chemistry for kids LINDA WOODWARD

The University 01 Southwestern Louisiana Lafayene, LA 70504

Toxicology for Middle School

The Effects of Common Substances on Daphnia

Ruth N. RUSSO Whitman College. Walla Walla, WA 99362

Susan Parrish Garrison Middle School, Walla Walla, WA 99362

Most seventh graders understand that "drugs" are harmful rmbstances, but they are surprised to learn that 'medicines" also can be hannful. With most medicines, however, poten- tiallv harmful characteristics of the substance have been ex- ploited for therapeuticends. In fact, researchers identify com- oounds that are lethal to emenmental subiects at hich doses. k d test them as medicines at lower dose; (1 ,Z) . ~s'Paracel: sus (1493-1541) said: "All things are poisons, for there is nothing without poisonous qualities. It is only the dose which makes the thing a ooison" (3).

we use the inve&brate~a~hnia to demonstrate that com- mon substances. whether or not the students think of them as medicines, &gs of abuse, or foods, have toxic effects on Da~hnia. The students oractice mixingsolutions and makim caieful obscn~ations. More importan;ly, the students lean; that the extent of ilaphnm toxicity is due lo both the conren- tration (amount per-unit volumejand the potency (extent of biological effect per amount) of a common substance. Highly potent substances are toxic at low concentrations, while weakly potent ones are not. The weakly potent substances are toxic, however, if present in high concentration. The take- home message for seventh graders is that the highly potent substances must be ingested with caution, if at all, because small amounts are toxic. Weakly potent substances are less risky but can be toxic in excess.

Using Daphnia Visible hearts, eyespots, and "babies" endear the Daph-

nia to middle school students. Because this experiment is performed in the spring, we collect Daphnia from a local pond and study them as part of our "Pond Water Life" unit. These Daphnia are then used for the bioassay. Daphnia cultures and growth media also can be purchased from supply houses (the cost is -520 from Ward's).

The Experiment We test substances that the students encounter every

day: coffee, aspirin, allergy capsules, cooking sherry, and chewing tobacco. Every group of students chooses one of the common substances and makes an aqueous solution.

Substances Used

coffee - use undiluted drip coffee; aspirin - dissolve a 500-mg tablet in 10 mL water; allergy capsules - dissolve a Benadryl caplet in 10 mL water; . cooking sherry - use undiluted; chewing tobacco - mash a pinch (- 1 g) in 10 mL warm water with a stirring rod until the liquid is amber. (Students

may wish to mimic the action of saliva by substituting spit far several millimeters of water.)

Each group then transfers five Daphnia into each of five test tubes and fills the tubes with pond water (or growth medium) to a volume of 10 mL. The students ohtain a medicine dropper full of the test solution and deliver one, two, three, or four drops of the test solution into each of four test tubes. The fifth test tube receives no test solution and serves to show that the Daphnia remain healthy throughout the experiment. If Daphnia should die in this tube, then the mortality counts from the test solution tubes cannot be trusted.

In each group, students observe each of the five test tubes every few minutes. One student describes macro- scopic changes in the Daphnia's movement, and two stu- dents observe changes in heart rate, gill movement, etc. under the microscope. Afourth student records all data in a notebook. The living Daphnia are counted just before class ends and again on the next day.

Results The students are easily able to observe the effects on

Daphnia. The chewing tobacco causes quick death at the lowest doses. The cooking sherry first slows the heart rate and then is lethal at higher doses. Aspirin and allergy caplets are lethal at the highest doses; and coffee causes a racing of the heart but usually is not lethal.

Discussion To interpret these results, three questions should be dis-

cussed.

What is the active ingredient in each complex substance?

The table lists the active ingredients that are in each of the substances used above. We assume that the active in- gredients listed in the table cause the death of the Daph- nia, but it's possible that Daphnia are sensitive to some other toxic component in these complex substances.

In some cases, further experiments could show that the active ingredient in the test solution is actually the toxic agent. For example, the group testing coffee could make an identical set of test tubes substituting water-processed de- caffeinated for caffeinated coffee. If caffeine is the toxic substance, then the Daphnia exposed to decaffeinated cof- fee should be unaffected. The groups studying alcohol or nicotine could compare tests of alcoholic versus non-alco- holic beers, or tobacco from regular versus low-nicotine cigarettes.

