Affect of road salt runoff on C. Vullgaris and D. Pulex

[AquaFish]

Hi,
I am doing a project on The Affect of road salt runoff on freshwater environments and have run into a few problems that I have been unable to solve. The first is the level of salinity I should use. I am thinking of going from 0 (control) to around 30000ppm (end limit of brackish water). I have not been able to find levels of salinity caused by road salt use, but this way you could use C. Vulgaris and D.pulex as a bio-indicator species. The second is how to culture daphnia and Chlorella. I have read some articles and they give conflicting information. Are they hard species to culture? I would need either a stable or rising population in my controls. Also, I am using a conductivity meter for testing salinity. Is this the optimal way?

Thanks,
AquaFish

[SciB]

Hi AquaFish,

That’s an appropriate topic for this time of year when winter snows will be coming soon to parts of the U.S. and Canada. Chlorella is a common alga and easy to grow. You just have to provide it with some minimal nutrients and light so it can photosynthesize. It is also a good idea to aerate the cultures with an aquarium air pump but this is not absolutely necessary. Here’s a youtube video that has some good general instructions on growing and harvesting algae in a high school lab: http://www.youtube.com/watch?v=iWyZlo1FWBg

If you buy Daphnia from Carolina Biologicals they include culture instructions. You can also buy the growth medium from them. http://www.carolina.com/catalog/search- ... SearchForm

You can also buy C. vulgaris and culture instructions from Carolina Biologicals: http://www.carolina.com/catalog/search- ... SearchForm

As to the amount of salt in a freshwater pond from road salt runoff, I really don’t have any idea what it might be in an actual environmental situation. One thing I would suggest is to try to get the actual salt compound used on roads. When you add this to your algae you’ll know they are being exposed to the same stuff as they would be in a pond near a highway. Otherwise, rock salt like what we used to use in ice cream freezers should be close enough. Since you are adding salts by weight to a known volume of water you can calculate the concentration yourself. If you have a salinity meter you can use it to check your concentrations.

I did a search for growing Chlorella in brackish water (0.5 to 30 ppt salinity) and discovered that there are some species of Chlorella that can tolerate higher concentrations of salt: http://www.google.com/url?sa=t&rct=j&q= ... XY&cad=rja

The paper is rather technical, but it does have some useful information about the ingredients in the algae culture medium and the growth curves of the algae under different salt concentrations. The authors isolated algae from a brackish pond and they apparently did not identify the individual species, so I don’t know how salt tolerant C. vulgaris would be.

The range you proposed, 0 to 30 ppt covers the salinity from no salt to the saltiness of the ocean. I would think contamination from road salt would not be higher than 30 ppt.

I hope this info is helpful. Post again with more specific questions and we’ll try to help you in planning your experiments.

Good luck!

Sybee

[AquaFish]

Thanks Sybee. I believe I have two options in my experimentation. I could have multiple containers of Daphnia/Chlorella and have each at a different level of salinity (i.e. Container 1: 0 ppt Container 2: 5 ppt etc.). My second option is to have one container (but multiples of this one container for greater reliability of data) and slowly raise the salt concentration until the population decreases to half its prior concentration. Speaking of population, I am still shaky about how to count the Daphnia. I have a few ideas for the algae but not many for the daphnia. For the algae, I read in a book that you make a grid below a slide and count the number of cells in a few of the boxes and extrapolate the number of cells from there. I believe this is called a hemocytometer. I am also thinking of getting Evans Blue so that I can get a percentage of non-viable cells. I am not sure if I should as 10 grams is around $40 . If this would be enough for a lot of tests this would be fine, but 10 grams doesn't seem like a lot. About the daphnia, I suppose I could do the same, but I do not have any daphnia, so I am not sure if they are small enough to do this. Also, they move around, so that would make counting even harder unless if I got a microscope camera adapter.
In regard to the type of salt that I will be using, the kind of road salt that the local place will be used in my experiment. What is the formula for salinity by weight of salt? Also, what intervals would you suggest for the levels of salt? Should I go by 5ppt? About the hemocytometer, here is a video that I found about it. https://www.youtube.com/watch?v=69T755- ... -9ATEPOaPg

AquaFish

[SciB]

Thinking about your two experimental options, my suggestion would be to have one salt concentration per culture and to run three cultures with the same salt concentration so you will be able to get an average and do statistical tests on your data. Scientists depend on statistical analysis to prove or disprove their hypotheses and if you do this it will really strengthen your data. Remember that the algae grow and divide over time so if you are changing the salt concentration at different times then the cell numbers in the later cultures will be different from the initial ones. This would I think introduce an uncontrollable variable into your experiments. Your dependent variable is the change in cell numbers and the starting number has to be the same for each culture in order for you to be able to compare them.

I would start an experiment using one single culture split into as many tubes as you need to test all the salt concentrations at one go with three replicates for each concentration. So, for example, if you tested 0, 0.5, 5 and 10 ppt you would need 4 x 3 = 12 tubes of algae all from the same culture so the starting number of algae cells is the same in each tube.

