Important data missing in toxicity study

rshimek said:
Hi Frank,

You presume I am or was attempting to test variability within the salts. As I made replicates from one bag each, the N with for such a test would 1. I did not run such a test. Instead I tested between the salts. Here the N was 6.

I am testing the difference between 6 different types of salt water and a control. For the ANOVA to test the difference in the variances the question was different than the one you presume. I am, in that case, asking whether the variance for each solution was different from all others, in this case the N = 6, with 10 replicates of each treatment. In this case each treatment was a different salt mix.

Now, I could have done this with each treatment being a different bag of the same salt mix. Answering that question was not of interest to me given my limited supplies, so I didn't do that.

Or I could have made this a two factor test and tested between and within salt mixes instead simply between them using several differing bags of salt per experimental treatment. I simply didn't have the resources or space to that.

My test was between 6 salt mixes in one test, and the N=6.

Individual tests of the samples against the control were done with 10 sample replicates of each solution. And a t-test was used. N here refers to the replicates of the same treatment, and was 10.
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I understand the question you asked. You weren't interested in variability within each batch of salt, just which was potentially the most toxic. What I am saying is that with your experimental technique (which is good, just incomplete) you can't answer that question. In order for your test to have any relevance, it must be a two factor test. The only question you can possibly answer without more samples of each particular salt is "Which particular batch of sea salt in my posession that I tested is the most toxic to urchin larvae?" Okay, so your resources and space are limited. Then so are your results. I don't disbelieve your results, I just want to see more experimentation to be convinced.
 
Originally posted by Frank Mularo
Click to expand...

Hi Frank,

You weren't interested in variability within each batch of salt, just which was potentially the most toxic. What I am saying is that with your experimental technique (which is good, just incomplete) you can't answer that question. In order for your test to have any relevance, it must be a two factor test.

Well and good, but that is not the question I asked. What I asked is simply is there a difference between each salt water that I tested and a control of NSW. I asked nothing about magnitude of the differences, although it is obvious. I simply used a simple oneway ANOVA to assess differences in the variances of the samples. These variances were different, and that's all I used that particular test for. Knowing that difference allows the correct choice of the t-tests for the differences in the means.

The only question you can possibly answer without more samples of each particular salt is "Which particular batch of sea salt in my posession that I tested is the most toxic to urchin larvae?"

And that is why the above IS the question I asked.

In point of fact, the question was even more limited. The basic question was is there any difference between NSW and each particular salt water that I tested on a one to one basis. That was asked, tested for, and the differences shown to be highly signficantly different.

:D
 
Hab

Are you suggesting that if journal's reviewer would disprove the hypothesis the answer be....

get off your butt and do the tests

No, I would never suggest that:D

But it does remind me of and old story when I was doing a Animal Behavior paper in college on the food habits and feeding behavior of the American River Otter 25 years ago. A bunch of researchers sat in the labs and had trappers bring in the stomachs and intestines with students collecting scats. The conclusion was Otters eat allot of insects. Well Hab, Otters don't eat insects.....:D So it goes like this :rolleyes:

Insects in Otters = Metals toxins in water

Otters eat insects = Metals killed larvae

Ron

The potential problem with publishing the study is that it doesn't say anything really new except that there is are artificial salt mixes that allow embryos to live when freshly mixed. The common presumption is that salts are toxic enough that no researcher will use them if they can avoid it.

I have no problem with this and is all your article proved, nothing more nothing less. I'm glad you did it and you may have a good case, but it must go to the next level before anyone can take it seriously that it was indeed the metals. Now think about that for a minute.......and all it would do to the above negative comments, if it actually was the metals. Your head would now swell exponentially........I told you so and I would be all :D :cool: :D for you.
 
Actually, I don't really care what caused the difference. I proposed the hypothesis that metals were the cause because it seems obvious. If you don't agree, fine. Propose another hypothesis and test it. Or test my hypothesis and disprove it. But do test it... All of the hooting and hollering obscures the real finding of the study, that being that other ASW did not kill the larvae. It doesn't make any difference to me, what the cause is. :eek2:

That some ASW has problems supporting delicate organisms was never a question as far as I was concerned. I have know for decades of the problems with some of these salts. However, I presumed, and I think a lot of other invertebrate zoologists presumed, that the all ASW was equally nasty.

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. I really don't care if the problem is due to excessive heavy metals, or excessive farting by workers in the plant as the ASW mixture is being made.

