Coral Coloration & Trace Element Experiment

[Dan_P]

All the values I used are corrected for the hanna baseline (I got around ~17ppb baseline for my water)
So the 26,27 are raw values of 43,44 on the checker.

Thanks. You might have guessed I started lab work with a quick baseline study.

 

[taricha]

So here's where I ended up on a dosing rate going forward.

Ignoring the early extreme drop of near 100ppb in a few hours and fitting to the more stable pattern over the next 3-4 days - I get a rate of about 8ppb per day for Fe. It's higher than a lot of other sources recommend but lower than the consequence of chasing Red Sea's target of 150ppb Fe, so I'm good with that for a week at a time.

And here's the situation for iodine.

I'm using the data in red from the early part to come up with a daily depletion/dose rate of ~22ppb per day. I'm choosing to use the faster early rate rather than the slower rate from days 2-5 because the target of 60ppb Iodine is reasonable, and the early fast uptake may well be mostly real biological uptake.
I suspect that after dosing 22ppb/day for a little while, the demand might slow down some and I might be okay to back off the dosing. We'll see.

Anyway, that's my dosing rate for Phase 3 and how I got there.

 

[Dan_P]

So here's where I ended up on a dosing rate going forward.
View attachment 3924621
Ignoring the early extreme drop of near 100ppb in a few hours and fitting to the more stable pattern over the next 3-4 days - I get a rate of about 8ppb per day for Fe. It's higher than a lot of other sources recommend but lower than the consequence of chasing Red Sea's target of 150ppb Fe, so I'm good with that for a week at a time.

And here's the situation for iodine.

I'm using the data in red from the early part to come up with a daily depletion/dose rate of ~22ppb per day. I'm choosing to use the faster early rate rather than the slower rate from days 2-5 because the target of 60ppb Iodine is reasonable, and the early fast uptake may well be mostly real biological uptake.
I suspect that after dosing 22ppb/day for a little while, the demand might slow down some and I might be okay to back off the dosing. We'll see.

Anyway, that's my dosing rate for Phase 3 and how I got there.

Will you occasionally confirm that the consumption is staying approximately the same during phase 3?

 

[taricha]

Will you occasionally confirm that the consumption is staying approximately the same during phase 3?

right. I'll test a couple of times a week to check how the system is processing the daily doses. My hunch is that the consumption of Iodine will drop after a while, and I'd like to catch it happening. Have no hunch on how Fe-edta consumption will evolve.

 

[Doctorgori]

Holy smokes, on the one hand I feel under qualified and overwhelmed even attempting to do anything close to this…
But Im also impressed and inspired…

I am going to take advantage of this and revisit/ readjust my experiment to attempt to compliment this one given my very limited skill set and equipment

Appreciate you even doing this, its important

 

[Dan_P]

So here's where I ended up on a dosing rate going forward.

Ignoring the early extreme drop of near 100ppb in a few hours and fitting to the more stable pattern over the next 3-4 days - I get a rate of about 8ppb per day for Fe. It's higher than a lot of other sources recommend but lower than the consequence of chasing Red Sea's target of 150ppb Fe, so I'm good with that for a week at a time.

And here's the situation for iodine.

I'm using the data in red from the early part to come up with a daily depletion/dose rate of ~22ppb per day. I'm choosing to use the faster early rate rather than the slower rate from days 2-5 because the target of 60ppb Iodine is reasonable, and the early fast uptake may well be mostly real biological uptake.
I suspect that after dosing 22ppb/day for a little while, the demand might slow down some and I might be okay to back off the dosing. We'll see.

Anyway, that's my dosing rate for Phase 3 and how I got there.

A thought about what is being measured with the Hanna low range iron tester. I have not contacted Hanna tech help on this.

The TPTZ method used by Hanna likely is testing total iron, both Fe (III) and Fe(II). This is accomplished by reducing Fe (III) to Fe (II) which can then combine with TPTZ to turn it blue. Have you noticed the sulfurous odor of the test solution? I think the “TPTZ” method is using a sulfide to reduce ferric to ferrous ions. This can mean that ferric compounds that are insoluble and possibly not bioavailable are being measured as iron in solution and bioavailable.

The light bulb came on when I was studying iron concentration in Instant Ocean with ferrous sulfate additions. A yellowish brown floc when resuspended in IO was detected as a ~200 ppb increase in iron.

