Important data missing in toxicity study

[Habib]

Here is one more of which I don't know yet which ASW was used.

Water Research
Volume 31, Issue 2 , February 1997, Pages 351-355

A simplification the bivalve embryogenesis and larval development bioassay method for water quality assessment

E. His2, *, M. N. L. Seaman1 and R. Beiras2

2 IFREMER, Quai du Commandant Silhouette 33120 Arcachon France
1 Institut für Meereskunde 24105 Kiel Germany



Abstract

A simplification of the standard bivalve embryo-larval bioassay method for seawater quality assessment is described. Methodological features aimed to increase reliability, sensitivity and accuracy, and to reduce time and costs, include: induced natural spawning to guarantee gamete quality, earlier exposure to the test solutions and direct observation without subsampling. In the first example of application, embryos of oysters (Crassostrea gigas) and mussels (Mytilus galloprovincialis ) were used to compare the adequacy of artificial and natural seawater for the incubation of bivalve larvae. An average of 91-93% of the embryos developed normally in all cases, and there were no significant differences between species or between the two waters tested, concluding that both species and both types of water are equally suitable for ecotoxicological monitoring purposes. In the second example the C. gigas embryo-larval bioassay was used for the assessment of the water quality in 15 tributaries of an oyster farming area: the Bay of Arcachon. Methods can be applied to freshwater ecotoxicological studies

 

[Habib]

Oh, I almost forgot.

To my question:
Could the results have been diffrent if collecting and fertilization was done in one of the salt mixes?

You said:
Certainly. If the animals were spawned into IO or Coralife, a goodly portion of the embryos would have likely died immediately. This is why in all bioassays of this nature fertilization is done in sea water.


From one of the above clippings I gave:

Both Neiheisel and Young (1992) and Jonczyk et al. (2001) reported
favourable results in echinoid (sea urchin) fertilization assays performed using Instant Ocean TM for adjusting sample
salinity and for preparing the control/dilution water.

Also see the abstract of Jonczyk's paper (see one of the previous posts).

TIA

 

[Boomer]

Ron

I proposed the hypothesis of metals poisoning to explain the consistent deaths in the two salts with the highest metals concentrations (and they were higher by in some cases several thousands of times). So... it seems reasonable to me.

Well, it would only seem reasonable to me if a tox screen was done on the urchins, as to why they died. Otherwise it is just a guess IMHO, to many variables.


Hab

That is some interesting stuff you found

 

[rshimek]

Hi Habib,

Just a comment for the future... how about trying to put all of your comments into one post. If I can do it, so can you and it will make it easier.

Comments on your posts:

REVISED PROCEDURES FOR ADJUSTING SALINITY OF
EFFLUENT SAMPLES FOR MARINE SUBLETHAL TOXICITY
TESTING CONDUCTED UNDER ENVIRONMENTAL EFFECTS
MONITORING (EEM) PROGRAMS
Method Development and Applications Section
Environmental Technology Centre
Environment Canada
December 2001
.

This is pretty much irrelevent. I did not do a test of fresh water effluent where I had to bring it up to sea water conditions, nor was this a fertilization test, and we are not discussing sublethal toxicity here. Both the tests and the parameters are different. Nor did I have to adjust the salinity of anything more than 0.01 sp. g. unit. But when I did, , I guess I followed their guidelines...

Testing laboratories should obtain the A best quality A of
commercial sea salts (e.g., Forty Fathoms TM Toxicity Test Grade) available from the supplier, and should evaluate its
ability to meet the test-validity requirements in preliminary toxicity tests using such new products or batches for
preparing salt controls (see Section 5.0).

Different strokes for different folks... Forty Fathoms = Crystal Seas Marinemix bioassay formulation.

then there is:
Sea urchin fertilization assay: an evaluation of assumptions related to sample salinity adjustment and use of natural and synthetic marine waters for testing.
Jonczyk E, Gilron G, Zajdlik B
Environ Toxicol Chem 2001 Apr 20:804-9

Well, here again a non-comparable test and on fresh water effluents.... The paper involved differences in results from adjusting fresh water sample salinities.

and then...

