Important data missing in toxicity study
- Thread starter Habib
- Start date
Ron:
I've been reading your article and this thread with a great deal of interest and I hope you will clarify a couple of things for me.
You have indicated here that the toxicity of freshly made ASW is well established. You also seem to acknowlege the fact that studies have shown that while freshly made ASW is toxic, as the mix ages, it ceases to be toxic.
In your article you mention that, "one day prior to the arrival of the test animals, I mixed up one gallon of each of the salt mixes." I take that to mean that the salt mixes had 24 hours or less to dissolve. How did you determine that this was enough time for the mixes to age? When you write:
What I tried to show, and what I did show, was that some ASW appears to be as good as NSW within the constraints of the study. Frankly, as far as I am concerned, that's all that's necessary. Whatever the problems with some salts are, they are not my concern, they should be the concern of the ASW manufacturers.
How can we be sure there is a problem with the salts? How can we eliminate the possibility that dissolution rates differ for the salts and some had ceased to be toxic in the time you allowed, but that others had not aged enough? How else can we explain that Hobbyist A water using IO seemed to be pretty good?
The other question I have is about your conclusion that there is a strong correlation between toxicity and metal content. To reach that conclusion you chose to use the Atkinson analysis of IO rather than your own because they show higher levels of metals. Were we to use your figures from earlier articles, the correlation would be much weaker, wouldn't you agree? If you don't trust your own data enough to use it here, doesn't it raise questions about your earlier use of the data?
Thanks for taking time out to explain your article.
I've been reading your article and this thread with a great deal of interest and I hope you will clarify a couple of things for me.
You have indicated here that the toxicity of freshly made ASW is well established. You also seem to acknowlege the fact that studies have shown that while freshly made ASW is toxic, as the mix ages, it ceases to be toxic.
In your article you mention that, "one day prior to the arrival of the test animals, I mixed up one gallon of each of the salt mixes." I take that to mean that the salt mixes had 24 hours or less to dissolve. How did you determine that this was enough time for the mixes to age? When you write:
What I tried to show, and what I did show, was that some ASW appears to be as good as NSW within the constraints of the study. Frankly, as far as I am concerned, that's all that's necessary. Whatever the problems with some salts are, they are not my concern, they should be the concern of the ASW manufacturers.
How can we be sure there is a problem with the salts? How can we eliminate the possibility that dissolution rates differ for the salts and some had ceased to be toxic in the time you allowed, but that others had not aged enough? How else can we explain that Hobbyist A water using IO seemed to be pretty good?
The other question I have is about your conclusion that there is a strong correlation between toxicity and metal content. To reach that conclusion you chose to use the Atkinson analysis of IO rather than your own because they show higher levels of metals. Were we to use your figures from earlier articles, the correlation would be much weaker, wouldn't you agree? If you don't trust your own data enough to use it here, doesn't it raise questions about your earlier use of the data?
Thanks for taking time out to explain your article.
Hi,
<img src="/images/welcome.gif"><br><b><i><big><big>To Reef Central</b></i></big></big>
How did you determine that this was enough time for the mixes to age?
I arbitrarily chose that time as being convenient for the study. I was not trying specifically to age the mixtures, but I wanted to have the containers and their contents ready to go when the urchins arrived the next day.
How can we be sure there is a problem with the salts? How can we eliminate the possibility that dissolution rates differ for the salts and some had ceased to be toxic in the time you allowed, but that others had not aged enough?
Well, the salts seemed completely dissolved with in a few minutes and were mixed pretty vigorously. But, frankly, that some salts may be toxic when mixed was rather the point of the study. Frankly, I think any salt that has any toxicity at any time after it is mixed has problems and is unsuitable for use, no matter what the cause of the toxicity.
How else can we explain that Hobbyist A water using IO seemed to be pretty good?
I suspect hobbyist A has some bacterial or other biological component in her system that is producing some material binding the metals in the tank. As I mentioned in the article this could be any number of things.