Let them figure it out. If other folks have made salts that produce non-toxic ASW, then it is feasible and I now have a salt to use that is safe for delicate organisms in a reef tank.

I published the test simply so that other folks could also treat their animals better. You don't have believe the data and you don't have to act accordingly. However, I do, and I will.

If anybody wants to figure out why it occured, go right ahead. Enjoy. :D However, such a determination is only going to be done with experimentation, it will not be determined by web surfing or argumentation. You are going to have to sit down design the tests, and then you are going to have to try to kill animals, because that is the only way you can be sure that materials are toxic or not.
 
Well said, Dr. Ron. I think your test and purpose of your test is now clear to us many non-Phds... If life were only that simple.
 
Man, you guys make my head hurt!

I can take two things away from all this.

1. This is a lot more complex that a few simple tests.

2. freshly mixed Crystal Seas Marinemix dosn't kill urchin larvae the way freshly mixed I/O does.

Now, heavy metals are a pretty good bet as the culpret but, we don't know that for sure. What puzzles me is why Steve Kempf finds aged artificial mix (like Instant Ocean) just fine? What happens to the nasties as the water ages?

Sigh, ignorance is bliss. I'm gonna stop reading these posts.

Fred.
 
Originally posted by Fredfish
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Hi,

1. This is a lot more complex that a few simple tests.

No, not really.

What happens to the nasties as the water ages?

The same thing that happened to Hobbyist A in my study, who also used IO and got moderately good survival. Probably something in the tank detoxed the metals (or what ever) by binding them and taking them out of solution. There are number of possibilities for pathways to do this. All of them speculative, but all with some supportive data somewhere in the literature.
 
1. This is a lot more complex that a few simple tests.

No, not really.

OK, Then why do experiments using NSW with heavy metals in lower concentrations than our tanks prove toxic to urchin larvae when aged ASW dosn't? At liest, that is the conclusion I draw from the information given so far.

Thanks
Fred.
 
Hi Ron,

We have never had success culturing nudibranch eggs or larvae using freshly mixed artificial sea water. Instead, all sea water for culture is drawn from established aquaria that contain sea water that is either natural or prepared from a commercial formula (e.g. Instant Ocean) with either de-ionized or distilled water

They don't mention that they had success using natural sewater after depriving of organics (nutrients?). So there is no control.
It even suggests that even NSW has to be used after being some time in an aquarium.

And that these organics can be important is mentioned by Kempf himself:

Lecithotropic development, such as that characterized by the embryos and larvae of Berghia verrucicornis, is generally thought to use maternally derived yolk reserves to fulfill energetic needs. Recent investigations (Jaeckle and Manahan, 1989; Manahan, 1989; Manahan et al., 1989; Shilling and Manahan, 1990) indicate that cellular endocytic systems in tissues other than those of the digestive tract may allow dissolved organic material from surrounding seawater to make a significant contribution to the energetic requirements of larval and possibly embryonic development. Although no conclusions with respect to the developmental importance of DOM can be drawn from this study, the decrease in size of teh right digestive diverticulum during embryonic development in B. verrucicornis suggests that yolk reserves stored in this organ are an important nutrient source used to support embryonic energetic needs.

Above can be found:

http://www.breeders-registry.gen.ca.us/Reprints/biolbull/v179/berghia_bb.htm

Which a reprint from a scientific journal.



The presence of DOM might be very essential and could be the major key to success in cultivation of Berghia.
 
Originally posted by Fredfish
Click to expand...

Hi Fred,

OK, Then why do experiments using NSW with heavy metals in lower concentrations than our tanks prove toxic to urchin larvae when aged ASW dosn't? At liest, that is the conclusion I draw from the information given so far.

Well, take a look at the discussions about some of the earlier components of these studies. In many tanks- but not all tanks - it appears that the heavy metals are detoxified by some factor or factors. It could be either binding of the metals with humic acids or binding in the lower layers of DSB with sulfides or whatever. Finding that answer is going to be a complicated.

The results of this study are pretty straight forward.

:D
 
Originally posted by Habib
Click to expand...

Hi,

The presence of DOM might be very essential and could be the major key to success in cultivation of Berghia.

By all means it is. When you culture them in fresh IO, they die before they reach the stage of needing it. In many cases they will die, even before hatching, it permeates the egg capsules and kills them.
 
Originally posted by Boomer
Click to expand...