Copper toxicity to larval Mercenaria mercenaria (hard clam).
Labreche TM, Dietrich AM, Gallagher DL, Shepherd N
Environ Toxicol Chem 2002 Apr 21:760-6

Environ Toxicol Chem ââ"šÂ¬Ã‚¢ Volume 21 ââ"šÂ¬Ã‚¢ Issue 4

And here they raised clams in ASW with and without more copper being added. Fine. And it tells us what?

Their controls were ASW, too. It says nothing about survival versus NSW. I got some survival of urchins in ASW, too. I could have done this had I wanted. But it is also irrelevent. That a few will survive was not the point. That most don't, was.

And then..

Water Research
Volume 31, Issue 2 , February 1997, Pages 351-355

A simplification the bivalve embryogenesis and larval development bioassay method for water quality assessment

E. His2, *, M. N. L. Seaman1 and R. Beiras2

Using a different test organism, you get different results. These are both organisms that are very tolerant of freshwater and quite pollution resistant. One might expect these results, and could use the organisms as some test in industrial situations. I used a sea urchin, specifically because they are less tolerant to salinity and metals variation.

And then your comment here:

Oh, I almost forgot.
To my question:
Could the results have been diffrent if collecting and fertilization was done in one of the salt mixes?
Also see the abstract of Jonczyk's paper (see one of the previous posts).TIA

I stand by my statement. I suggest you read the protocols for this type of test. In all cases the gametes are collected in NSW unless previous work has been to relate subsequent results to the changes that occur in ASW. There would have been changes in results and many cases I would bet the sperm that go into straight freshly mixed IO or day old IO will either die or have their fertilization rates affected.

Now, all of this is well and good. And irrelevent. The tests are different and in some cases the organisms are different.

If you don't think that metals concentrations are the cause of the differences, well and good. Do some tests of your own and show an alternative.

If you want to spend some time reading, go to a library and read the whole papers describing the various sea urchin tests and their use. Perhaps, spend some time reading the papers describing how copper kills reef organisms, including corals. I have included several in the article to give you a bit of a heads up.

I am certain I can surf the net and find numerous article showing how various metals in various concentrations can kill organisms.

And we can have a pis*s*ing match of the references.

That is also irrelevent.

The only way you can disprove my hypothesis is by actually testing it. So, unless you either get off your butt and do the tests, or by ponying up the money and having them done, all the veribage in the world will not make a bit of difference; nor will it mean squat.

 

[rshimek]

Originally posted by Boomer

Hi,

Well, it would only seem reasonable to me if a tox screen was done on the urchins, as to why they died. Otherwise it is just a guess IMHO, to many variables.

The urchins await. I have told you where to get them. Now do the tests you propose.

So, to repeat my comment to Habib,

"The only way you can disprove my hypothesis is by actually testing it. So, unless you either get off your butt and do the tests, or by ponying up the money and having them done, all the veribage in the world will not make a bit of difference; nor will it mean squat."

Hab

That is some interesting stuff you found

Interesting, yes. Relevent... no.

 

[Boomer]

"The only way you can disprove my hypothesis is by actually testing it. So, unless you either get off your butt and do the tests, or by ponying up the money and having them done, all the veribage in the world will not make a bit of difference; nor will it mean squat."

And all the veribage you toot doesn't mean squat about your hypothesis either, as your test proves nothing also. As far as butts go I think you are the one sitting down. I see you are up to your normal arrogants, when somene disagrees with you, a very poor chartater I migh add.

The urchins await

:lolspin: :lolspin:

You would think you would want to do the testing yourself to prove your theroy, isn't that the way things _should_be done.It is also a cop-out in your part

Interesting, yes. Relevent... no.

Umm, that would be your opinion

 

[rshimek]

I have done the tests...

