Re: How much of a substance do I use to test on Daphnia?

Hi Jo. Thanks for your question. You asked: "How much of a substance do I use to test on Daphnia? I am doing a science fair project on the effect of various substances on the heart rate of daphnia. I am testing ethyl alcohol, mineral oil, glucose, aspirin, and acetylcholine. How much should I use on the daphnia? I've tried to find dosage information for daphnia but can't find anything."

The reason you’re unable to find information for Daphnia is because the amount of any substance you want to test on your Daphnia will depend on the volume of water your Daphnia are in. Also, the information you want is the concentrations, not the dosages. A dose is the amount of a substance per unit of body weight, such as milligrams per kilogram (mg/kg). A concentration is the amount of a substance per a given volume of solution, such as milligrams per milliliter (mg/mL). Since you will be dumping your substances into a volume of water which houses your Daphnia, you will be working with concentrations. Now, you will need to know the amount of water that will house each Daphnia group and you will need to determine, by performing experiments, the concentrations of your substances that you will get when you add them to the water. I’ll help you with that after this important note:

AN IMPORTANT THING TO KNOW ABOUT ASPIRIN: Aspirin (ASA) does not dissolve in plain old water. ASA has a pH of around 5.0 and it, which we take orally, is designed to be processed by our stomach, which has a pH between 1-3. In order to get ASA to dissolve in water, we need to knock a hydrogen of its uncharged carboxyl group (this is what helps ASA to get absorbed by the stomach) to make the whole complex ionized (charged). OK, so how do we do that?? Increase the pH of the water, making it more alkaline! You could do this by adding some baking soda to the water. Notice that once you start adding other things to the water, though, you introduce other variables. You wouldn’t know if the results you’d see would be from the ASA, OR from the more alkaline water, OR from something else in the baking soda... I also spoke with a chemist at Sigma-Aldrich (www.sigma-aldrich.com) to see how water-soluble acetylcholine is, and their information says that it’s soluble in water at 100mg/mL.

Ignoring the fact that you will have to increase the pH of the water to work with ASA, let’s use it anyway as our example for determining how to set up various concentrations:

If your Daphnia are living in a bowl containing 500mL water and you dissolve one 81mg tablet of ASA (“baby aspirin”) into it, your resulting concentration, in mg/mL, will be 81mg/500mL, or 0.16mg/mL ASA. <--(This means that for every 1mL of water in your tank, there is 0.16mg ASA). You could start with this concentration and see if you notice any changes in your Daphnia.

If your Daphnia all die, you may want to add water to dilute their environment in order to obtain physiological changes without killing them. For example, you could add another 500mL water, doubling the volume. The ASA concentration would then come out to be 81mg/1000mL, or 0.08mg/mL ASA. ***Notice that you can also achieve this same concentration by keeping the volume of water at 500mL but only adding half of the 81mg tablet (40.5mg/500mL = 0.08mg/mL ASA).

Likewise, if you’re seeing nothing at these concentrations, you could go the other direction and try a more concentrated solution to see if anything happens. For example, you could use 250mL water and then add your 81mg ASA, giving you a concentration of 0.32mg/mL ASA solution. *** Again, you can also achieve this same concentration by keeping the volume of water at 500mL but add TWO 81mg tablets (162mg/500mL = 0.32mg/mL ASA).

You will probably have to try all these concentrations, and maybe more, for EACH substance you plan on looking at to determine the most practical concentration to use before you can even begin your experiment. Write everything down as you go so that once you find the best concentrations for ethyl alcohol (EtOH), mineral oil (MO), glucose (Glu), aspirin (ASA), and acetylcholine (Ach), you can re-create the most optimal one of each later when you’re ready to perform your main experiment. It would also more accurate and more scientific if you could keep all the volumes of water the same for every experimental group to avoid introducing other variables (such as temperature or pressure changes) that may occur if one group was in 800mL of water while another group was in only 20mL of water.

I would set up a working table like this:

mL H2O + Daphnia_______________EtOH_____MO______Glu_____ASA_____Ach

500mL + 10 Daphnia each_________100mL____100mL____10g_____162mg____100mg

500mL + 10 Daphnia each_________50mL_____50mL_____5g______81mg_____50mg

500mL + 10 Daphnia each_________25mL_____25mL_____2.5g____40.5mg____25mg

500mL + 10 Daphnia each_________12.5mL___12.5mL___1.25g____20.25mg___12.5mg

Notice how the top row starts with the most concentrated solutions of each substance and halves its way down until we get to the last row, the least concentrated solutions (you will also need a control group of Daphnia with nothing added to their water). Unfortunately, in order to carry out this table of experiments, and you will need to perform the 20 little experiments the table contains (5 different substances x 4 different test concentrations each), and you will use 200 more Daphnia (10 each x 20 experiments), plus the control group.

The coolest thing about setting up your concentration experiments like how I have it above is that you should see some really great trends in the Daphnia as the concentrations decrease. For example, let's look at ethyl alcohol (EtOH): at the most concentrated level (100mL), you might see that 60% of your Daphnia died and the rest had extremely low heart rates. At the 50mL level, you might see that all your Daphnia lived but all have slow hearts rates. At the 25mL level, you may see that all their heart rates are a little slow but almost normal. At the 12.5mL level, they may be normal and completely identical to your control Daphnia. You could then plot a graph of heart rate versus concentration and get some sort of inverse linear or curved response curve, and you would do this for each of your substances. YAY!

The thoroughness is totally worth it. This is how science is done. This is what I do when I'm in the lab and I need to determine concentrations for my next experiments. At the end, you will know exactly the best concentrations for each of your substances because you will have obtained this knowledge through true experimentation, not through guessing. I think your science fair would be very impressed to see the results/graphs of your test experiments as well. It shows the amount of work it took for you to finally get to your main experiment, and it shows that you settled on your final concentrations based on your preliminary experimental data. :)

Best of luck! Jo, feel free to email me at monreal.1@osu.edu if you come up with more questions as you go!

Sincerely,

G. Monreal