It's Still in the Water!

[Randy Holmes-Farley]

Eric:

I greatly appreciate all of the papers that you have sent me, and I also greatly welcome your input in this thread.

The primary reason that I am here in this thread is to try to provide some counterbalance to Ron's assertions that "we are killing corals" and that I am personally doing so. It could be true. We may be killing corals with metal toxicity. But we also may not be. An ICP measurement is not definitive enough, IMO, to start telling people that it is a demonstrated fact, as Ron has done.

I've provided several reasons why his test may not demonstrate that we are killing corals. Which one, if any, is true? I don't know. There is just no allowance in his articles or posts that there is any possibility that we are not killing corals. I'm allowing for tha possibility, and in the case of some corals, it really has to be a likely probability since many of us have kept many corals thriving for many years. Are these the ones that are especially resistant? Maybe. Or maybe they even exemplify the majority of corals.

ut, (and I do ask that you bring me up to speed on this thread if I repeat what's been said, fail to cover issues addressed, miss a point, etc., because as I said, I couldn't/wouldn't/shouldn't read it all), and excuse me for being presumptuous, but so what? Chelators themselves may have their own set of effects....

Hmmm. I seem to have suggested this as well. That organics themselves may be more toxic than metals in our tanks. Even beyond chelators, many organsims release organics specifically to KILL other things. Ron tossed out this hypothesis as ridiculous since I have not personally measured the levels of these organics in my tank.

 

[F4]

F4: Start a new thread some place for your argument with Eric. Better yet
take it off line. If it continues in this thread, I will close and delete the
thread.

I find it interesting that you allow Eric to bring the subject up, but threaten me for asking him for clarification of his comments.

I had hoped that the response would not be more attacks, but rather an acknowledgement of the real facts. I'm dissapointed in the reaction. My real hope was that the attempts to discredit would stop, but I see that is not going to happen.

But, never fear there is no more I can accomplish in this vein, there is no need to close the thread on my account.

 

[Randy Holmes-Farley]

Eric:

No offense taken on the publications. It would be safe to look for most of my publications with just the last name Holmes-Farley, since there are only 4 of us, and my wife has only two searchable publications on ethics (my 2 and 5 year old daughters have yet to publish (well, maybe they published in China before I adopted them, but those don't show up in normal searches )).

My papers end up with all combinations of first and middle names and initials, as my full name is Stephen Randall Holmes-Farley.

The majority of my recent publications have been patents. Here's a link to 44 of them for those that don't have a good database available, but Patent Office documents are hard to read this way:

http://patft.uspto.gov/netacgi/nph-...ley&FIELD1=INZZ&co1=AND&TERM2=&FIELD2=&d=pall

I am especially proud of my work on organic polymers that bind inorganic and organic materials (like iron, phosphate, bile salts, and fat), hence my keen interest in metal binding in this thread. My inventions have led to two FDA-approved pharmaceuticals (Renagel and WelChol) and have won several American Chemical Society awards for those research efforts. These awards were presented to me at symposia in my honor (co-honor with one other inventor in most cases) at regional and national ACS meetings.

 

[Randy Holmes-Farley]

Eric:

Irrespective of all this, and if corals can survive in the presence of all this garbage, why even postulate on why unless it is an area of research interest? Why even thinkabout adding more?

Were only postulating on why because Ron tells us that they aren't surviving. It just seems to us that many corals are, and we'd like to understand how Ron's data and assertions about toxicity can be compatible with the experience of hordes of hobbyists.

I don't advise people to add any heavy metals except iron, and Ron detected none. If anyone has evidence that there is "enough" iron around in our tanks, I'd gladly back off on that opinion. But I've seen many people benefit from additions. They just don't typically post such in Ron's forum. I've also NEVER seen anyone who had a toxicity problem that they attributed, rightly or wrongly, to iron additions.

Still, I'd have to say that unless one knows the bioavailability of the metals present in reef tanks, they could be in too short of supply EVEN IF the total metal concentration were higher than NSW.

That's where the tox tests come in. I think it would be great to see some good quality experiments run on corals that we keep (or want to keep) to know if this is an issue or not.