I have posted the data...

I have proposed an hypothesis that is consistent with the results and consistent with published literature, and I have cited that literature...

If you want to disprove the hypothesis, you have to do the experiments that do so.

You may not like it, but that is the basis of science. So...

In essence, put up or ....

 

[Frank Mularo]

Very interesting study. I also hope that you let us know if it's accepted into a peer reviewed journal, to see if your tests stand up to more rigorous standards.

I am a research technician in the fiels of molecular biology and genetics (15 years) and while your results are compelling, I'd have to say that I'd need to see more numbers to be convinced. An n of 1 (11 replicates notwithstanding, you still only tested a gallon of each mix) wouldn't meet the standard in my lab.

Let us know if you get published elsewhere, as it would add weight to your arguments.

 

[rshimek]

Originally posted by Frank Mularo

Hi,

. An n of 1 (11 replicates notwithstanding, you still only tested a gallon of each mix) wouldn't meet the standard in my lab.

6 different solutions gives an N of 6.

Better than the N of 0 that is used elsewhere....

I would love to see the result from tests where various batches of the salts were tested. That would ask a different question than I did.

Sounds like you have a lab... so go for it. Shouldn't cost more then a couple of kilobucks and it would be quick, no more than a week or two.

 

[Boomer]

I also hope that you let us know if it's accepted into a peer reviewed journal

:lol:

And the first thing they would ask is "what killed the urchins" and the answer would be .....I don't know, you figure it out

 

[Habib]

Boom,

ROFL!!!

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

get off your butt and do the tests

 

[Habib]

Hi Ron,

If you don't think that metals concentrations are the cause of the differences, well and good.

I even didn't go that far. But I must confess that I didn't see any correlations with metals.

There can't be any correlation because the metals were NOT measured. You just did assume that they had a certain concentration. Or is there a protocol (besides yours) which approves this?

Better than the N of 0 that is used elsewhere....

I guess that is the N=0.



According to your results IO performed very bad and that is a result which can not be neglected IMO. Afterall you did the tests and that was the outcome.

It is however surprising that results published in scientific journals are in favor of using e.g. Instant Ocean to bring the salinity of a fresh water effluent to that required for the various sea urchin bio assays.

DI water is a fresh water effluent emerging from a DI cartridge.

So basically it is being approved to use a salt mix as hobbyists use it and make it (if DI water is used) for e.g. sea urchin fertilization assays.

Furthermore from one of the previous abstracts:

Moreover, these procedures also allow for the use of either natural or synthetic marine water for culturing/holding test organisms and for full-scale testing.


Now, all of this is well and good. And irrelevent. The tests are different and in some cases the organisms are different.

So basically what you are saying is that you have found bad results with IO and good results with some other salts but the results can be totally different and perhaps totally the opposite because an aquarium is a different test and the aquarium inhabitants are different.

 

[rshimek]

Hi Habib,

There can't be any correlation because the metals were NOT measured. You just did assume that they had a certain concentration.

Yes, I assumed that the concentrations were as indicated in the published values. Using those data, one could certainly do a correlation analysis. Simply put when the metals are as high as they presumeably were in IO and Coralife relative to the other salts, it would seem to be a pretty good first estimate of the cause of mortality.

It is however surprising that results published in scientific journals are in favor of using e.g. Instant Ocean to bring the salinity of a fresh water effluent to that required for the various sea urchin bio assays.

No, it isn't surprising. People publish all sort of things. But this is only the first step, and in such protocols after the water is adjusted for the fertization bioassay it has to be normalized to assess the amount of change relative to NSW otherwise the test is done with the survival assessed relative to the ASW. As the ASW doesn't kill everything, you still can get acceptable results with a fertilization test.

All of the studies you read the abstracts of list the sea urchin fertilization test which is not the test I ran. Rather like comparing regular and decaf coffee... Not the same thing.

