Important data missing in toxicity study

[Habib]

Ron,

First of all many thanks for your article

But...
I could not find the metal concentrations and other parameters for the saltmixes, natural seawater and water obtained from the hobbyists.

You give values supplied by manufacturers one could wonder if they are correct. Even if they were correct then variations in concentration caused by the production process and variations in the commodities are missing.

And IIRC there was a major difference in IO analysis carried out by Marlin and Craig and the one done by you.

[Habib]

One more thing...

Once fertilization was noted, approximately one milliliter of the fertilized egg suspension was pipetted into each of the replicates (resulting in each replicate having between 50 and 80 fertilized eggs).


And

Table 1. Number of Arbacia punctulata larvae (early pluteus) found after 48 hours.

-How sure are you that it was always between 50 and 80 fertilized eggs?

-If the number of eggs varied by almost a factor 1.6 (and perhaps even more if not verified) then how is one allowed to compare the absolute number of larvae?

[Habib]

Ron,

These measurements were made with a hydrometer with a reference temperature of 60 deg F; and adjusted to compensate for the difference between the calibrated and ambient temperatures. Information about hydrometer calibration and use is available online in several sites.


Which one did you use?
Temp variations was 10 F right?

[Habib]

Eggs were collected by inverting the spawning urchins over beakers full of natural sea water.

So there was already an acclimatisation to the NSW sample.

Furthermore they were transferred to a water with a different composition such as osmotic pressure, pH,......

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

Could the results have been different if the NSW was modified in whatever way besides heavy metal concentration.
I think in particular about differences in pH and +/- 10% differences in salinity.

TIA

[rshimek]

Originally posted by Habib

I could not find the metal concentrations and other parameters for the saltmixes, natural seawater and water obtained from the hobbyists.

Please read the study. All sea water used in the test were made to a common sp. g. I did not test for metals or any other parameters.

You give values supplied by manufacturers one could wonder if they are correct.

Surely one could.

And IIRC there was a major difference in IO analysis carried out by Marlin and Craig and the one done by you.

What does IIRC mean?

I used the data from Atkinson and Bingman for the comparisons in the study.

How sure are you that it was always between 50 and 80 fertilized eggs?

I diluted the embryos to a known concentration and pipetted with a volumetric pipette a know volume of fluild. I did a few preliminary pipettings and counts. This is standard bioassay techiques as it is quite impossible to count all the embryos one by one. On the other hand when you mix a chemical solution, I suspect you don't count atoms, either.

-If the number of eggs varied by almost a factor 1.6 (and perhaps even more if not verified) then how is one allowed to compare the absolute number of larvae?

One is comparing the number of survivors, and if the number of embryos were sufficiently different one would expect the final data to be inconsistent. The data are not.

Which one did you use?

I used my own tables. But it immaterial, as all the samples were the brought to the same reading.

Temp variations was 10 F right?

Yes

So there was already an acclimatisation to the NSW sample.

Of course. This is standard technique in both embryology and bioassay work.

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

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.

Had I wanted to test fertilization efficiency or sperm motiliity I would used the other mixes directly. Both of those kinds of tests are done, but I think larval development for two days is a better indicator. By the way, I did not mention in the study, but a number of the embryos in the IO and Corallife were aslo deformed, while none were in any of the other mixes. I didn't present these data as the amount of deformation is somewhat hard to quantify.

Could the results have been different if the NSW was modified in whatever way besides heavy metal concentration.
I think in particular about differences in pH and +/- 10% differences in salinity.

Well, the salinity was as close as I could get it, and pH was that for one day old mixed sea water of several differnt manufacturers. Generally, pH differences that are minor don't effect embryos much.

[Ninong]

What does IIRC mean?

If I remember correctly.

[eAlex]

Originally posted by Habib
...
I could not find the metal concentrations and other parameters for the saltmixes, natural seawater and water obtained from the hobbyists.

You give values supplied by manufacturers one could wonder if they are correct. Even if they were correct then variations in concentration caused by the production process and variations in the commodities are missing.

And IIRC there was a major difference in IO analysis carried out by Marlin and Craig and the one done by you.

First of all I also want to thank Dr Ron for this article and for all other studies hi did for this hobby!

This study may be a good start point for as to find some good salt mix in Europe, but I have some questions on it.

Here is description of Table 4: Constituents of the salt mixes examined, in ppm. The data for Instant Ocean and Coralife salts are from Atkinson and Bingman, 1999. The data for Marinemix–Bioassay Formula were provided by the manufacturer. The data for Bio-Sea Marinemix are the average of two samples in the advertising literature from the manufacturer. The values for sea water are from Pilson, 1998.
I find that values for Bio-Sea Marinemix are from this test: http://www.aquacraft.net/w0002a.gif text in S-15 test have no sense, but ICP data are probably even true, because it show no benefits of AquaCraft products. From same test you can find also data for:
IO: http://www.aquacraft.net/w0014b.gif
Coralife: http://www.aquacraft.net/w0004b.gif
and Crystal Sea (normal mix?): http://www.aquacraft.net/w0008b.gif
I guess all tests done with same methodology, and they show very little difference in heavy metal concentration between that salts.