Likewise, we should keep our minds open enough that if Habib or someone else publish results that show metal additions to be beneficial, that we should at least read it and see if it is good science, or see if it is easily refuted (not by whining about stastics, but by showing a genuine flaw in the test or showing it to not be reproducible). If the stastical numbers are believed to not be high enough, then the response is to test more tanks, not to state that it can't be happening because the author only tested 65 tanks, and not the 154 necessary to attain a p of 0.05.

 

[rshimek]

Originally posted by Randy Holmes-Farley

Hi

I did a 2 x 2 chi square test and got the result posted. If you don't like the result, then I suggest you run your own survey.

I don't have to. Your data are conclusive and show that you are incorrect.

A 2 x 2 test, is a chi-squared test with 1 df. And such a test shows non-significant difference.

Well, I guess the contingency tests aren't as well known as they should be.

So...

Your data:

....................dose...................no dose.......total
spores............1...........................20.............21
no spores.......9............................31............40
Total..............10............................51............61

Chi-square statistitic = (61( abs val((1 x 31) - (9-20)) - 61/2)^2)/(10 x 51 x 40 x 21)

or ..... = (61 (abs val (31-180) -30.5 )^2)/(510 x 840)

or ..... = (61( 149-30.5)^2)/428400

or ..... = (61 (118.5)^2)/428400

or ...... = (61 x 14042.25)428400

or ...... 856577.52/428400 = 1.999

From tabled values of the chi squared statistic, with 1 df, such a value or greater one has a probability of occurring randomly in a sample of your size with a probability between 10 and 25 percent of the time.

Standard signficance in biological tests is a probablility of 5 percent or less.

Your data are therefore not statistically significant.

Test formulation from pp 64-66, and tabulated chi squared value from p. 479 of:

Zar, J. H. 1984. Biostatistical Analysis. Prentice-Hall, N. Y. 718 pp.

 

[Randy Holmes-Farley]

How abundant are these things in our tanks?

Ron, this isn't rocket science. They are very adundent in many tanks. Far higher than in seawater. That's why tanks are more yellow than seawater: because humic substances have built up. Spotte discusses these facts in detail. I'd suggest you read his discussion and those references before continuing to assert that this is an imaginary process or not seen in reef tanks.

 

[rshimek]

Originally posted by Randy Holmes-Farley

(not by whining about stastics, but by showing a genuine flaw in the test or showing it to not be reproducible).

Statistical analyses allow us to distinguish valid results from "noise." The only people who whine about statistics are those who can't understand them, or those whose data are not supported by them.

Your data, charitably, might be said to show a "tendancy." If you had done some actual experimentation with replication, used controls, and had a decent sized number of samples, you might be able to say something. As you didn't, didn't, and didn't, you really can't.

 

[rshimek]

Originally posted by Randy Holmes-Farley

They are very adundent in many tanks. Far higher than in seawater.

So you claim. Why not show me some numbers?

That's why tanks are more yellow than seawater: because humic substances have built up.

That's nice, Randy, you are a true master of generalizations.

Now what humic substances?

Which ones are there? Are they toxic in their own right? Or if they bind metals are they more-or-less toxic then?

Which ones bind toxic metals?

What is their fate in tanks?

What are their concentrations with time? If they vary signficantly, should we not be adding some of these to our systems to detoxify our salt mixes?

 

[dchisenh]

Hey Everyone , I've been following this thread for quite some time (along with half of the reefing community it seems) and something has caught my attention. Why is it the question is always about "tolerance" of metals and organics? Don't we want our inverts (and fish, and everything else alive in our tanks) to thrive and be "happy"?

I definately think the "chemistry" side of the debate has it's points on the ability of organisms to detoxify metals, sequester them, etc. and it's definitely good science for someone to play devil's advocate in order to continue the research in the area to find out definitively (well, as best as we can) the effects of these chemicals along with many others on all aspects of marine life. As valuable as it is for us keeping aquaria on a hobbyist level, it may end up doing the world a favor as our reefs face more and more problems in the years to come.