I don’t think this matters much to your experiment because chelated iron and iron sulfate dosed to aquarium water has measurable iron content even when filtered through a 0.45 μ syringe filter, i.e., no rust being measured as soluble iron.

 

[taricha]

A thought about what is being measured with the Hanna low range iron tester. I have not contacted Hanna tech help on this.

The TPTZ method used by Hanna likely is testing total iron, both Fe (III) and Fe(II). This is accomplished by reducing Fe (III) to Fe (II) which can then combine with TPTZ to turn it blue. Have you noticed the sulfurous odor of the test solution? I think the “TPTZ” method is using a sulfide to reduce ferric to ferrous ions. This can mean that ferric compounds that are insoluble and possibly not bioavailable are being measured as iron in solution and bioavailable.

The light bulb came on when I was studying iron concentration in Instant Ocean with ferrous sulfate additions. A yellowish brown floc when resuspended in IO was detected as a ~200 ppb increase in iron.

I don’t think this matters much to your experiment because chelated iron and iron sulfate dosed to aquarium water has measurable iron content even when filtered through a 0.45 μ syringe filter, i.e., no rust being measured as soluble iron.

Yeah, the sulfurous odor is super obvious.
This makes clear sense, and it explains the filterable detected iron quite simply.

 

[taricha]

If anybody is interested in letting me borrow a PAR meter, I think that would be helpful.
Reply or PM me if interested.
I'm not changing any lighting, so it's a constant through the experiment rather than a test variable. I already have a spectral profile of what my lighting is - but it being T5s, it might be unusually low. So people might want to know, to decide how relevant it would be to their system.
Not a big deal, but it'd be nice.

Edit: y'all are awesome. thanks, a member is sending me a PAR sensor to use.

 

[taricha]

A couple of updates on how the experiment will progress.
I wasn't sure what I'd do for phase 4

Phase 0: "do nothing" normal tank operation - initial data collection
Phase 1: 6 weeks algal trace element supplementation (red algae - palmaria palmata, and phytoplankton - phytofeast feeding)
Phase 2: 6 weeks of greater than the recommended 10% weekly water changes - 2 to 3 5% water changes per week
Phase 3: 6 weeks of Red Sea Trace A (halogens: I and F) and Trace C (Fe, Mn, transition metals) dosing
Phase 4?: TBD?

Phase 4: possibly more detailed element management? possibly a different nutrient strategy (ammonia supplementation)?

I've opted for a more detailed element management for phase 4. This will follow phase 3 where the Red sea trace mix is added, to see if managing more traces individually has any detectable advantages.
I'll cross reference Randy's list of possibly worthwhile elements with what ICP-MS says about my water and what Moonshiners says target levels / input rates should be for the ppb level trace elements. I'll use the captiv8 single element minor and trace pack. The missing ones I'll use other sources.
This will be the list of elements managed...

After that, I'll have a phase 5: "do nothing - again" where I go back to maintaining the tank with minimal trace inputs, and see if whatever benefits were seemingly observed during the interventions would be observed to decrease.

 

[taricha]

Math details, but here's an update about statistics for deciding if fluorescent pigments have varied from one phase to the next.

I take samples in pairs from each coral tested after each phase of interventions. Each sample pair gets tested on 3 optical parameters - fluorescence with 402nm excitation, with 496nm excitation, and Chlorophyll A fluorescence (402nm excitation). So at this point I have a couple of dozen pairs of data points that vary (mostly) due to the variation of one coral piece to the next and due to (a lesser extent) the variation from the sample measurement process. If I calculate how far each sample in a pair is from the pair-average, I can use this as a measure of the variation in my measurements of similar coral tips at a certain point in time.
This gets us to some sort of standard deviation and lets us say how big (as a %) 2 standard deviations from the average would be. This works fine as a meter stick for "did anything change enough to be a real effect?" That is, if the pair-average is more than 2 standard deviations from what the pair average was during the last phase, I'll feel like it's probably a real effect (p<.05)
In these charts, you see the pairs (so one is + and the other of the pair - from the average)

The +-2 standard deviations for each of these measure groups is similar, +-25% to +-45%, If I lump them all together, I get +-34% for 2 standard deviations.

I'll just use this as the +-2 S.D. value for now, and at the end of the experiment when I have more data, I'll revisit if it makes more sense to use a different standard deviation for the different optical measurements.

 

[taricha]

On to Phase 2: "Water Changes Fix Everything?"
Here's some theory behind it and setting some expectations. The resulting Data from phase 2 will be in a subsequent post.

