ocean acidification on crab shells

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ocean acidification on crab shells

Postby surferg1 » Tue Oct 01, 2013 8:14 am

I want to do an experiment on crab shells from the Gulf of Mexico to see the effects of ocean acidification. I have seen the UNC study and the Cheasapeake Bay study that crabs grow giant when exposed to lower ph levels, unlike other invertabrates whose shells disinegrate. I only have about 3 weeks, do you know I way that I could test the shells in that amount of time?
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Re: ocean acidification on crab shells

Postby SciB » Tue Oct 01, 2013 5:19 pm

Hi SurferG,

In three weeks? That's a tough one. You mentioned a possible effect of pH on the animal's size. Is this because they undergo more molts to make a bigger shell? The size would depend on the age of the crab and I don't see how you could compare sizes unless you had some way to determine the age.

I think your best plan would be to purchase a variety of crabs from as many different locations--Chesapeake Bay, Gulf of Mexico, Atlantic, Pacific, Arctic and deep water and test their shells for calcium content relative to chitin. I looked for a method to do this, but found only one paper that mentions using citric acid to extract calcium (http://www.ncbi.nlm.nih.gov/pubmed/22108289) and a patent for a method to extract chitin from crab shells (http://ip.com/patfam/en/40571043).

You could try extracting calcium from a piece of crab shell with citric acid, which you can buy in the canning section of the supermarket, or with vinegar. If all the minerals are extracted by the acid, what you are left with is primarily chitin. You could weigh a piece of shell, extract with acid then weigh again. The difference between the two weights would be the mineral content of the shell. I know the mineral content of clam shells is changed by the presence of excess CO2 in the ocean, and since crab shells also contain calcium carbonate, I would expect their shells to also reflect changes in CO2, pH and bicarbonate levels relative to carbonate in seawater.

Let us know what your ideas are for crab shell vs pH experiments and we will help you get your project underway.

Best regards,

Sybee
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Re: ocean acidification on crab shells

Postby surferg1 » Tue Oct 01, 2013 6:27 pm

wow, thanks for that. do you think that if I modified the "Swimming in Acid" experiment, it would also work? I would just measure the weight of the crushed crab shells before and after immersing them in a ocean & vinegar solution of around 6.5 ph? I don't think I understand the citric acid and calcium and chtitin measuring, what does that prove? Maybe that's better than what I was thinking. I'm now a little confused.
I also have more time than I thought, a little over 4 weeks for the experiment.And, I'll use either blue crabs or stone crabs.Thanks so much.
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Re: ocean acidification on crab shells

Postby SciB » Wed Oct 02, 2013 9:37 am

Hi,

Crab shells are made up mostly of a biopolymer called chitin--basically a long chain of sugar subunits--plus some protein and calcium. If you dissolve away the calcium by soaking the crab shell in acid--either citric acid or vinegar (acetic acid)--what you are left with is chitin. If you weigh the shell before you soak it then weigh it again afterwards, the difference is approximately equal to the amount of calcium in the shell, assuming the acid treatment dissolves all of it.

My hypothesis is that the pH of the ocean in which the crab grew up will determine how much calcium is in its shell. In order to test the hypothesis, you need a way to determine how much calcium is in a crab shell. One way to do that is using the acid treatment described above. The problem with this is that it is difficult or impossible to know the pH of the water where the crab was caught, and to be able to see differences you would need to have crabs from oceans that had different pH's. That's why i said to try and get crabs from as many different oceans, bays, etc as you could.

If you just want to do the Swimming in Acid demonstration, that's fine. Do you have an electronic scale that is accurate to milligrams and a pH meter? I would do several pH's--5, 6, 7 and 7.5 for example, so you could plot the change in weight vs pH and show that the lower the pH, the greater the weight lost. Remember that the pH scale is logarithmic so each point change means a 10-fold change in H+ in the water. If the ocean pH dropped as low as 5 I think everything in it would die.

If you need help with the details, let us know.

Good luck!

Sybee
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Re: ocean acidification on crab shells

Postby surferg1 » Wed Oct 02, 2013 5:52 pm

Ok. I'm getting the crabs out of the gulf of mexico, so i can definately use the water the crab grew in as my control. How long should I run the experiment? How long should I keep the crab shells in the lower pH solutions before I get a reliable result? Should I crush the shells into tiny peices or leave them in big pieces?
I'm trying to find a reason why the crabs turn into giants while other invertabrates' shells disentigrate (or become weaker). Is it the crab growing bigger inside the shell or is the shell gowing bigger or stronger too?
You are being so helpful! Thanks!
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Re: ocean acidification on crab shells

Postby SciB » Wed Oct 02, 2013 7:05 pm

Hi! I'm happy to help. It's a very interesting subject and one of real importance because if we mess up the oceans, we could kill the phytoplankton that produce our oxygen supply!

I read the article in Chesapeake Quarterly about crabs getting larger in tanks through which CO2 was bubbled to lower the pH, but i don't understand why they do. Remember crabs have to molt their shells and rebuild them to get bigger, so maybe they are molting more often. The researchers said crabs can maintain their internal pH at a normal level, but they still have to use carbonate from the water to rebuild their shells after molting and the CO2 converts it to bicarbonate which they can't use [??]. Need to read more about it...