Also, the "biology" side of the debate makes a great point on the fact that these metals aren't naturally found in these concentrations in NSW and they most likely (no one can be 100% sure all the time) that they are a major suspect in "mysterious" coral and invert deaths that are all too common in the aquarium supply chain. I can't remember the exact percentage of species harvested which end up dead in the process even before they get to the consumer (which the consumer himself is most often responsible for a LOT of the subsequent deaths), but I know that if we were dealing with dogs, cats, birds, ferrets, marmosets, pigmy elephants (just kidding) or any other cute and cuddly animal, all hell would break loose with animal rights activists and the entire public in general. Funding for the prevention of all of these causes of mortality would sky-rocket and the world could sleep at night thinking they've done their good for the day. Unfortunately, corals and inverts aren't exactly cute and cuddly (have you ever tried to hug a frag of fire coral or kiss a mantis shrimp?) and so their mass deaths go un-noticed and un-investigated. The biologists, and everyone on this planet, should be rightfully inflamed over this and should be actively looking for and investigating as many causes as possible.

Perhaps these metals are the killers they're made out to be, perhaps it's something else AND the metals, and maybe something else and the metals and something else..and so on, and so on. Either way, I think we're on the right track and in just this thread alone I see reason to continue research with metals, and organics, and chelators, etc. Hopefully, we'll figure this thing out and greatly reduce the mortality of corals coming into this trade, and perhaps help out the natural reefs in the process. But, in the meantime, I think we should be continuing to look for ways to allow our corals that have made it through the gauntlet and end up in our tanks to thrive and "enjoy" life, not just "tolerate" it. If reducing metals helps, great. If reducing organics helps, great. If ANYTHING helps, great, let's try it. I'm sure if the critters could talk, they'd thank us if we try.

That's just my thoughts on the subject,

Dan

Also, as a senior biology major working on options in biotechnology and ecology, it's an honor to engage in a conversation with researchers with the credentials we have here on this website. I think all of us reefers, professional and hobbyist alike, owe you all a great deal of thanks for sharing your knowledge and expertise with us. Thank you once again.

 

[Randy Holmes-Farley]

Ron:

"Statistical analyses allow us to distinguish valid results from "noise." The only people who whine about statistics are those who can't understand them, or those whose data are not supported by them.

This (below) was the conclusion that I made in the paper. If you'll note, the conclusion was that the results were intriguing and worthy of additional study. If I thought that the result was a definitive fact, I would not have written that. Please don't exaggerate what I said and make it seem as if I stated that I had found and demonstrated something beyond all doubt.

"In other words, there is a 96% chance that there is a real difference between the iron dose group and the no iron group with respect to Caulerpa undergoing a sexual event. However, we must recognize that those who dose iron may be the same folks who dose other things (like iodine, which also was statistically significant against no dosing), or do something else in common, potentially confounding a statistical test. Nevertheless, the difference is intriguing, and worthy of additional study. In my opinion, it is also good enough evidence for those plagued with such events to try iron dosing. "

 

[Habib]

Ron,

ASW is universally thought to be toxic to invertebrate embryos invertebrate embryologists.

Don't you know that various invertebrate stages require nutrients which they derive from the surrounding water? That these nutrients are a.o amino acids or other nitrogen compounds.

ASW will normally be devoid of such compounds. So toxicity could be in fact malnutrition?????

Does anybody know the exact nutrient requirement for corals not to mention the early stages of corals?

WHY ARE THE SALT WATER MIXES MADE WITH TOXIC CHEMICALS IN THEM?

Just look at the NSW given by you in the febr article. You corrected it later thanks to IIRC Tatu and Randy.

GP2 is perhaps crap and Crystalsea Marinemix can be perhaps and perhaps after some slight modifications mimic NSW.
But I want to have it confirmed by analyses. I am about ready to have them done and I am paying for them. And if somebody want to make donations that is fine; I only accept beer or californian pistache nuts



When I tested aquarium waters, these chemicals were found in the waters in lethal amounts. If they are chelated, fine. They are likely also chelated in the natural systems when the test mortality occurs. It simply doesn't really matter whether they are ionic or chelated; sooner or later unless they are removed, they will be effecting the animals in our systems.