The other question I have is about your conclusion that there is a strong correlation between toxicity and metal content. To reach that conclusion you chose to use the Atkinson analysis of IO rather than your own because they show higher levels of metals. Were we to use your figures from earlier articles, the correlation would be much weaker, wouldn't you agree?
No, I don't agree, the total metals concentrations in the toxic salts would still be exceptionally high and much higher than NSW and the other salts.
If you don't trust your own data enough to use it here, doesn't it raise questions about your earlier use of the data?
By using the A-B analysis, I was able to get comparable data for at least 2 or the 4 salts. If I chose to use the IO data from my earlier study, I would have had 4 different salt analyses to deal with. I relied on the A-B data simply to both reduce the potential variation in the salt analyses and to rule out any potential variation caused by mixing of the salt in my previous study (it was mixed by a hobbyist and submitted by him to be analyzed). I think it was quite likely that the single sample of IO mixed for my previous study was mixed in a regular mixing vat which could have had some detoxifying bacteria in it. Alternatively, of course, is the possibilty that IO has changed their formulation since the A-B study, nonetheless, cobalt, nickel, titanium and zinc all range from the hundreds to thousands of times more concentrated in the mix compared to NSW.
However, the bottom line is that I proposed the heavy metal hypothesis to explain the results. It seemed reasonable to me at the time, and it still does. However, that hypotheses remains untested, and certainly the mortality caused by these mixes could be due to some other cause.
In point of fact, the cause of the mortality is immaterial. Some salts definitely were not toxic, regardless of the cause. These salts should be the ones we are using in our systems.
Last edited:
Ron,
Thanks for the quick response. It seems the information is of great value to aquaculturalists who are raising larvae in laboratory conditions, but most of us are using ASW for water changes. That seems a very different set of circumstances.
When you write that:
The use of such "good" artificial sea water will promote the health of coral reef organisms
and
These salts should be the ones we are using in our systems
Aren't you going well beyond what you can safely conclude from your work? If there are detoxifying elements in a reef tank, how can we be sure that "good" salt is any better than "bad" salt.
Your work has raised some intriguing questions, but so far all the dangers are theoretical. Don't we need more direct connections between reef tank health and heavy metals before we can say that (for a reef tank) there are good and bad salts, the sand needs to be periodically thrown out, and all the other changes you've advocated?
Thanks for the quick response. It seems the information is of great value to aquaculturalists who are raising larvae in laboratory conditions, but most of us are using ASW for water changes. That seems a very different set of circumstances.
When you write that:
The use of such "good" artificial sea water will promote the health of coral reef organisms
and
These salts should be the ones we are using in our systems
Aren't you going well beyond what you can safely conclude from your work? If there are detoxifying elements in a reef tank, how can we be sure that "good" salt is any better than "bad" salt.
Your work has raised some intriguing questions, but so far all the dangers are theoretical. Don't we need more direct connections between reef tank health and heavy metals before we can say that (for a reef tank) there are good and bad salts, the sand needs to be periodically thrown out, and all the other changes you've advocated?
Frank Mularo
New member
Statman said:
Oh, oh, oh! (waving my hand frantically) I know this one:
"Sure. Go ahead and do the experiments and let us know what happens."
Right, Ron?
:rolleye1:
Hi Ron. First off, thanks for doing the studies on tank water and ASW. No one else has spent the $$ or made the effort to find out what is going on with the water in our tanks.
Now, several questions relating to your latest study.
1. However, that hypotheses remains untested
How does one go about confirming your heavy metal hyothesis? Are the pathways of metal toxicity in salt water animals well defined?
2. Is it known if there a base level of organic "contaminants" in the various ASW brands That could contribute to toxicity?
Thanks.
Fred
Now, several questions relating to your latest study.
1. However, that hypotheses remains untested
How does one go about confirming your heavy metal hyothesis? Are the pathways of metal toxicity in salt water animals well defined?