We can not do test like Ron did, but we can test salts what ever we want for trace elements, if they are key for salt toxicity (it can not be done in Finland - very expensive, but maybe Habib can show as some good lab in Europe, or maybe it can be done for reasonable price at his lab, no Salifert product involved this time )

Because we (poor hobbyist ) must pay that expensive tests by itself, we need to know for sure, that heavy metals are responsible for salt toxicity, so is there any other data for salts involved in this test, that may help to find possible relation between heavy metals and salt toxicity? Maybe Habib know some?

[rshimek]

Hi,

Keep in mind while you do all this testing of metals concentration, it won't tell squat about toxicity.

You need actually to do experiments where you kill some animals (as I just did).

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.

If it was the metals, propose another hypothesis, and test it.

[eAlex]

Thank you for fast response!

I will not waste my money on testing of metals concentration, if it won't tell me squat about toxicity

I guess we can only hope that you or someone else will test salt brands available in Europe for toxicity, using living organisms placed in the waters.

[rshimek]

Originally posted by eAlex

Hi,

I guess we can only hope that you or someone else will test salt brands available in Europe for toxicity, using living organisms placed in the waters.

I won't be doing it, but it is not hard to do. I will be glad to discuss it with anybody who wants to. It does take access to gravid sea urchins (sold here as sources for embryological material), and a good microscope.

[Habib]

Ron,

Thanks for answering the above.

I don't doubt that there was a major difference between some of the salts used in your study.

The R.S.D. was high and there was no significant difference found between NSW and two of the saltmixes. I assume now, for the sake of simplicity, that in the 10 replicates the number of fertilized eggs were as an average about the same for the different water samples.

So it seems that in this particular set of experiments (conditions, organisms, etc ) two saltmixes performed just as good as NSW.

One of the salt mixes would have, if the that batch was representative and the data supplied by the manufacturer were correct, about 40 ppb of Cu but was just as good as NSW.

The experiment with 10 ppb of Cu added as cupric chloride gave very bad results.

Do you have an explanation for this discrepancy?



I know you did not aerate the beakers but were the prepared salt mixes aearated prior to use? If so how long were they aerated?


This is standard bioassay techiques as it is quite impossible to count all the embryos one by one. On the other hand when you mix a chemical solution, I suspect you don't count atoms, either.




Well actually I count them indirectly by a.o. weighing.

[Habib]

eAlex,

IO available in Europe is probably made in France and might be different from what Ron used.

Marine Enterprise's salt is available in at least some European countries.

IMO I would not use the Marine Environment salt.

But beware. My opinion could be biased because I do some business with Marine Enterprise. And before you ask, no I don't sell their salt.

[simonh]

Originally posted by Habib
IO available in Europe is probably made in France and might be different from what Ron used.

You mean apart from costing twice as much

[rshimek]

Originally posted by Habib

Hi Habib,

So it seems that in this particular set of experiments (conditions, organisms, etc ) two saltmixes performed just as good as NSW.

That was my conclusion as well.

Do you have an explanation for this discrepancy?

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.

I know you did not aerate the beakers but were the prepared salt mixes aearated prior to use? If so how long were they aerated?

All mixes and the NSW were mixed vigorously with a power head for an hour (by timer) in a bucket cleaned and rinsed between mixes. It is possible that the power head introduced something into the mixes, but if so, it was likely the same in all cases.

I would not use the Marine Environment salt.

I don't think I would either, actually, and it has to do with some of the interactions I had with the owner of the firm some years ago at a meeting.

[big red]

Excellent article and very informative.

Are there other organisms that could be used in the test as an indicator species other than the urchin? Is there a saltwater standard (freshwater = daphnia) for tox studies? I'm asking out of curiosity and not from a perceived gap in the study.

All mixes and the NSW were mixed vigorously with a power head for an hour (by timer) in a bucket cleaned and rinsed between mixes. It is possible that the power head introduced something into the mixes, but if so, it was likely the same in all cases.

Did you use RO water to make all of your fresh salt mixes? I assume so but did not catch it in the article.

Thanks for the time and energy!

[rshimek]

Originally posted by big red

Hi,

Are there other organisms that could be used in the test as an indicator species other than the urchin?

Yes, all sorts. For 10-14 day studies, polychaete worms could be used. Also amphipods, and bacterial tests are available.

Is there a saltwater standard (freshwater = daphnia) for tox studies?

Yes, generally there is an urchin test of some sort, either larval survival as I have done, or sperm motility or fertilization success. These are tests for acute problems. This type of test is often coupled with a longer term test with polychaetes or amphipods to test some chronic problems. Finally bacterial tests such as the "Microtox" test panel are used to check mutagenicity.

Did you use RO water to make all of your fresh salt mixes? I assume so but did not catch it in the article.