They are, if present in elevated concentration, more likely to be chelated in the aquarium then in NSW. Especially when food is added in the aquarium. For example copper can bind strongly to the highly abundant amide groups in polypetides (proteins).


If they form any kind of particulate organic material, they will be eaten.

If the particles are very small and there is sufficient water movement then they will not be eaten. This is plain physics and rheology. If the particle size is larger it might be eaten; ever looked at metal concentration in naturally ocurring algae. They greatly magnify the metals concentration of NSW; upto 100,000x ?!?

You have asked a lot more questions

So lets say that if the heavy metals measured in the concentrations as presented in Reefkeeping etc etc when present as free ions have the potential to be (highly) toxic to at least some marine organisms.

It still remains to be answered if they are present as free ions or are in any way bound to organics or other inorganics and present as e.g. ion-pairs or as colloids.

Toxicity to atleast some marine organisms depends on the (meta)stability of these compounds in the water column or in the organism if ingested directly or indirectly.

Further research on the speciation of these metals in aquaria should be conducted and if possible simple methods of monitoring be devised. The search towards better synthetic seasalts should be continued in the meantime.

Can you agree on this as a further basis for discussions?

 

[Randy Holmes-Farley]

That's nice, Randy, you are a true master of generalizations.

Actually, for the immediate section that you are so up in arms about, I'm simply the master of copy and paste. All of the discussion of humic substances is directly from the detailed description in Spotte and references included therein.

I haven't yet seen you name a specific instance where I incorrectly genralized something into a fact that wasn't, and I'm still waiting. Simply because you don't know the fact, doesn't mean that it doesn't exist.

Now what humic substances?

I'm afraid that even asking that question shows that you know little about seawater chemistry and humic substances.

According to references given in Spotte, humic materials are "amorphous, brown or black, hydrophilic, acidic, polydisperse substances of molecular weight ranging from hundreds to tens of thousands". It is a big class of many different compounds. So it is not a list of 1 or 34 or even 23,456 compounds. It is a continuum of different products that evolve chemically with time in each tank. They are typically studied as a class or at least in sub groups (like humic acids and fulvic acids).

Are they toxic in their own right?

Yes, in some stages of their formation they are (the polyphenol stage has an LC50 of 0.3 mg/L to plaice larvae). In some chemical stages they are not toxic to these same larvae.

Or if they bind metals are they more-or-less toxic then?

I already addressed this, but in the case of certain organsims the toxicity of copper goes down. In others, it stays the same as the free copper.

Which ones bind toxic metals?

All are polyphenolic and all polyphenols bind metals at the pH of seawater. Some bind more strongly than others.

What is their fate in tanks?

Some humic acids stay in solution forever. That's why they build up in both tanks and in seawater. Some have been floating around in seawater for thousands of years. These are termed the refractory substances. In a reef tank with certain types of filtration (skimming or carbon) they may be removed that way, which would not happen in the ocean. This is likely a great export mechanism for toxic metals, whether we want them exported or not.

What are their concentrations with time? If they vary signficantly, should we not be adding some of these to our systems to detoxify our salt mixes?

If you didn't mind a yellow tank, knew where to buy some, and believed that you had excess metals, then I think that is a wonderful idea Now were getting somewhere!

Of course, it isn't really the salt mix that is at fault, is it? The natural seawater samples in your study were among the worst. Perhaps if metal toxicity is a real issue, we ought to look to other sources, like calcium and alkalinity additives.

 

[Randy Holmes-Farley]

I can't track them all down right now, but several times in this thread it has been asked: Why have all these metals in the salt mixes?

I can think of one critical reason (cost) but that aside, I think before beating the war drum for low metal salt mix, we need to ask whether it will help.

In Ron's test the answer really appears to be: Not a teeny tiny bit.

The natural seawater samples are among the worst in terms of metals content. In some instances, they are the absolute worst.