In this 6-week phase, I did 15% change each week (week 1 was only 10%). Technically it was as 5% change increments, so 3x5% changes each week. With 17 total 5% changes, it might be tempting to call that an 85% water change, but it's not - only 58% - because math 1-(.95^17) = .58
Anyway, many people - myself included have observed that after a period of no water changes, a modest water change can spur a flush of microalgae growth on the glass and surfaces. It's thought that this is likely due to trace limitation and that water changes can bring in some trace metals needed for photosynthetic growth. Iron and Manganese are top candidates, as they get depleted quickly and a small influx can allow photosynthetic stuff to resume growth.
Water changes also can alter the ionic balance if your water has gotten shifted to some degree far from what your new salt mix gives you. That's not really relevant in my situation - my tank water is pretty close to what instant ocean mixes to in terms of major elements.
Water changes don't just add Traces - they can also dilute things in the water - pollutants, undesirably high nutrients, and organics.
Let's look at what tests say about my water (before water changes), and what a fresh input of Instant Ocean would do.

The left two columns are my water as tested by Fauna Marin and Oceamo - the middle two columns are from results by ATI from @rtparty ultimate salt test, and from Oceamo ICP-MS on a batch of IO. The right three columns are what might be expected with a hypothetical 15% weekly water change: the resulting value, how much of a change, and that change as a %.

(as for the major element discrepancies between the two sets of I.O. data, the ATI data was done on full buckets, the Oceamo was done on a small subsample out of a bucket - so for major elements I'll lean toward the ATI data)

Sr and F in my water have drifted much lower than new IO and we can expect water changes to raise that.
The nutrients Si, NO3, PO4 being basically absent in new mixed saltwater will be lowered by around 15% - the amount of the water change.
Iodine in my water is quite low, and my testing of IO agrees with the ~0.100ppm from oceamo which means each water change will give nearly a double the value.

And here's the PPB elements...

since clean IO is mostly lacking or low in Al,Cu, and Sn water changes will lower these by dilution - which in the case of my high Cu and modestly high Al, is good.
It also seems to have lower levels of Ni, Mo, and V than my water, so those possibly useful traces would be lowered rather than supplemented by water changes.
Fe would seemingly be unchanged by the water changes.
Mn present in the new salt mix would be far larger than what remains in my depleted water - so that one is likely a beneficial input.
(the 10x Mn descrepancy between the ATI and Oceamo data, I believe reflects a temporary change in IO salt, other data we have back from '21 showed 4 ICP vendors agreeing that IO had 25-30ppb Mn, the ATI data in '22 shows it - and reef crystals - low, and the oceamo data in '23 shows it back at 25-30ppb)

So what can we expect from water change data? It certainly won't fix everything - some desirable things will actually be diluted lower, but some important Traces will clearly be supplemented by the water changes and maybe those end up having noticeable effects.

 

[taricha]

Chemical and Coral color data from after Phase 2: "Water changes fix everything" (6 weeks of 15% Water changes)

Phase 2 Chemical Data - note, I goofed and the salinity was 36.8 (the drift happened in the few days before sampling). I'd already taken and sealed the ICPs before I realized, so we'll just go with it and all values shown are mathematically corrected from 36.8 back to 35.0 salinity for comparison with other phases.

page 2

On major elements / parameters:
the Abs at 254nm (measure of organics) dropped significantly from the water changes.
K mostly constant, ticked down just a bit, bringing it inside the 400-420 recommended values.
Sr went up as expected, and the nutrients Si, NO3 & PO4 went down as expected - based on the measurements of new saltwater with instant ocean in the last post.
Interestingly, Fluorine and Iodine are totally unchanged, remaining just as low as when they weren't supplemented before - even though we have good evidence that water changes were bringing in significant amounts of both of these each week. So the mechanisms for Fluorine (maybe deposition on calcium) and Iodine (maybe biological) are consuming these new inputs as fast as they are added.

On the "ppb elements"....
Cu is high and diluted lower by water changes while Al stays near 20ppb, with water changes or not.
Sn, Ni, and Mo are lowered by dilution as expected from prior measurements of IO.
Fe remains constant, as expected due to IO and my water having similar concentrations.
Zn went up a small unexpected amount.
Mn like F and Iodine discussed earlier, is just as depleted as before even with known significant weekly inputs (I should try to confirm that current IO has the ~20ppb Mn it typically has.) Given the known necessity to photosynthetic organisms, and how thoroughly depleted it remains in my water, this seems to me the best candidate for a trace element limiting growth of photosynthetic stuff. (my hunch going into this experiment was Fe as my prime suspect, but looks like the data favors Mn.)