2. Is it known if there a base level of organic "contaminants" in the various ASW brands That could contribute to toxicity?
Thanks.
Fred
Hi
That seems a very different set of circumstances.
It surely is. You need non-toxic sea water even more than aquaculturists.
there are detoxifying elements in a reef tank, how can we be sure that "good" salt is any better than "bad" salt.
Anything that detoxifies the salt in a tank, leaves the toxin in tank subject to some re-solution later.
Your work has raised some intriguing questions, but so far all the dangers are theoretical.
Not to the animals that died in the tests. These types of tests are standard tests for toxicity. You will also note the references included in the study, which showed toxic effects on reef organisms with metals at elevated levels.
Don't we need more direct connections between reef tank health and heavy metals before we can say that (for a reef tank) there are good and bad salts, the sand needs to be periodically thrown out, and all the other changes you've advocated?
What I wonder is why anybody would want to put any toxic material in their tanks?
Nobody's forcing you to use less toxic salt mixes, stick with the ones you like. Personally, I would like to see my animals under the lowered stresses of low metals loads. They will grow better, and live longer.
Hi Fred,
How does one go about confirming your heavy metal hyothesis?
One needs to kill more animals with toxicity testing, and do some tissue analyses to see if their has been an accumulation of metals, or if some other material seems to be the cause.
Are the pathways of metal toxicity in salt water animals well defined?
For some animals, yes. For the majority of them, no. As I mentioned in the article, this research is "human" driven, relatively few people have looked at, for example, corals for corals sake. They look at corals because death of the corals in an area may impact the fisheries, or have other economic effects.
In these sorts of studies, all that is determined is the levels at which the animals die, relative to pollution or some other cause. Generally, the actual cause of the mortality is not determined.
2. Is it known if there a base level of organic "contaminants" in the various ASW brands That could contribute to toxicity?{/b]
Not as far as I know. One can test for some organics, but it is a lot harder than testing for metals, and significantly more expensive. There simply are so many more of them to test for, and some will be toxic, others will not. I suppose one could start with a test panel of the EPA's priority pollutant list - these are mostly pesticides, PCBs, and petroleum derivitives. It would be a place to start.
Boomer,
Yup, that will be the answer :spin2: It is always the answer
:rolleye1:
Yeah, it will have to be. You can wave your hands all you want (and you seem to like to do that) but without any data, it will just generate wind....
If you can't do the experiments, find a bunch of like souls, pool your $$$ and hire some one who can do the work.
:lolspin: :lolspin:
it will just generate wind....
I like to generate wind, but gee Ron, you are like a hurricane
You left that wide open, not like you
I do appreciated what you are doing and have done in this hobby
I doubt that seriously
No Ron I do, but I do need to keep you in line once in awhile
If you can't do the experiments, find a bunch of like souls, pool your $$$ and hire some one who can do the work
Well, I actual did. I thought it would be a good idea to get an XRD on a few salts, to see what is really in them....no takers and cheap, just a donation to UMD Geology depatment, $500. Of course an XRD won't tell you what is actually going to end up in solution but it will tell you what is exactly in the bag:eek1:
it will just generate wind....
I like to generate wind, but gee Ron, you are like a hurricane
You left that wide open, not like you
I do appreciated what you are doing and have done in this hobby
I doubt that seriously
No Ron I do, but I do need to keep you in line once in awhile
If you can't do the experiments, find a bunch of like souls, pool your $$$ and hire some one who can do the work
Well, I actual did. I thought it would be a good idea to get an XRD on a few salts, to see what is really in them....no takers and cheap, just a donation to UMD Geology depatment, $500. Of course an XRD won't tell you what is actually going to end up in solution but it will tell you what is exactly in the bag:eek1:
Let me repeat a few things from above posts:
I:
So it seems that in this particular set of experiments (conditions, organisms, etc ) two saltmixes performed just as good as NSW.