Here's the link:

http://reefkeeping.com/issues/2002-02/rs/feature/index.htm

and here's Ron's conclusion:

"but even aquarists using NSW as the initial medium don't have tanks where the chemical composition bears much similarity to Natural Sea Water. "

In fact, the copper concentration only varied by a factor of two between the lowest tank and the highest tank. That seems odd to me, but still it is what we have to work with. Is there some other metal that we think is a problem in salt mixes that would be "cured" if only we used natural seawater types of mixes?

So why the tremendous angst about salt mix?

If metal toxicity really is an issue, we must look deeper into the problem Since the media used for reactors have been studied by many people, we can see how much metal is there (lots, in some cases):

http://www.animalnetwork.com/fish/l...iers+--+Biochemistry+of+Aquaria&RecordNo=1571

http://www.animalnetwork.com/fish2/aqfm/1997/aug/bio/default.asp

Craig first reported this problem years ago, but few seemed to care.

 

[dchisenh]

Hey Randy or Habib, do you think the sodium EDTA found in Kent's Marine Coral-vite additive has any appreciable chelating effect for a reef tank? It's listed as the fifth ingredient on the list on the bottle, but it doesn't mention a concentration. Just wondering.

Dan

 

[Randy Holmes-Farley]

Sure, but it may be already chelated to something besides sodium in the mix, and that something may not come off until the EDTA is photochemically degraded in the tank. Still, if added with just sodium, it will undoubtedly chelate metals. That's why it is added to the product to begin with (I expect).

 

[dchisenh]

So...if the EDTA is in the tank in any appreciable amount with normal dosing of that particular product (I'm guessing it's in others too, but that's the only one I have on my desk in front of me right now ), would that create a "pool" of EDTA available in the tank that combined with the typically intense lighting reefkeepers employ, be routinely available via photochemical degradation? If so, would that help explain the seemingly successful tanks some people have with high metal concentrations, or has that already been taken into consideration when doing assays? I've taken my fair share of O. Chem through my academic career, but I'm not sure how it would react in a typical aquarium setting and I apologize if this has already been addressed earlier, I must have missed it.

Thanks,

Dan

Just realized this, are you saying that the metal the EDTA may be bound with initially won't be released until the bond with the EDTA is degraded or the EDTA itself is degraded?

And Ron, if this isn't in keeping with the theme of your main discussion and is too much of a chemistry related side-issue, just let me know and I'll address any other questions about it directly to Randy in the chemistry forum, I just thought it sounded like chelators were part of the original discussion.

 

[Randy Holmes-Farley]

If so, would that help explain the seemingly successful tanks some people have with high metal concentrations,

It could, yes. It could effectively lower the free (and possibly more toxic) copper (II) ions, and hold it in a less toxic form.

At the same time, the EDTA may help maintain the solubility of iron that is typically very, very insoluble (it may be the least soluble metal ion in seawater).

Yes, it is when the EDTA is degraded photochemically that iron gets loose and is available to animals and plants for consumption. So in that sense, EDTA helps maintain a steady but low concentration of bioavailable iron.

 

[rshimek]

Originally posted by Habib

Habib,

Don't you know that various invertebrate stages require nutrients which they derive from the surrounding water? That these nutrients are a.o amino acids or other nitrogen compounds.

No, sir, they don't.

In early development any nutrition is dependent upon yolk. They may absorb some compounds from the water, but they don't absorb meaningful amount of amino acids, or any other food.

If they feed, they do it by suspension-feeding.

They may require and absorb certain other things, including, in small amounts trace elements. In larger amounts, these elements kill and and this is the basis for bioassays of non-feeding larvae.

ASW will normally be devoid of such compounds. So toxicity could be in fact malnutrition?????

Not a chance. The larvae die before needing any food.

Does anybody know the exact nutrient requirement for corals not to mention the early stages of corals?

Yeah, for some corals some parts of the puzzle are known; it is out there in the literature; at least for a few. However, most corals are "black boxes" nutrient of some source goes in, coral tissue comes out. Pocillopora has been raised as "lab rat" since 1975 or there abouts and there is a lot written about it. You may be able to track down some of these data if you need it.

But....regarding the larvae and embryos...