I used store bought distilled water.

[Habib]

Ron,

I came across the following which appear, at the first glance, not in favor of your findings. I would appreciate if you could comment on them TIA.

The first one is from:
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

Rationale
A recently-published study (Jonczyk et al., 2001) has shown that, using Environment Canada ˜ s
echinoid fertilization assay (EC, 1992), the sublethal toxicity of a number of samples of industrial
effluent did not differ when their salinity was adjusted by either the direct addition of dry salt or
hypersaline brine (HSB) to the samples. This study also showed that sample toxicity was similar
when either artificial seawater or natural seawater was used as the control/dilution water in the test.
These findings, together with a desire to enable testing of the sublethal toxicity of effluent samples at
concentrations up to and including 100% (i.e., undiluted effluent), has resulted in a revisiting of
Environment Canada = s existing guidance for adjusting salinity for marine sublethal toxicity tests
performed under Environmental Effects Monitoring (EEM) programs (EC, 1997).



Following are revised procedures for salinity adjustment, now recommended by Environment Canada
for this purpose. These revised procedures offer additional options for adjusting the salinity of
effluent samples as well as the associated control/dilution water used for the sublethal toxicity test.
Depending on the options chosen, sample toxicity may now be determined at concentrations up to
and including 100%.1 Also, these revised procedures now offer the option of using artificial seawater
(prepared using artificial HSB or by the addition of dry salt directly to deionized or other water) as
the control/dilution water.2

Included here is the reamrk #2:
2 The earlier guidance (EC, 1997) recommended that HSB should be prepared using a source of
uncontaminated natural seawater, and that this (natural) HSB should be used both as the control/dilution water and for
adjusting sample salinity.

[Habib]

From the same report:

Commercially-available mixtures of dry sea salts include products marketed in aquarium supply stores under
trade names such as Instant Ocean TM , Forty Fathoms TM , and HW Marinemix TM (EC, 1992; Neiheisel and Young, 1992;
USEPA, 1994, 1995; Jonczyk et al., 2001). 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. 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).

[Habib]

Here is the abstract of the Jonczyk's 2001 paper:

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

Environ Toxicol Chem • Volume 20 • Issue 4




Abstract
Most industrial effluents discharged into the marine coastal environment are freshwater in nature and therefore require manipulation prior to testing with marine organisms. The sea urchin fertilization test is a common marine bioassay used for routine environmental monitoring, investigative evaluations, and/or regulatory testing of effluents and sediment pore waters. The existing Canadian and U.S. Environmental Protection Agencies test procedures using sea urchin (and sand dollar) gametes allow for sample salinity adjustment using either brine or dry salts. 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. At present, it is unclear to what extent these variables affect test results for whole effluents. The test methods simply state that there are no data available and that the use of artificial dry sea salts should be considered provisional. We conducted a series of concurrent experiments aimed at comparing the two different treatments of sample salinity adjustment and the use of natural versus synthetic seawater in order to test these assumptions and evaluate effects on the estimated end points generated by the sea urchin fertilization sublethal toxicity test. Results from these experiments indicated that there is no significant difference in test end points when dry salts or brine are used for sample salinity adjustment. Similarly, results obtained from parallel (split-sample) industrial effluent tests with natural and artificial seawater suggest that both dilution waters produce similar test results. However, data obtained from concurrent tests with the reference toxicant, copper sulfate, showed higher variability and greater sensitivity when using natural seawater as control/dilution water.


But in you latest test results there was not a direct relationship with copper.

[Habib]

Here is one more but I don't know which ASW they did use. The full article might tell us more.

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 • Volume 21 • Issue 4



Abstract
Preset larval Mercenaria mercenaria were exposed to nominal concentrations of 1 (control) to 495 microg Cu/L in artificial seawater and monitored for mortality, activity, development, and metamorphosis in sealed 30-mm plastic petri plates containing 1.5 ml of artificial seawater or toxicant solution. The plastic petri plates sorbed only about 2.6 microg/L at any dose and allowed direct observation of larval clams under a light microscope for a period of two weeks; control survivorship was in excess of 60% at 400 h. The dose-response curve for mortality for clams exposed to copper and fed Isochrysis galbana was characterized by survival similar to or better than controls at doses of 5 and 14 microg Cu/L, while doses of 7 and > or = 29 microg Cu/L exhibited mortality greater than controls. Values of lowest concentration at which 50% of the organisms died (LC50) were 62.4, 21.2, and 11.7 microg Cu/L, and the lowest observed adverse effect concentration values of 57, 29, and 29 microg Cu/L were determined at 48, 96, and 192 h, respectively. In contrast, activity, as judged by swimming, exhibited a typical exponentially decreasing response at these same concentrations. Experiments on the uptake of dissolved copper by I. galbana confirmed literature reports that these algae concentrate copper. Ingesting copper-containing algae was demonstrated to be a source of copper toxicity for larval clams

[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 • Volume 21 • 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.