Ron:
That was my conclusion as well.
I (with respect to a salt performing well despite high Cu):
Do you have an explanation for this discrepancy?
Ron:
No, not really, as you and Randy have pointed out many things can happen to these metals; and I think particularly where they are found in complex mixtures all sorts of compound effect occur.
Ron did the experiments and there were differences. It could be that if they were repeated by him or any one else that the same results, close to the results or even totally different results might be found for whatever reason.
Only further experiments can show this and would have to be performed first. In the mean time Ron's experiments are as far as I know the only one with the given conditions.
That was one of the reasons to search for other assays using NSW and ASW and ask Ron for his opinion. This can be found in above posts. Basically the references I came up with had other conditions and not fully comparable but IMO still interesting.
Regarding the metals as the cause I see it as Ron's working hypothesis and IMO there is nothing wrong about it.
Further experiments would be required to test if the hypothesis is still correct, has to be modified slightly or has to be changed completely. Such further experiments, if they can not be found in the literature, would have to be done first.
I personally would use as a working hypothesis that the combination of concentration AND the speciation of metal(s) has (very likely) been the cause of toxicity towards the urchin embryos.
It is IMO a bit safer.
I think that the last article by Ron might have a huge impact either directly or indirectly in the near future (and seems to have an impact already).
That is a compliment, Ron!
I:
So it seems that in this particular set of experiments (conditions, organisms, etc ) two saltmixes performed just as good as NSW.
Ron:
That was my conclusion as well.
I (with respect to a salt performing well despite high Cu):
Do you have an explanation for this discrepancy?
Ron:
No, not really, as you and Randy have pointed out many things can happen to these metals; and I think particularly where they are found in complex mixtures all sorts of compound effect occur.
Ron did the experiments and there were differences. It could be that if they were repeated by him or any one else that the same results, close to the results or even totally different results might be found for whatever reason.
Only further experiments can show this and would have to be performed first. In the mean time Ron's experiments are as far as I know the only one with the given conditions.
That was one of the reasons to search for other assays using NSW and ASW and ask Ron for his opinion. This can be found in above posts. Basically the references I came up with had other conditions and not fully comparable but IMO still interesting.
Regarding the metals as the cause I see it as Ron's working hypothesis and IMO there is nothing wrong about it.
Further experiments would be required to test if the hypothesis is still correct, has to be modified slightly or has to be changed completely. Such further experiments, if they can not be found in the literature, would have to be done first.
I personally would use as a working hypothesis that the combination of concentration AND the speciation of metal(s) has (very likely) been the cause of toxicity towards the urchin embryos.
It is IMO a bit safer.
I think that the last article by Ron might have a huge impact either directly or indirectly in the near future (and seems to have an impact already).
That is a compliment, Ron!
Hi Habib,
That is a compliment, Ron!
Yes, I know, thank you.
But the rude and offensive remarks, many of them posted in this as well as other threads by people who have no conception of even what an experiment is, have had their effect. Unless something changes, as it presently stands, while I will likely be doing more research into topics I consider important for my personal reef aquarium hobby, I doubt I will be doing much in the public venue.
Hi, from Macedonia
I carefuly reed your topics, because I,m new in the hoby, and in my country it is inposible to make choices on salt mixes. But reading Mr. Ron topic I ask my self one question: " When we import such amounts of metals trough salt mixes and food, why then at all we need to buy RO or RO/DI units."
Kindest Regards
Sory for bad English
I carefuly reed your topics, because I,m new in the hoby, and in my country it is inposible to make choices on salt mixes. But reading Mr. Ron topic I ask my self one question: " When we import such amounts of metals trough salt mixes and food, why then at all we need to buy RO or RO/DI units."
Kindest Regards
Sory for bad English
Mr_Quality
Premium Member
I am a professional scientist at a major biotech company. I have been watching this thread for weeks. Firstly, I want to thank Ron for his performing and posting his study. We need this kind of data and there are too few out there who are able and willing to help us hobbyists out.