As for counting cell numbers, the hemocytometer is the best way to do it. Can you borrow one of these from a lab? Most every lab that I know of that works with cells has several of these and someone can teach you how to use it. They would also have a 0.4% solution of trypan blue which is what most people use for counting viable cells by distinguishing live from dead cells (http://en.wikipedia.org/wiki/Trypan_blue). Since you are only counting the cells in a volume of about 12-15 microliters, you need very little dye. You could use Evans blue, but I would buy a solution rather than solid powder.

Why don’t you work on setting up the experiments with Chlorella first and then test Daphnia later if you have time. Daphnia are multicellular with actual organs and a reproductive cycle--much more complex than Chlorella. I would think that the effects of higher salt levels on their physiology would not be so easy to measure. I don’t think straight counting of numbers would work. You would have to do some more reading on Daphnia to find out what other scientists measure.

I hope this information is helpful. Do let us know when you have more questions.

Good luck!

Sybee

[deleted-242961]

This is a great study you are performing. I have completed similar experiments for different materials including salt. My suggestion is to use a log scale of salt concentrations 1,2,4,8,16,32 g/L.

good luck
Matthew

[AquaFish]

Hello again,
I have hit another problem in my experimentation. I purchased my algae at ( http://www.carolina.com/algae/chlorella ... %2C+living ) and put in a Erlenmyer flask with a bubbler and a florescent aquarium bulb above it. It is not growing and I suppose it is because I did not get a growth medium. I could not find out if it would mess up my testing. Do you know of any growth mediums that would not effect my testing?

Thanks,
Aquafish

[AquaFish]

And just so you know, the due date is a typo. It is due around February.

[SciB]

The Chlorella algae will not grow unless you supply it with nutrients as well as light. I suggest that you buy the growth medium from Carolina Bio and grow your algae with that. It does contain a small amount of salts (get the composition from Carolina Bio) but the amount of salt you will be adding to imitate road-salt runoff should be much greater. Also, all the algae cultures will be grown in the same medium so any differences have to be due to what you add.

Try this and let us know what happens.

Good luck!

Sybee

[AquaFish]

Thanks a lot! I will try it.

[24601]

Hi,
I locked myself out of the other account so I made another one . I just wanted to show you the results.


Initial Measure 1 Measure 2 Measure 3 Measure 4 Measure 5 Measure 6 Measure 7 Measure 8 Measure 9 Measure 10
Day/Time 2/3/2015 19:45 2/4/2015 20:33 2/5/2015 15:30 2/6/2015 17:00 2/7/2015 14:40 2/8/2015 11:00 2/9/2015 17:15 2/10/2015 19:25 2/11/2015 18:58 2/13/2015 17:30 2/14/2015 17:50
Days 0 1.033333333 1.822916667 2.885416667 3.788194444 4.635416667 5.895833333 6.986111111 7.967361111 9.90625 10.92013889
0 ppt
256 235 0 149 213 171 64 149 107 149 32
Bottle Two 107 171 405 48 59 213 192 128 107 43 37
Bottle Three 341 128 192 128 64 149 85 85 128 21 59
Average 235 178 199 108 112 178 114 121 114 71 43
Change 0 -24.24% -15.15% -53.79% -52.27% -24.24% -51.52% -48.48% -51.52% -69.70% -81.82%

Day/Time 2/3/2015 20:45 2/4/2015 20:44 2/5/2015 15:50 2/6/2015 17:15 2/7/2015 14:55 2/8/2015 11:08 2/9/2015 17:15 2/10/2015 19:26 2/11/2015 18:58 2/13/2015 17:30 2/14/2015 6:06
Days 0 0.999305556 1.795138889 2.854166667 3.756944444 4.599305556 5.854166667 6.945138889 7.925694444 9.864583333 10.38958333
5ppt
Bottle One 171 192 213 48 149 192 107 128 192 53 37
Bottle Two 171 171 128 128 85 107 235 235 448 69 37
Bottle Three 213 171 85 235 21 128 107 107 32 48 27
Average 185 178 142 137 85 142 149 156 224 57 34
Change 0 -3.85% -23.08% -25.96% -53.85% -23.08% -19.23% -15.38% 21.15% -69.23% -81.73%

Day/Time 2/3/2015 21:02 2/4/2015 20:56 2/5/2015 16:04 2/6/2015 17:36 2/7/2015 15:07 2/8/2015 11:32 2/9/2015 17:34 2/10/2015 19:47 2/11/2015 18:10 2/13/2015 17:50 2/14/2015 18:16
Days 0 0.995833333 1.793055556 2.856944444 3.753472222 4.604166667 5.855555556 6.947916667 7.880555556 9.866666667 10.88472222
10ppt
Bottle One 192 107 256 32 149 37 171 64 32 21 59
Bottle Two 171 149 128 59 235 149 256 85 21 32 16
Bottle Three 277 171 64 107 107 128 235 69 235 32 32
Average 213 142 149 66 164 105 220 73 96 28 36
Change 0 -33.33% -30.00% -69.17% -23.33% -50.83% 3.33% -65.83% -55.00% -86.67% -83.33%