The takeaway from this round of element data is that water changes don't actually change very much - numerically. They may be an important source of limited traces, but in my case measurements say only a very few elements are actually being added in notable amounts from the water changes, compared to my water:
Sr, F, I, Mn.
My gorgonians likely care a lot about the I, but most everything else would be more concerned about Mn. Does anything care much about the Sr, F ? who knows.

Phase 2 Coral Color Data- (later post)

 

[Dan_P]

Chemical and Coral color data from after Phase 2: "Water changes fix everything" (6 weeks of 15% Water changes)

Phase 2 Chemical Data - note, I goofed and the salinity was 36.8 (the drift happened in the few days before sampling). I'd already taken and sealed the ICPs before I realized, so we'll just go with it and all values shown are mathematically corrected from 36.8 back to 35.0 salinity for comparison with other phases.

page 2

On major elements / parameters:
the Abs at 254nm (measure of organics) dropped significantly from the water changes.
K mostly constant, ticked down just a bit, bringing it inside the 400-420 recommended values.
Sr went up as expected, and the nutrients Si, NO3 & PO4 went down as expected - based on the measurements of new saltwater with instant ocean in the last post.
Interestingly, Fluorine and Iodine are totally unchanged, remaining just as low as when they weren't supplemented before - even though we have good evidence that water changes were bringing in significant amounts of both of these each week. So the mechanisms for Fluorine (maybe deposition on calcium) and Iodine (maybe biological) are consuming these new inputs as fast as they are added.

On the "ppb elements"....
Cu is high and diluted lower by water changes while Al stays near 20ppb, with water changes or not.
Sn, Ni, and Mo are lowered by dilution as expected from prior measurements of IO.
Fe remains constant, as expected due to IO and my water having similar concentrations.
Zn went up a small unexpected amount.
Mn like F and Iodine discussed earlier, is just as depleted as before even with known significant weekly inputs (I should try to confirm that current IO has the ~20ppb Mn it typically has.) Given the known necessity to photosynthetic organisms, and how thoroughly depleted it remains in my water, this seems to me the best candidate for a trace element limiting growth of photosynthetic stuff. (my hunch going into this experiment was Fe as my prime suspect, but looks like the data favors Mn.)

The takeaway from this round of element data is that water changes don't actually change very much - numerically. They may be an important source of limited traces, but in my case measurements say only a very few elements are actually being added in notable amounts from the water changes, compared to my water:
Sr, F, I, Mn.
My gorgonians likely care a lot about the I, but most everything else would be more concerned about Mn. Does anything care much about the Sr, F ? who knows.

Phase 2 Coral Color Data- (later post)

Ever wonder if there is more to the trace element availability story? Could microorganisms, particularly bacteria, be the main source of trace elements, e.g., cobalt from vitamin B-12 releases?

 

[taricha]

Ever wonder if there is more to the trace element availability story? Could microorganisms, particularly bacteria, be the main source of trace elements, e.g., cobalt from vitamin B-12 releases?

Sometimes, yes. I feel like water measurements are the tips of icebergs with some elements showing us 10% above the water and others only showing us 1% or 0.01% above the water and the rest frozen up in all the organisms and processes in our tank.
But that's not what this data makes me think. This data makes me think a surprising number of elements are actually boring. I mean look at the 12 elements from Al down the list to Se. You could have predicted on that list who would be stable and who would go up or down just based on the data for IO that other people already recorded. And you would have been right on almost all of them.
The final values just look a lot like the start values modified by the amounts in the water changes. weirdly un-complex.

 

[taricha]

Phase 2 Coral Color Data- (later post)

So here is the coral color data from Phase 2 - "water changes fix everything"
The sinularia and sarcophyton show no color differences to the eyeball or to the camera over phases 0,1,& 2.
sinularia....

sarcophyton....

Next is the orange monti cap and green pocillopora, these also show no differences noticeable to the eye or camera.

and the pocillopora....