Reading this reminds me of being at work because almost invariably, no matter how good the experimental design nor how compelling the results, there is always debate (euphemism for argument lol). But generally, thatââ"šÂ¬Ã¢"žÂ¢s a good thing. Sometimes are egos get singed though and thatââ"šÂ¬Ã¢"žÂ¢s a bad thing.
No experiment is perfect, but this I a damn good one and Iââ"šÂ¬Ã¢"žÂ¢ll take the data to heart next time I buy salt!
Thanks Ron!!!
Mike
Reading this reminds me of being at work because almost invariably, no matter how good the experimental design nor how compelling the results, there is always debate (euphemism for argument lol). But generally, thatââ"šÂ¬Ã¢"žÂ¢s a good thing. Sometimes are egos get singed though and thatââ"šÂ¬Ã¢"žÂ¢s a bad thing.
No experiment is perfect, but this I a damn good one and Iââ"šÂ¬Ã¢"žÂ¢ll take the data to heart next time I buy salt!
Thanks Ron!!!
Mike
Ron,
I know you don't like to follow links such as:
http://reefs.org/phpBB2/viewtopic.php?t=27367&postdays=0&postorder=asc&start=120
So I am pasting what I did post over there. I am sure you would not like me to paste the whole thread over here. :lol:
I agree with Eric.
If toxicity is the issue then a salt analysed for copper showing say 5 ppb could be more toxic than a salt having a much higher copper concentration of say 40 ppb.
Or is there somewhere proof that a certain metals concentration higher than a certain value is ALWAYS toxic? Don't forget that techniques such as ICP and ICP-MS cannot determine the metal's speciation.
The bioassay as done by Ron Shimek was able to discrimate between saltmixes and even between two tankwaters. This is IMO fantastic.
The biosensors told us which water they liked and which not.
They might have not liked it because of just a single metal, a combination of metals, a plasticizer (thanks Jerel aka Bomber), an anticaking agent, ammonia, highly oxidixing substances in the salt, ........
The bioassay can provide us IMO very useful and important information.
Bioassay done using water of tanks using just NSW and a good ASW might tell us if there is a difference after a certain period of time.
FWIW, and I think it is worth a lot, tanks using NSW acquire a metal concentration comparable to tanks using IO or other salt mixes. That is at least true for the tanks Ron has analysed and the tanks analysed by us.
An ICP-MS analyses would tell us that all the tanks are (approximately) the same while we know that they are not. We need something which can discrimate between those tanks which according to one technique (e.g. ICP-MS) are the same.
The bioassay technique done by Ron Shimek has IMO the potential to discrimate between such tanks but also between virgin salt mixes.
I know you don't like to follow links such as:
http://reefs.org/phpBB2/viewtopic.php?t=27367&postdays=0&postorder=asc&start=120
So I am pasting what I did post over there. I am sure you would not like me to paste the whole thread over here. :lol:
I agree with Eric.
If toxicity is the issue then a salt analysed for copper showing say 5 ppb could be more toxic than a salt having a much higher copper concentration of say 40 ppb.
Or is there somewhere proof that a certain metals concentration higher than a certain value is ALWAYS toxic? Don't forget that techniques such as ICP and ICP-MS cannot determine the metal's speciation.
The bioassay as done by Ron Shimek was able to discrimate between saltmixes and even between two tankwaters. This is IMO fantastic.
The biosensors told us which water they liked and which not.
They might have not liked it because of just a single metal, a combination of metals, a plasticizer (thanks Jerel aka Bomber), an anticaking agent, ammonia, highly oxidixing substances in the salt, ........
The bioassay can provide us IMO very useful and important information.
Bioassay done using water of tanks using just NSW and a good ASW might tell us if there is a difference after a certain period of time.