Hi,

I would suppose you will say it is irrelevant and they don't know what they are doing, so go for it

No, this one may well be relevent, but it would interesting to test. Pfiesteria, the organism they used, is one of the strangest organisms on the planet (read up on it if you wish) and it is not even in the same kingdom as the urchins I used. Its metabolism is very odd.

Nonetheless, the presence of plasticizers may be causing problems.

So... prove that it is them rather than heavy metals.

The heavy metal hypothesis is still consistent with all the data.
 
B]Nonetheless, the presence of plasticizers may be causing problems[/B]

So, it maybe these or it may be ammonia or it maybe HM or it may dissolution problems, or it may be other things not looked at yet That's the point Ron, it may be other things also. No one is disputing that salt X, Y or Z killed more urchins and salt A, B and C was less.

So... prove that it is them rather than heavy metals

I don't have to prove anything, it is your theory and you don't want to seem to prove that is the HM, which is the bases of science, try and prove your own theory. You have done nothing to prove it is HM, which is your theory, only that they are there, based on assays years old, which may have changed in recent years. Larvae just died for some unknown reason is all that you have shown, which is nice and even you have more or less said so. No matter how one looks at your results, you have shown something. However, is real big of you to tell an average very day hobbyist, who doesn't have access to lab, who may not have the background, who ever he or she may, be........ prove me wrong.... I'm Dr Ron ... it even rimes

You can have the finally word, have a nice weekend and yes I do appreciated what you are doing and have done in this hobby, just so you know :D
 
Originally posted by Boomer
Click to expand...

So... prove that it is them rather than heavy metals

I don't have to prove anything, it is your theory and you don't want to seem to prove that is the HM, which is the bases of science,[/b]

Actually, it isn't. We can't ever prove anything using the scientific method; it is fundamentally and logically impossible. Instead, we disprove alternative hypothesis. So... If you or anyone wishes to try to disprove my hypothesis, go for it. If you can't do the work personally, hire someone who can.

I do appreciated what you are doing and have done in this hobby

I doubt that seriously.
 
Habib said:


And that these organics can be important is mentioned by Kempf himself:

Lecithotropic development, such as that characterized by the embryos and larvae of Berghia verrucicornis, is generally thought to use maternally derived yolk reserves to fulfill energetic needs. Recent investigations (Jaeckle and Manahan, 1989; Manahan, 1989; Manahan et al., 1989; Shilling and Manahan, 1990) indicate that cellular endocytic systems in tissues other than those of the digestive tract may allow dissolved organic material from surrounding seawater to make a significant contribution to the energetic requirements of larval and possibly embryonic development. Although no conclusions with respect to the developmental importance of DOM can be drawn from this study, the decrease in size of teh right digestive diverticulum during embryonic development in B. verrucicornis suggests that yolk reserves stored in this organ are an important nutrient source used to support embryonic energetic needs.
Click to expand...

As mentioned (I think), the eggs will not hatch at all in unconditioned ASW so they never get to the larval stage. I have no reference link for this since I lived it instead. ;) At one point I had well over 200 Berghia scattered throughout my house and office. I would not be surprised if they relied on organics a little after hatching, but it is most likely for a VERY brief period of time. Once hatched, it takes Berghia less than 3 days to become very efficient predators. Within 2 weeks of hatching they are pretty good sized juveniles and at around 40 days they begin reproducing themselves. If Ron can't lasso Morgan into doing some experimenting with his Berghia population I could do so, although the culturing tanks etc. are long gone and I would not really look forward to it. :D Previously, I was much more interested in their community feeding behavior and did not focus on trying too many different ways to hatch them out. I tried fresh ASW which of course doesn't work, I tried aged ASW which should remove NH4 and stabilize the pH-didn't work, I tried ASW from a tank that had some sand, algae, and cyanobacteria where I held the aiptasia population which was fed and had some organics as there wasn't even a filter on the tank-didn't work, ASW from my reef system with the whole boat of goodies-well that water worked. When I say didn't work, I mean truly did not work as in no eggs even hatched. It was either an all or nothing proposition so it seemed. HTH
 
Justin (Saltshop):

HTH

Yes! :)

FWIW I tried to reach Kempf twice today but got only his voice mail and the Berghias started to crawl in my ear. :D
 
Guys,

This topic is well off anything to do with my article. The urchins did all right in most cultures and died in others. They don't worry seem to used dissolved organics, and in any case we have no data about them.

I suggest you start a different thread in the main reef discussion forum for any continuation of this tack.

:D
 
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