Cnidarians, by and large, have large yolky eggs and do not feed or take nutrients from the water until after they settle and metamorphose into juveniles.

If the particles are very small and there is sufficient water movement then they will not be eaten. This is plain physics and rheology.

The operative part of your statement is very small. Once they aggregate to anything over about 0.01 micrometer they will be eaten. I suspect that happens fairly rapidly.

Also our tanks have an enormously high surface area to volume ratio compared to the ocean. With all of that suface these particles will find their way there, be adsorbed and then be eaten.

If the particle size is larger it might be eaten; ever looked at metal concentration in naturally ocurring algae. They greatly magnify the metals concentration of NSW; upto 100,000x ?!?

Yes, I know. Substance like iodine in particular (probably because it makes a great defensive factor against herbivores) and I have data from aquaria, on Caulerpa which I hope to publish soon. They don't concentrate the levels of tank water metals much.

You have asked a lot more questions

Yes, and most of them are not answerable with the data at hand.

It still remains to be answered if they are present as free ions or are in any way bound to organics or other inorganics and present as e.g. ion-pairs or as colloids.

Yes, agreed. It would be interesting find out what way they are found, and how stable the systems are.

Toxicity to atleast some marine organisms depends on the (meta)stability of these compounds in the water column or in the organism if ingested directly or indirectly.

Yes.

Further research on the speciation of these metals in aquaria should be conducted and if possible simple methods of monitoring be devised. The search towards better synthetic seasalts should be continued in the meantime.

Can you agree on this as a further basis for discussions?

Absolutely.

 

[Randy Holmes-Farley]

The search towards better synthetic seasalts should be continued in the meantime.

Can you agree on this as a further basis for discussions?

Absolutely.

What will that do for us? Tanks using such seawater (natural seawater) had just about as much copper and other metals in your test.

Wouldn't we still be having all these same concerns if every tank used natural seawater or low metal salt mixes? This is, if every tank looked like the natural seawater ones in your study?

 

[rshimek]

Originally posted by Randy Holmes-Farley

Randy,

"In other words, there is a 96% chance that there is a real difference between the iron dose group and the no iron group with respect to Caulerpa undergoing a sexual event.

And this should have been,

In other words, there is between a 75% and a 90% chance that there is a real difference between the iron dose group and the no iron group....

As Regards the NSW tank in the original study...

Yes, it had high levels of heavy metals. But that is immaterial to the study comments at hand. The salt water in the tank was polluted from the accumulation of heavy metals in foods, more than likely.

Toxic metals enter our tanks by a number of path ways.
Apparently, they don't exit the tanks much, although I will have more to say about that.

The net result is accumulation.

If we can develop some way of filtering the toxic material out, or otherwise exporting it, or rendering it non-toxic we will have animals that will live. I suggest that some folks are able to do this some of the time, maybe a few most of the time.

Nonetheless, we start out the game at a decided disadvantage; we start out with salt that is toxic.

We pay for a medium to keep animals alive. It makes no sense whatsoever that it should be so poorly and cheaply made as to be potentially toxic from the get-go.

Now... maybe, if I run these bioassays I intend to run I will find that not all salts are created equally bad. Maybe some will be pretty good. Then it makes sense to use those salts to start with in our tanks, all things being equal.

On the other hand, if they are all bad = significant larval mortality, done in an experiment with replication and controls, then what does that mean?

Well, it means that surrogate animals died in the salts. Not corals. I don't have either the money nor the space to do this experiment with corals. However, the use of delicate animal stages as surrogates is well-documented and well-supported in the ecological literature as well as in the environmental regulatory community. It is an understood and useful technique.

If there is mortality in these bioassays, will it prove corals will die? No. Most assuredly not. However, it will say that the water in the container kills animals in very short periods (4 days or less). The assumption is that any animals in such toxic soups for any long period of time will show stress and that many of them will die.

The fact that my bioassays will be directly comparable to bioassays run with coral larvae which already show that corals die at metals concentrations far lower than what we find in our tanks will allow a rather good assessment, I think, of the liklihood of long-term survival of many corals and other reef animals in our tanks.