Finally, here's the monti digitata - again the only noticeable eyeball change is a very slight increase in green coloration, being more eyeball-visible on the skin in one spot.

here's a blue LED+yellow filter picture to highlight the section being talked about.

this spot of green skin is not new - it was visible under the LED+filter conditions in phase 1 as well, but it became more eyeball-apparent under normal lighting during this phase.
This green spot is notable in that it is the exception on the coral - generally new growth tips are associated with the brightest coloration, and this green spot is a new growth section, but there are many other spots on this coral that also showed new growth during Phase 2, but they had no green skin coloration. So this coral continues to demonstrate that there is plenty of unrealized potential to generate fluorescent pigments that current conditions in the system seem not to support.

Phase 2 pigment extraction data

Here's the fluorescence of the coral extracts when illuminated by 402nm violet light. The 0, 6 and 12 corresponds to time in weeks - 6 weeks being after phase 1 and 12 weeks after phase 2. Each bar represents measurement from one extract of a coral cutting. Next to each bar-pair is an error bar with an estimate of +- 2 standard deviations, +-34% from the 2-sample average. (method for +- 2 stdev estimate in post 70 here)

The sinularia is the best candidate for evidence of increased coral fluorescence, followed by sarcophyton. Pocillopora and monti digi don't show anything statistically significant.

Here's the fluorescence of the coral extracts when excited by 496nm cyan light.

monti cap and monti digi show no increase over these phases, and plausibly a decrease in the monti cap samples measured. Note that the sarcophyton pigment tracked here is the same pigment in the chart above with 402nm excitation, but in this chart (with a more rubust emission measurement - see y-axis for the relative strength) the evidence for an increase is still present but weaker. This suggests that the 402nm data for phase zero with the lowest emissions are very noisy due to how low the detected fluorescence was, and we should lower our confidence in the amount of sarcophyton pigment increase.

And here is the emission at 675nm (402nm excitation) - a.k.a. symbiont Chlorophyll A measurement.

This data set presents the best evidence of some system effect from the two interventions (algal inputs - phase 1, and water changes - phase 2).
With the exception of the monti digi - which is only n=1 and the hardest to sample - all corals show an increase in the average symbiont pigment from one phase to the next to the next. Some increases - phase 2 vs phase 0 - are large enough to escape overlapping error bars (sarcophyton, monti cap, and pocillopora).

Other Tank observations:
During the Phase 2 - 15% weekly water changes, the most notable eyeball observation of a biological system change was an increase in the flushes of microalgae growth on the glass (dots pictured below).

This is despite the NO3 (5-9ppm) and PO4 (0.08-0.15ppm) and Si (0.25-0.40ppm) remaining fairly stable (post 72) at modest levels that should allow growth. In fact, all those nutrients decreased during phase 2 - in large part from simple dilution by water changes. So the water changes allowed greater microalgae growth despite nutrient stability or slight decreases.


Commentary on Phase 2:
A few takeaways for me on this. From the chemical data (post 72), using my normal Instant Ocean to do lots of water changes actually moved very few trace elements, arguably the only inputs that are significant (meaning good evidence that inputs were larger than what was in the water) were Iodine, Fluorine, and Manganese. Each of these, at the conclusion of the 6 week 15% water changes phase remained almost exactly as depleted as they were before.
The notable increase in the amount of photosynthetic growth on the glass is interesting given how few elements seem to have actually been brought in by the water changes. The strongest candidate element for the increased photosynthetic growth is Manganese, which is against my expectation of Fe. Iron can't be ruled out, but the amount of Fe in the water has remained remarkably stable through phase 0,1,and 2 and it matches closely ICP-MS data for what instant ocean Fe levels are upon initial mixing. So it's hard to make the case for much of an Fe input effect.
The flush of microalgae growth on the glass is also interesting paired with the symbiont pigment extraction data - that also shows an increase across almost all coral samples, in spite of largely stable nutrients. I hadn't considered the possibility that the trace limitations and additions that are likely responsible for the flushes of microalgae on the glass might also have a role in regulating coral internal symbiont density, but that may be what we are seeing - this is the most interesting part of phase 2 for me.
The coral coloration remains largely lackluster, although likely the sinularia and maybe the sarcophyton increased extractable fluorescent pigment, and a small patch of green fluorescent skin on the monti digitata became more apparent - the overall impression is that at least some of the corals could have much higher pigments than they have shown under these conditions. (I'll cheat chronology here and say future data confirms this assertion.)