Anyway, i would follow the Swimming in Acid project and grind the shells into bits. Just be sure you clean the shells REALLY well before you grind them, because if there are any bits of tissue left on the shells, bacteria or other microbes could grow in the solution and that would be bad for your experiments!

I really don't know how long the shells have to be soaked in the pH solutions. The project says one month, so i would try to let it go that long if you have time, otherwise just do the longest you can. Keep the eggshell solutions covered and inside where the temperature is fairly constant away from sunlight.

To make your experiments more interesting, you can use several different pH's as i suggested before, just be sure to include the one that is closest to the prediction for the ocean pH in 2020 or 2050 or by the end of this century if CO2 levels continue to rise at the current rate. Another thing you might do that i just thought of is to take pieces of shell from the claws and compare their calcium content with that of pieces of shell from the top of the crab. I read that different parts of a crab or lobster have different amounts of calcium and therefore may lose calcium at different rates.

Good luck! Hope this helps. Use your phone to take pix of the crabs and your work as you proceed with the experiment as a visual record of what you did. And be sure to write down everything in your lab notebook.

Keep posting to let us know how it is going.

Best wishes,

Sybee
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Re: ocean acidification on crab shells

Postby heatherL » Thu Oct 03, 2013 6:56 pm

Hi surferg1,

You have already gotten some great advice from the other SciB. I just wanted to give you a clue that might help you understand why crabs react differently to low pH than molluscs do. Mollusc shells disintegrate in acid because they are made of calcium carbonate, and hydrogen ions tend to bond with the carbonate ion (pulling it out of the shell and into solution). Crabs are arthropods, and their shells are made of a substance called chiton. It is made of entirely different molecules, which is a likely reason that crab shells react differently in acidic environments.

I hope this helps as you continue working on your project. Please keep us posted with your progress!

Heather
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Re: ocean acidification on crab shells

Postby SciB » Fri Oct 04, 2013 4:42 am

Hi,
While it is true that mollusc shells do not contain chitin, it does not necessarily follow that this is the reason for the apparent size increase in crabs exposed to lower pH. Please read my previous posts. Crab shells contain BOTH calcium and chitin. Here is a reference from PubMed:

PLoS One. 2013 Apr 4;8(4):e60959. doi: 10.1371/journal.pone.0060959. Print 2013.
Effects of ocean acidification on juvenile red king crab (Paralithodes camtschaticus) and Tanner crab (Chionoecetes bairdi) growth, condition, calcification, and survival.
Long WC, Swiney KM, Harris C, Page HN, Foy RJ.
Source

Kodiak Laboratory, Resource Assessment and Conservation Engineering Division, Alaska Fisheries Science Center, National Marine Fisheries Service, NOAA, Kodiak, Alaska, United States of America. chris.long@noaa.gov
Abstract

Ocean acidification, a decrease in the pH in marine waters associated with rising atmospheric CO2 levels, is a serious threat to marine ecosystems. In this paper, we determine the effects of long-term exposure to near-future levels of ocean acidification on the growth, condition, calcification, and survival of juvenile red king crabs, Paralithodes camtschaticus, and Tanner crabs, Chionoecetes bairdi. Juveniles were reared in individual containers for nearly 200 days in flowing control (pH 8.0), pH 7.8, and pH 7.5 seawater at ambient temperatures (range 4.4-11.9 °C). In both species, survival decreased with pH, with 100% mortality of red king crabs occurring after 95 days in pH 7.5 water. Though the morphology of neither species was affected by acidification, both species grew slower in acidified water. At the end of the experiment, calcium concentration was measured in each crab and the dry mass and condition index of each crab were determined. Ocean acidification did not affect the calcium content of red king crab but did decrease the condition index, while it had the opposite effect on Tanner crabs, decreasing calcium content but leaving the condition index unchanged. This suggests that red king crab may be able to maintain calcification rates, but at a high energetic cost. The decrease in survival and growth of each species is likely to have a serious negative effect on their populations in the absence of evolutionary adaptation or acclimatization over the coming decades.


Your project involves exposing ground up shells to pH solutions in the range of 5-8 and measuring a change in weight after soaking them and drying them again. This soaking will dissolve calcium in the shells, not chitin. There could be a change in the chitin because of the lower pH but your weight measurement would not detect it. The change in size that the researchers observed in their lab experiments could be a result of altered calcification, some effect on chitin, a combination of these factors or something else entirely. So, be very careful what you conclude from your experiment.

