I'm not a math quiz, but I did have a gram scale on hand. So I used the internet and found that 5.7grams is the same as 5.7 ml. So, to create a 1500ppm solution for a 1 gallon saltwater tub, you would add 5.7mLs of clove oil.

Be aware that 1500ppm made from a 100% clove oil extract is safe with exceptions; some brands may have added ethanol to their clove oil products to make the oil more soluble. Unfortunately, ethanol is a toxic factor to be aware of as it can have an adverse effect on marine fish (at least, according to Google).

According to the related, linked study, the Müge Aliye Hekimoğlu*, Cüneyt Süzer, Şahin Saka and Kürşat Firat (2017) conducted the following dosages of clove oil when investigating the sedative and recovery effect of the drug:

"Melanosis and not melanosis groups were exposed to 3 doses of clove oil (0.5, 1 and 1.5 ml/l)" (Hekimoğlu, et. al 2017).

They found that "In the group to which was applied clove oil, the fish did not show abnormal behaviors and induced following the normal swimming behavior when put in water" (Hekimoğlu, et. al 2017).

So, theoretically, by comparing the dosages between the studies, I found that 5.7mL of clove oil in 1 gallon of water is the same as 5.7mL / 3.78 L, which when calculated can be considered as ~1.5mL per 1L of saltwater.

But, will it treat urenoma, as a remedy for fish health? I honestly cannot say, as that specific protozoan was not found in my reef tank. But, it did eliminate the other three species of cilliates from what I can tell from the corals, and there have been no other signs of ciliate infections since their second dip.

I hope this helps explain; let me know if you have any other questions! :)
 
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OceanPup

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Excellent study!

Do you have a link for the source of your 100% clove oil?
Thank you for the support!

To answer your inquiry of a link to the clove oil, I do not; but, as long as the clove oil from one’s local pharmacy (humane grade) is acceptable/considered safe as long as it’s pure and not mixed with ethanol-based ingredients.

I hope this helps!
 

MartinM

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Clove oil treatment of coral infections is well document in the scientific community. But most hobbyists aren’t trained to be able to understand scientific publications. I’m glad it worked for you, I also use it.
 

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This is an interesting read. Has anyone tried this with brown jelly disease?
 

MartinM

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This is an interesting read. Has anyone tried this with brown jelly disease?
No luck the one time I tried. Lots of evidence that is bacterial, and lots of evidence of people using antibiotics to treat at successfully.
 
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