While water changes clearly input a few depleted elements, and brought down the undesirable copper and high organics, and at least some of those inputs seem to have been critically limiting for photosynthetic organisms, water changes do not "fix everything" from a trace element perspective - it actually input only a small handful of elements, and those that were depleted remained exactly as depleted as before despite the inputs. As a trace element "fix", water changes in my case are a weak effect and do not achieve most of the desired chemical or biological (coral color) changes.

On to Phase 3: Actual trace element dosing with Red Sea Trace parts A and C.

 

[Randy Holmes-Farley Verified Community Expert]

Nice study. :)

“I'll cheat chronology here and say future data confirms this assertion.”

My grad school professor would sometimes communicate what he hoped we would accomplish in the lab by drawing a graph for us, and “all” we had to do was collect the data to match it. Much easier when the idea matched reality, than when the idea crashed and burned.

 

[Dan_P]

So here is the coral color data from Phase 2 - "water changes fix everything"
The sinularia and sarcophyton show no color differences to the eyeball or to the camera over phases 0,1,& 2.
sinularia....

sarcophyton....

Next is the orange monti cap and green pocillopora, these also show no differences noticeable to the eye or camera.

and the pocillopora....

Finally, here's the monti digitata - again the only noticeable eyeball change is a very slight increase in green coloration, being more eyeball-visible on the skin in one spot.

here's a blue LED+yellow filter picture to highlight the section being talked about.

this spot of green skin is not new - it was visible under the LED+filter conditions in phase 1 as well, but it became more eyeball-apparent under normal lighting during this phase.
This green spot is notable in that it is the exception on the coral - generally new growth tips are associated with the brightest coloration, and this green spot is a new growth section, but there are many other spots on this coral that also showed new growth during Phase 2, but they had no green skin coloration. So this coral continues to demonstrate that there is plenty of unrealized potential to generate fluorescent pigments that current conditions in the system seem not to support.

Phase 2 pigment extraction data

Here's the fluorescence of the coral extracts when illuminated by 402nm violet light. The 0, 6 and 12 corresponds to time in weeks - 6 weeks being after phase 1 and 12 weeks after phase 2. Each bar represents measurement from one extract of a coral cutting. Next to each bar-pair is an error bar with an estimate of +- 2 standard deviations, +-34% from the 2-sample average. (method for +- 2 stdev estimate in post 70 here)

The sinularia is the best candidate for evidence of increased coral fluorescence, followed by sarcophyton. Pocillopora and monti digi don't show anything statistically significant.

Here's the fluorescence of the coral extracts when excited by 496nm cyan light.

monti cap and monti digi show no increase over these phases, and plausibly a decrease in the monti cap samples measured. Note that the sarcophyton pigment tracked here is the same pigment in the chart above with 402nm excitation, but in this chart (with a more rubust emission measurement - see y-axis for the relative strength) the evidence for an increase is still present but weaker. This suggests that the 402nm data for phase zero with the lowest emissions are very noisy due to how low the detected fluorescence was, and we should lower our confidence in the amount of sarcophyton pigment increase.

And here is the emission at 675nm (402nm excitation) - a.k.a. symbiont Chlorophyll A measurement.

This data set presents the best evidence of some system effect from the two interventions (algal inputs - phase 1, and water changes - phase 2).
With the exception of the monti digi - which is only n=1 and the hardest to sample - all corals show an increase in the average symbiont pigment from one phase to the next to the next. Some increases - phase 2 vs phase 0 - are large enough to escape overlapping error bars (sarcophyton, monti cap, and pocillopora).

Other Tank observations:
During the Phase 2 - 15% weekly water changes, the most notable eyeball observation of a biological system change was an increase in the flushes of microalgae growth on the glass (dots pictured below).

This is despite the NO3 (5-9ppm) and PO4 (0.08-0.15ppm) and Si (0.25-0.40ppm) remaining fairly stable (post 72) at modest levels that should allow growth. In fact, all those nutrients decreased during phase 2 - in large part from simple dilution by water changes. So the water changes allowed greater microalgae growth despite nutrient stability or slight decreases.