FWIW, and I think it is worth a lot, tanks using NSW acquire a metal concentration comparable to tanks using IO or other salt mixes. That is at least true for the tanks Ron has analysed and the tanks analysed by us.
An ICP-MS analyses would tell us that all the tanks are (approximately) the same while we know that they are not. We need something which can discrimate between those tanks which according to one technique (e.g. ICP-MS) are the same.
The bioassay technique done by Ron Shimek has IMO the potential to discrimate between such tanks but also between virgin salt mixes.
Ron:
Just one more question to make sure I'm reading your tables and the references correctly. You wrote:
You will also note the references included in the study, which showed toxic effects on reef organisms with metals at elevated levels.
If I've read your tables correctly, your IO copper figure was .018 ppm and Atkinson's was .114 ppm. According to the Alutoin reference, Porites was uneffected at 10 ppm and effected at 30 ppm. The Negri article tested copper at 110 ug/l and found it toxic. If I'm reading these numbers correctly, aren't the scientists finding toxicity at much higher levels than you found?
It seems the copper found in IO higher than NSW, but still only 4 tenths of one percent of Alutoin's toxic level. Am I reading the numbers correctly?
Just one more question to make sure I'm reading your tables and the references correctly. You wrote:
You will also note the references included in the study, which showed toxic effects on reef organisms with metals at elevated levels.
If I've read your tables correctly, your IO copper figure was .018 ppm and Atkinson's was .114 ppm. According to the Alutoin reference, Porites was uneffected at 10 ppm and effected at 30 ppm. The Negri article tested copper at 110 ug/l and found it toxic. If I'm reading these numbers correctly, aren't the scientists finding toxicity at much higher levels than you found?
It seems the copper found in IO higher than NSW, but still only 4 tenths of one percent of Alutoin's toxic level. Am I reading the numbers correctly?
Great Job!
Great Job!
Thanks, Ron, for the great work!
It doesn't surprise me that your findings have generated such a heated discussion. Similar reactions can be found in most hobbies (and, unfortunately, science in general) whenever one dares challenge long held beliefs. The US EPA in their research guidelines recommends two salts for standization, one being the Marinemix formula (http://www.epa.gov/waterscience/WET/disk1/ctm1-3.pdf), which lends further credibility (at least face validity) to your report.
Based on your findings, I have switched to the bio assay formula and will report any changes I observe. For those who are having difficulty finding the bio assay formula, I orderd mine from www.aquaticeco.com ($26 for 150 gal mix when ordered in quantity).
Take care.
Tim
-----------------
Timothy Lowe, Ph.D.
Great Job!
Thanks, Ron, for the great work!
It doesn't surprise me that your findings have generated such a heated discussion. Similar reactions can be found in most hobbies (and, unfortunately, science in general) whenever one dares challenge long held beliefs. The US EPA in their research guidelines recommends two salts for standization, one being the Marinemix formula (http://www.epa.gov/waterscience/WET/disk1/ctm1-3.pdf), which lends further credibility (at least face validity) to your report.
Based on your findings, I have switched to the bio assay formula and will report any changes I observe. For those who are having difficulty finding the bio assay formula, I orderd mine from www.aquaticeco.com ($26 for 150 gal mix when ordered in quantity).
Take care.
Tim
-----------------
Timothy Lowe, Ph.D.
Hi,
You asked, "Am I reading the numbers correctly?"
No. You are off by a factor of 1000.
Autoin was finding effects at 30 micrograms Cu/l; this = 30 ppb, not 30 ppm.
I have attached a reduction of a graphic I made for my talk at the last MACNA from a number of different sources regarding copper toxicity.
You asked, "Am I reading the numbers correctly?"
No. You are off by a factor of 1000.
Autoin was finding effects at 30 micrograms Cu/l; this = 30 ppb, not 30 ppm.
I have attached a reduction of a graphic I made for my talk at the last MACNA from a number of different sources regarding copper toxicity.
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