So basically what you are saying is that you have found bad results with IO and good results with some other salts but the results can be totally different and perhaps totally the opposite because an aquarium is a different test and the aquarium inhabitants are different

I said nothing of the sort. You are saying a fertilization bioassay designed to test viability of sea urchin sperm in potentially toxic fresh water effluents is the same as a test showing the suvivability and growth of embryos in full strength sea water. The test is different, the media are different, and the questions asked are different.

I didn't say anything about specific aquarium animals or the aquarium situation with the specific results of the test. I gave references that discussed the results of metals toxicity to corals and other common aquarium inhabitants. All I did was, once more, show that fresh IO is toxic to embryos. You like to search the web, do a search on Steve Kempf (who studies nudibranchs and their embryology and ecology). You will find statements where he discusses the same survivability problem with IO, and other salts, with regard to Berghia, the nudibranch that eats Aiptasia.

http://www.breeders-registry.gen.ca...erghia_azaa.htm

quote:
--------------------------------------------------------------------------------
"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."
--------------------------------------------------------------------------------

Delve even deeper into the embryological or larval ecological literature and you will find more supportive data.

The larvae are like canaries in a mine. They indicate poisoning. And they die with these salt mixes, and have for a long enough time, that it has become part of standard embryological protocol not to use the salts.

Now, if we have a salt that is gentle enough on the organisms to allow survival, it will likely aid the survival of other animals as well.

 

[Frank Mularo]

Hi,

6 different solutions gives an N of 6.

Actually, it is an N of 1 for six different solutions. If, for example, I am testing a stimulant on various cell lines in culture, each time I apply that stimulant and record the results, it is an N of 1. If I stimulate another sample of the same cell line and record the results, that gives me an N of 2. If I stimulate a different cell line and record the results, that does not give me an N of 3, it gives me an N of 1 for that cell line. (Dr. Shimek already knows this, this is for people not versed in scientific technique) You have a PhD, you should know that no conclusion can be based on testing one sample. You mixed up one sample and tested it 11 times. Had you tested samples from six different bags of each type of mix (which of course you know would give you an N of 6 for each sample) and gotten the same or similar results, your conclusions would definitely carry more weight. And, just based on experience with accepted experimental methods, I'd have to say that you can throw out the results from the hobbyists' tanks because it is impossible to control for variables such as parasites, pollutants, etc, that without a doubt have contaminated those samples. In fact, those variables make the results from the hobbyists' tanks worthless.

More samples would be interesting. You started the debate by basing your conclusions on limited data. Had you submitted this for peer review for a journal or for a grant, they most certainly would have asked for more data to back up your contentions. A response of "I did enough, do it yourself" would guarantee no publication or grant money.

 

[rshimek]

Originally posted by Boomer

And the first thing they would ask is "what killed the urchins" and the answer would be .....I don't know, you figure it out

Actually the answer would be that those particular salts killed the organisms. This is rather old news and has been around for some time; researchers have known for years that fresh ASW kills animals.

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.

 

[saltshop]

Trying to lighten the mood....

Steve Kempf-Faculty Director, Department of Biological Sciences Auburn University

If he was just a post doc when you met him some 20 years ago...that would make you....Oh My!!

In case you ever want to get a hold of him...and yes he will take the time to talk to aquarists. About Berghgia anyway, I would not be confident that he would like to join this discussion though.

kempfsc@auburn.edu

 

[rshimek]

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.

 

[rshimek]

Originally posted by saltshop

If he was just a post doc when you met him some 20 years ago...that would make you....Oh My!!

About the same age...

He has more hair than I do now, and I seem to recollect way back then (early 1980s) he had a whole LOT more hair.

 

[Habib]

Saltshop:

Trying to lighten the mood....

I am always in a good mood. Right now I am enjoying my beer (it's evening over here).

How many decades ago was that picture taken?

 

[rshimek]

Originally posted by Habib

How many decades ago was that picture taken?

About 0.5, I believe.