Commentary on Phase 2:
A few takeaways for me on this. From the chemical data (post 72), using my normal Instant Ocean to do lots of water changes actually moved very few trace elements, arguably the only inputs that are significant (meaning good evidence that inputs were larger than what was in the water) were Iodine, Fluorine, and Manganese. Each of these, at the conclusion of the 6 week 15% water changes phase remained almost exactly as depleted as they were before.
The notable increase in the amount of photosynthetic growth on the glass is interesting given how few elements seem to have actually been brought in by the water changes. The strongest candidate element for the increased photosynthetic growth is Manganese, which is against my expectation of Fe. Iron can't be ruled out, but the amount of Fe in the water has remained remarkably stable through phase 0,1,and 2 and it matches closely ICP-MS data for what instant ocean Fe levels are upon initial mixing. So it's hard to make the case for much of an Fe input effect.
The flush of microalgae growth on the glass is also interesting paired with the symbiont pigment extraction data - that also shows an increase across almost all coral samples, in spite of largely stable nutrients. I hadn't considered the possibility that the trace limitations and additions that are likely responsible for the flushes of microalgae on the glass might also have a role in regulating coral internal symbiont density, but that may be what we are seeing - this is the most interesting part of phase 2 for me.
The coral coloration remains largely lackluster, although likely the sinularia and maybe the sarcophyton increased extractable fluorescent pigment, and a small patch of green fluorescent skin on the monti digitata became more apparent - the overall impression is that at least some of the corals could have much higher pigments than they have shown under these conditions. (I'll cheat chronology here and say future data confirms this assertion.)

While water changes clearly input a few depleted elements, and brought down the undesirable copper and high organics, and at least some of those inputs seem to have been critically limiting for photosynthetic organisms, water changes do not "fix everything" from a trace element perspective - it actually input only a small handful of elements, and those that were depleted remained exactly as depleted as before despite the inputs. As a trace element "fix", water changes in my case are a weak effect and do not achieve most of the desired chemical or biological (coral color) changes.

On to Phase 3: Actual trace element dosing with Red Sea Trace parts A and C.

The results haven’t quite reached the level of difference seen in the before-and-after photos associated with diets or exercise advertisements. Anyway, my hats off to you for performing all this work.

If this were my experiment, this would be about the time I’d start wishing I had done XYZ. In this case I would have wished for a control where nothing was done to a set of identical corals receiving your standard care. I would wish that because just maybe the changes you are seeing would have happened anyway. How are you dealing with this sort of ambiguity?

 

[taricha]

If this were my experiment, this would be about the time I’d start wishing I had done XYZ.

...like realizing some of the wrong corals were focused on, and that much better photography methods could've been used :)

In this case I would have wished for a control where nothing was done to a set of identical corals receiving your standard care. I would wish that because just maybe the changes you are seeing would have happened anyway. How are you dealing with this sort of ambiguity?

Agreed. At this point to feel better about whether effects are real or not, I'll need to see a) a color effect for some Phase that's significantly greater than the ambiguous results of earlier phase interventions that preceded it, and b) I'll need that phase to be followed up by a "do nothing" phase where the supposedly helpful interventions are removed, and see the significant color effects reverse to at least some extent. I'm a little concerned that a color-up that might happen in 4-6 weeks could take much longer to wash out once the supposedly helpful interventions are removed. But that may not be true, and even if it does take a longer time - that would just be annoying, not actually problematic if it took 8-10 weeks to lose color gained in 4-6 weeks.

 

[bubbgee]

So here is the coral color data from Phase 2 - "water changes fix everything"
The sinularia and sarcophyton show no color differences to the eyeball or to the camera over phases 0,1,& 2.
sinularia....

sarcophyton....

Next is the orange monti cap and green pocillopora, these also show no differences noticeable to the eye or camera.

and the pocillopora....

Finally, here's the monti digitata - again the only noticeable eyeball change is a very slight increase in green coloration, being more eyeball-visible on the skin in one spot.

here's a blue LED+yellow filter picture to highlight the section being talked about.

this spot of green skin is not new - it was visible under the LED+filter conditions in phase 1 as well, but it became more eyeball-apparent under normal lighting during this phase.
This green spot is notable in that it is the exception on the coral - generally new growth tips are associated with the brightest coloration, and this green spot is a new growth section, but there are many other spots on this coral that also showed new growth during Phase 2, but they had no green skin coloration. So this coral continues to demonstrate that there is plenty of unrealized potential to generate fluorescent pigments that current conditions in the system seem not to support.

Phase 2 pigment extraction data

Here's the fluorescence of the coral extracts when illuminated by 402nm violet light. The 0, 6 and 12 corresponds to time in weeks - 6 weeks being after phase 1 and 12 weeks after phase 2. Each bar represents measurement from one extract of a coral cutting. Next to each bar-pair is an error bar with an estimate of +- 2 standard deviations, +-34% from the 2-sample average. (method for +- 2 stdev estimate in post 70 here)

The sinularia is the best candidate for evidence of increased coral fluorescence, followed by sarcophyton. Pocillopora and monti digi don't show anything statistically significant.

