176 The american biology Teacher volume 72, no. 3, march 2010Ab s t r A c tStudents commonly test the effects of chemical agents on the heart rate of the crusta-cean Daphnia magna, but the procedure has never been optimized. We determined the effects of three concentrations of ethanol, nicotine, and caffeine and of a control solution on heart rate in Daphnia. Ethanol at 5% and 10% (v/v) reduced mean heart rate to ~50% and ~20% of its initial value, respectively. Recovery was rapid after removing 5% ethanol, but recovery from 10% ethanol took 20–30 minutes. Nicotine at 100 μM revers-ibly increased mean heart rate by ~20%. Higher concentrations produced varied and sometimes irreversible effects. Caffeine at 0.1%, 0.5%, and 2% (w/v) had no convincing effect on heart rate. Of the three agents tested, nicotine’s peculiar effects make it the least useful in an educational setting. Caffeine could be used to emphasize the need for blind observers because it does not increase heart rate in Daphnia. If students find that it does, their bias is revealed. Ethanol produces unambiguous effects at 5% and 10%. Heart rates recover quickly after removing 5% ethanol, which allows students to explore reversibility as an alternative to having a separate control group.Key Words: Heart; caffeine; nicotine; ethanol; Daphnia.Daphnia are small crustaceans that are inexpen-sive, easy to care for, and transparent. The latter property allows students to easily observe the beating heart (Figure 1). Students commonly investigate the effects of chemical agents on heart rate in Daphnia. Protocols are available in published laboratory manuals (see references in Gray, 1996). More can be found on the Internet. A kit for the lab is sold by Ward’s Natural Sci-ence. Results of the exercise are often inconsis-tent and unanticipated. Gray (1996) stated “that even if data were collected from all biology sec-tions, results were still too inconsistent to draw definite conclusions…it was not unusual for nearly half of the animals tested to show increased heart rates in alcohol while the rest showed decreases.” Similarly, when this paper’s first author used Daphnia in a course on research design, he found that several commonly tested com-pounds failed to generate statistically significant effects on heart rate. What compounds and concentrations should be tested to produce con-sistent effects that can be distinguished from those of chance?Three commonly tested agents are nicotine, caffeine, and ethyl alcohol. Scholarly, peer-reviewed reports of how these compounds affect heart rate in Daphnia are scant. Baylor (1942) described effects of nicotine but provided no sample sizes or graphs. Campbell et al. (2004) found that 2 mM caffeine caused a slight initial increase in heart rate fol-lowed by a subsequent decrease starting 30 minutes later. Foster (1997) studied a range of caffeine concentrations but only determined heart rate immediately after the caffeine was applied. Sollmann and Webb (1941) briefly mentioned the effects of 1% ethanol. Clearly, the scholarly lit-erature provides little guidance as to what concentrations of these com-pounds should be tested and what effects to expect.Here, we report on the effects of ethanol, nicotine, and caffeine on heart rate in Daphnia. Each compound was tested at three concentrations. We determined their effects, the time necessary to achieve those effects, and whether or not the effects could be reversed. Doing so required a lengthy procedure and statistical analyses that are unsuitable for high school students. We do not recommend that teachers ask students to repeat our procedure. Our intent is to help teachers modify their existing protocols to make the most of the Daphnia heart rate lab.Materials & MethodsJJJDaphnia magna were shipped overnight from Ward’s Natural Science (Rochester, NY). They were maintained at room temperature in the medium in which they were shipped. Animals were fed Daphnia growth food daily (Ward’s cat-alog number 88W5950). Natural light provided a summer photoperiod. Feeding and overnight shipping are essential for long-term maintenance of Daphnia cultures. With standard shipping, cul-tures invariably die within a few days, even when they are fed. With overnight shipping, our cul-tures typically survived for 2 weeks.The procedure was based on that of Baylor (1942). A small amount of cotton was placed into a 1.5-mL glass well. A Daphnia was added with enough aged tap water to immerse the animal. The cotton pre-vented the Daphnia from swimming but still allowed some movement. Heart activity was recorded directly to iMovie by a Sony Handycam camera that was mounted on a dissecting microscope and connected to an Apple computer. Heart rate was determined in iMovie by playing a 15-second portion of the recording in slow motion and counting the beats.[The] scholarly literature provides little guidance as to what concentrations of these compounds should be tested and what effects to expect.The American Biology Teacher, vol. 72, no. 3, pages 176–179. iSSn 0002-7685, electronic iSSn 1938–4211. ©2010 by national association of biology Teachers. all rights reserved. request permission to photocopy or reproduce article content at the university of california Press’s rights and Permissions Web site at www.ucpressjournals.com/reprintinfo.asp. Doi: 10.1525/abt.2010.72.3.9 INQUIRY & INVESTIGATION Making the Most of the Daphnia Heart Rate Lab: Optimizing the Use of Ethanol, Nicotine & CaffeineFrank Corotto, Darrel Ceballos, aDam lee, linDsey VinsonThe american biology Teacher maKing The moST oF The DaPhnia hearT raTe lab 177Initial experiments focused on the effects of ethanol (1%, 5%, and 10% v/v), nicotine (0.1 mM, 1 mM, and 50 mM), and caffeine (0.1%, 0.5%, and 2% w/v). All three compounds were obtained from Sigma-Aldrich (St. Louis, MO). Solutions were made with aged tap water and frozen before use. Aliquots were coded to prevent observer bias. Six ani-mals were tested with each concentration of each compound. Eighteen animals were tested with aged tap water to serve as controls. Each ani-mal’s heart activity was recorded three times at 10-minute intervals to establish a baseline. Then, the solution bathing the animal was replaced with the test solution. Heart activity was recorded immediately and then three more times at 10-minute intervals.