I hope this clarification helps,

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Re: ocean acidification on crab shells

Postby surferg1 » Sat Oct 05, 2013 3:13 pm

I am going to follow the procedures in the Swimming with Acid experiment, but use blue crabs from the gulf of mexico for the shells and the water they were caught in for the control and to make the pH altered solutions.
how much liquid should I use in the glass container, and can it be plastic instead of glass?
Thank you so much!
Last edited by surferg1 on Sun Oct 06, 2013 5:52 pm, edited 1 time in total.
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Re: ocean acidification on crab shells

Postby surferg1 » Sun Oct 06, 2013 5:51 pm

and.i'm having trouble with my hypothesis. i'm trying to prove that crabs will not have the same results as shells/mussels based on the fact that crabs grow larger in lower pH, higher CO2 ( according to some studies).so should i say that if i put crabs shells in a lower than sea water pH solution there will be no change in their weight? or will they weigh more?
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Re: ocean acidification on crab shells

Postby heatherL » Sun Oct 06, 2013 8:23 pm

Hi surferg1,

Let me answer your questions one by one.

1) Using the water in which the crabs were caught is a great control. Make sure to measure the pH of that water!

2) You need to use enough liquid to completely cover the shells, and keep it the same for every treatment. The exact amount does not matter, as long as you follow those criteria.

3) Glass would be a better bet than plastic, to ensure there is no interaction between the container and the liquid (especially at extremely low or high pH levels).

4) I am assuming you will be using the crab shells and not live crabs. (You may run into issues with animal use if you try to use live crabs.) Because it is likely that crabs get bigger due to growth, which requires the crab to be alive, it is probably safer to expect no change in the weight of the shell rather than an increase. But keep in mind that your hypothesis does not have to be supported by your data, as long as it makes sense going into the project. In other words, it's okay if your hypothesis is disproved. Just make sure your prediction agrees with the background information you know.

Best,
Heather
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Re: ocean acidification on crab shells

Postby surferg1 » Mon Nov 11, 2013 7:52 pm

I've finished the experiment but I'm really not sure what to do with the results. Each sample lost relatively the same amount of mass after 30 days, regardless of the pH of the solution it was soaking in. Each sample started with 28grams of crushed crab shell and ended with approx 23 grams. ( The samples from the 6.5 pH solution averaged 22.6g; the samples from the 7.0 pH solution averaged 23.3g; the samples from the control sample of 7.5 pH averaged 23.4g)I have to assume that a negligable amount of mass could have been lost in the sieve, but I didn't see any, I was extremely careful, so I doubt it.

Not that it was part of the original experiment, but as part of my observations I remeasured the pH of each sample and they each went down about .5 from their starting pH.
And, the shell were fizzing like carbonation sometimes throughout the experiment- which I think could be CO2 being released as the shells decayed?
Also,the water in all of the jars smelled horrible!
So basically what did I prove, anything? And are there any practical applications for this experiment or it's outcome?
Help!Please!
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Re: ocean acidification on crab shells

Postby JMP » Wed Nov 13, 2013 7:45 am

Hi surferg1,

I think you have a number of things you can say about your experiment. For one thing, although you are correct that your final values are quite close (and possibly within normal experimental error), you can mention that you observed a small trend of greater loss of weight in the more acidic samples. Then I would consider and mention some possible explanations for this loss of weight (in all samples), and why you didn't see more of a difference between your samples. This easily transitions into what you would do differently next time. Some obvious options that occur to me are: 1) a wider range of pH (i.e. down to 5.5 and up to 8.5), 2) a longer amount of time in the acid (I'm not sure how long you left them in so it might have been plenty long, but I would think a minimum of a month would be necessary). See if you can come up with any others.

Another thing you might consider is that the normal pH of seawater is 8.5 to 7.5, so all of your samples were somewhat acidified compared to normal seawater, which might explain why all of the shells lost a fair amount of weight.

As for your other observations (fizzing, smell), those are great observations to mention in your report.

Finally, you should re-read the the introduction/background to the Swimming in Acid project for some ideas on real world applications.

Hope this helps!
JMP
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Re: ocean acidification on crab shells

Postby surferg1 » Wed Nov 13, 2013 10:47 am

Thanks jmp.
For clarification, the samples were left in the water for 31 days and the 7.5 pH was the control sample of the Gulf of Mexico water that the crabs actually came from. So let me know if this changes anything in your mind.
I'm not sure why the shells lost any mass, because they shouldn't have broken down the same way the mollusks do.That is contrary to what the U of NC Chesapeake bay studies about crabs getting giant in high co2, low pH waters proved (I think)
and, why the same loss in every sample, especially the control from their "home" water. i really dont have an explanation for the loss or the differences.

I appreciate any help with anything from anyone so much.
Last edited by surferg1 on Wed Nov 13, 2013 7:55 pm, edited 1 time in total.
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Re: ocean acidification on crab shells

Postby SciB » Wed Nov 13, 2013 7:48 pm

Hi surfer-g1,

I would just point out one thing you could talk about in your experimental discussion and that would be the difference between a shell from a dead crab and a shell still attached to a living crab. Also, the fact that you increased the surface area by crushing the shell would certainly make a difference in the effect of acid on it. The sea is a very complex system, and it is dynamic, meaning it is changing all the time. You separated out one factor--the pH of the water--to study and that is the way scientists do model complex systems. Putting all the facts and observations back together again is the real challenge.

Report the data as you found it and explain the results in terms of your artificial system, but point out the ways it differs from real life and how this might change the results.

If you have any questions about the presentation, discussion or conclusions just send us another post and we will be happy to help.

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