Here's the fluorescence of the coral extracts when excited by 496nm cyan light.

monti cap and monti digi show no increase over these phases, and plausibly a decrease in the monti cap samples measured. Note that the sarcophyton pigment tracked here is the same pigment in the chart above with 402nm excitation, but in this chart (with a more rubust emission measurement - see y-axis for the relative strength) the evidence for an increase is still present but weaker. This suggests that the 402nm data for phase zero with the lowest emissions are very noisy due to how low the detected fluorescence was, and we should lower our confidence in the amount of sarcophyton pigment increase.

And here is the emission at 675nm (402nm excitation) - a.k.a. symbiont Chlorophyll A measurement.

This data set presents the best evidence of some system effect from the two interventions (algal inputs - phase 1, and water changes - phase 2).
With the exception of the monti digi - which is only n=1 and the hardest to sample - all corals show an increase in the average symbiont pigment from one phase to the next to the next. Some increases - phase 2 vs phase 0 - are large enough to escape overlapping error bars (sarcophyton, monti cap, and pocillopora).

Other Tank observations:
During the Phase 2 - 15% weekly water changes, the most notable eyeball observation of a biological system change was an increase in the flushes of microalgae growth on the glass (dots pictured below).

This is despite the NO3 (5-9ppm) and PO4 (0.08-0.15ppm) and Si (0.25-0.40ppm) remaining fairly stable (post 72) at modest levels that should allow growth. In fact, all those nutrients decreased during phase 2 - in large part from simple dilution by water changes. So the water changes allowed greater microalgae growth despite nutrient stability or slight decreases.


Commentary on Phase 2:
A few takeaways for me on this. From the chemical data (post 72), using my normal Instant Ocean to do lots of water changes actually moved very few trace elements, arguably the only inputs that are significant (meaning good evidence that inputs were larger than what was in the water) were Iodine, Fluorine, and Manganese. Each of these, at the conclusion of the 6 week 15% water changes phase remained almost exactly as depleted as they were before.
The notable increase in the amount of photosynthetic growth on the glass is interesting given how few elements seem to have actually been brought in by the water changes. The strongest candidate element for the increased photosynthetic growth is Manganese, which is against my expectation of Fe. Iron can't be ruled out, but the amount of Fe in the water has remained remarkably stable through phase 0,1,and 2 and it matches closely ICP-MS data for what instant ocean Fe levels are upon initial mixing. So it's hard to make the case for much of an Fe input effect.
The flush of microalgae growth on the glass is also interesting paired with the symbiont pigment extraction data - that also shows an increase across almost all coral samples, in spite of largely stable nutrients. I hadn't considered the possibility that the trace limitations and additions that are likely responsible for the flushes of microalgae on the glass might also have a role in regulating coral internal symbiont density, but that may be what we are seeing - this is the most interesting part of phase 2 for me.
The coral coloration remains largely lackluster, although likely the sinularia and maybe the sarcophyton increased extractable fluorescent pigment, and a small patch of green fluorescent skin on the monti digitata became more apparent - the overall impression is that at least some of the corals could have much higher pigments than they have shown under these conditions. (I'll cheat chronology here and say future data confirms this assertion.)

While water changes clearly input a few depleted elements, and brought down the undesirable copper and high organics, and at least some of those inputs seem to have been critically limiting for photosynthetic organisms, water changes do not "fix everything" from a trace element perspective - it actually input only a small handful of elements, and those that were depleted remained exactly as depleted as before despite the inputs. As a trace element "fix", water changes in my case are a weak effect and do not achieve most of the desired chemical or biological (coral color) changes.

On to Phase 3: Actual trace element dosing with Red Sea Trace parts A and C.

And here I was going to buy Instant Ocean salt.. lol...
Maybe I should switch to AF Hybrid Salt and try out this probiotic thing..

 

[taricha]

And here I was going to buy Instant Ocean salt.. lol...
Maybe I should switch to AF Hybrid Salt and try out this probiotic thing..

Nothing wrong with IO, it just isn't a source of significant variety of traces.

Check out rtparty's salt survey. You might find a salt that provides good amounts of traces that you would choose to dose otherwise.

Thread 'The Ultimate Salt Test' https://www.reef2reef.com/threads/the-ultimate-salt-test.949805/