Day/Time 2/3/2015 21:22 2/4/2015 21:07 2/5/2015 16:15 2/6/2015 17:47 2/7/2015 15:19 2/8/2015 11:18 2/9/2015 17:45 2/10/2015 19:53 2/11/2015 20:18 2/13/2015 17:58 2/14/2015 18:27
Days 0 0.989583333 1.786805556 2.850694444 3.747916667 4.580555556 5.849305556 6.938194444 7.955555556 9.858333333 10.87847222
15ppt
Bottle One 149 64 149 128 75 53 107 85 27 48 16
Bottle Two 448 128 64 128 213 48 128 128 32 16 37
Bottle Three 192 107 21 149 53 43 213 37 85 32 21
Average 263 100 78 135 114 48 149 84 48 32 25
Change 0 -62.16% -70.27% -48.65% -56.76% -81.76% -43.24% -68.24% -81.76% -87.84% -90.54%

Day/Time 2/4/2015 18:04 2/5/2015 16:38 2/6/2015 18:00 2/7/2015 15:32 2/8/2015 11:40 2/9/2015 17:53 2/10/2015 19:53 2/11/2015 20:27 2/13/2015 18:21 2/14/2015 18:33
Days 0 0.940277778 1.997222222 2.894444444 3.733333333 4.992361111 6.075694444 7.099305556 9.011805556 10.02013889
20ppt
Bottle One 171 171 171 48 107 107 149 107 48 21 103
Bottle Two 235 21 16 64 21 107 27 21 80 16 41
Bottle Three 192 192 53 171 43 128 64 149 32 21 95
Average 199 128 80 94 57 114 80 92 53 20
Change 0 -35.71% -59.82% -52.68% -71.43% -42.86% -59.82% -53.57% -73.21% -90.18%

Day/Time 2/4/2015 19:43 2/5/2015 16:50 2/6/2015 18:08 2/7/2015 15:45 2/8/2015 11:50 2/9/2015 18:07 2/10/2015 19:53 2/11/2015 20:35 2/13/2015 19:14 2/14/2015 18:41
Days 0 0.879861111 1.934027778 2.834722222 3.671527778 4.933333333 6.006944444 7.036111111 8.979861111 9.956944444
25ppt
Bottle One 256 32 69 37 16 21 69 21 27 16 57
Bottle Two 277 171 85 27 75 59 123 32 21 27 90
Bottle Three 171 75 85 75 21 53 32 27 37 16 59
Average 235 92 80 46 37 44 75 27 28 20
Change 0 -60.61% -65.91% -80.30% -84.09% -81.06% -68.18% -88.64% -87.88% -91.67%

Day/Time 2/4/2015 20:11 2/5/2015 17:00 2/6/2015 18:24 2/7/2015 16:00 2/8/2015 12:04 2/9/2015 18:47 2/10/2015 20:13 2/11/2015 20:50 2/13/2015 19:27 2/14/2015 18:49
Days 0 0.867361111 1.925694444 2.825694444 3.661805556 4.941666667 6.001388889 7.027083333 8.969444444 9.943055556
30ppt 111
Bottle One 213 128 69 101 59 363 96 21 53 5 74
Bottle Two 213 80 48 59 37 59 37 21 171 16 71
Bottle Three 341 48 43 53 43 75 16 43 32 16
Average 256 85 53 71 46 165 50 28 85 12
Change 0 -66.67% -79.17% -72.22% -81.94% -35.42% -80.56% -88.89% -66.67% -95.14%

[24601]

I hope I am not breaking any forum rules

[SciB]

Hi and thanks for sending us your data so we could see how the Chlorella experiment turned out.

Let me see if i got what you did ok. You started a culture of Chlorella algae in water containing some algae nutrients then split it up into a bunch of separate bottles to which you added either no runoff water or various amounts to groups of three bottles for each 'dose'. Am I right so far?

I had suggested using a hemocytometer to count the number of algae cells in each culture. Is that what you did? What are the numbers that you report for each bottle? They should be number of cells for a particular volume of culture, usually one milliliter.

The measurements for each bottle on successive days seem to change erratically--up one day down the next, up, down, without any obvious reason. The overall trend seems to show a decrease from the first day to the last. Did you plot the measurements over time? You could use Excel or whatever program you like to plot your data for each bottle over time and try to fit a curve to the points. I am sure some of the readings are outliers that can be discarded with statistical certainty. Excel has built-in curve-fitting modules and you can learn how to use them by finding an appropriate Excel tutorial on youtube.

How were you planning to show the data? As a comparison of the changes with various ppt runoff on a certain day? I really think you need to plot the data and try to get a reasonable curve fitted to it. If you can get a straight line then Excel can calculate the slope of the line for you and you can use that to compare treatments.

You did a lot of work on this project. Congratulations! Now you need to process the data in some way that allows you to do a meaningful comparison. Let us know if you have more questions.

Good luck!

Sybee