Volume 72 Number 1 January 1995 49

Concentration and Rodent LD5o of Active Ingredients

Common Active ingredient Amount Active Concentration in Rodent L D ~ O ~ Substance Ingredient in First Test Tubea

Common Substance

aspirin acetylsalicylic acid 500 mgltablet 0.25 mg/mL 1.5 @kg

allergy capsule diphenhydramine 50 mgltablet 0.03 m@mL 0.5 g\kg

chewing tabacco nicotineC 3% (wlw) 0.01 mg1mL 0.23 @kg

coffee caffeined 150 mg/5 02. cup 0.005 mg/mL 0.13 @kg

cooking sherry ethanole 17% (vlv) 0.7 mg1mL 10.6 glkg

'Assume 20drops = 1 mL. Test tube received 1 drop of wmmon substance. %DW values from (4). Reported as g wmpound per kg rodent body weight. 'Percent nicotine in leaves from (5). Assume students extract 80% of available nicotine. d~afeine data from Coffee and Caffeine'. a brochure from Sta!buck's Coffee Company, Seahle, WA, 1993. Assume

1 oz. = 30 mL. 'Assume density ethanal = 0.8 glmL.

chewing tobaccn I Active Ingredient Concentration (mg/mL)

Comparison of Lowest Toxic Concentrations. For each common sub- stance, students note the test tube, containing the fewest number of drops of solution, in which Daphnia died. in the experiment shown above, three drops of cooking sherry, four drops of aspirin solution, four drops of allergy capsule solution, and one drop of chewing to- bacco solution were sufficient to kill Daohnia. (Coffee is not shown becatse it was not lox c at any dose.) ~imoer of drops m~lliplleo by 'Concentrat on in Fmt Test T~be" (See tao e) yle os !he owest lox c concentranon for eacn act ve ngreoent.

Why are some of these active ingredients more toxic than others?

There are two possibilities. A toxic compound is either present i n high concentration (i.e., present in a large amount in the test tube), or is a potent substance (i.e., even small amounts, compared with the other active ingredi- ents, are lethal to Daphnia).

The table lists the approximate concentration of each ac- tive ingredient in the test tube that received 1 drop. (Mul- tiply this concentration by the number of drops to obtain the final concentrations in each of the other test tubes.) To determine potency, the students graph the lowest active ingredient concentration that is toxic to Daphnia. (See fig- ure.) If all of the active ingredients were of equal potency, then they would have the same lowest toxic concentration. The figure shows, however, that some substances are more potent than others. For example, the nicotine in the chew- ing tobacco solutionis toxic toDaphnia a t a low concentra- tion (0.01 mgImL); therefore, nicotine is very potent. The ethanol in cooking shelly, on the other hand, is not very

potent, because it is toxic only a t higher concentrations (2.1 mglmL and over). Consequently, this comoound is toxic in this ex- periment because of its high con- centration. Accordina to the fie- ure, students can ra& the active ingredients by decreasing po- tency: nicotine > diphenhydra- mine > acetylsalicylic acid > ethanol. The caffeine in coffee is left out of the figure because it was not toxic to Daphnia even at its highest dose (0.025 mgImL). We know only that i t is less po- tent than nicotine.

Can we assume that what is toxic to Daphnia is toxic to humans?

If the students compare the conditions of the Daphnia ex-

oeriment with how humans encounter these substances. the biggest difference is that the Daphnia are swimming id solutions of these substances, whereas humans eat these substances or absorb them through the skin. (Additionally, the Daphnia experiment is short term, whereas we worry about long-term human toxicities and the development of cancer.)

Because we cannot perform toxicity bioassays in hu- mans, the best information we have on short-term toxicity is based on other mammals. The table shows the rodent LD50 (Lethal Dose to 50%) values for the active inmedi- ents. If a large group of rats or mice are fed the compound a t the level of the LD5a value, 50% of them will die, usually within a week. LD~o values indicate relative potencies: the smaller the LDm value, the more toxic the compound is. Based on rodent LDKn values. the students can rank the *"

active ingredients in order of decreasing rodent potencies: caffeine, nicotine, diphenhydramine, acetylsalicylic acid, and ethanol. This is the same relative potency ranking as in the Daphnia experiment. Thus, these compounds may affect mammalian and invertebrate physiology in similar ways. Caffeine, the only exception, seems to be the most toxic compound to ma&mals but is less toxic than nicotine in the Daphnia bioassay. Perhaps it influences inverte- brate and mammalian p h y s i o l o ~ differently.

Conclusion This activity combines an inexpensive hands-on experi-

ment with extensive discussion to help the students think about chemical concentration, potency, and the difference between harmful and beneficial substances. In this survey, students see that even useful substances are toxic a t high concentrations and that the most dangerous substances are those that are lethal a t low concentrations. An addi- tional benefit of this exercise is that when students watch Daphnia struggle under the influence of substances like chewing tobacco and cooking sherry, the powerful message of deterrence reinforces other drug abuse lessons.

Acknowledgment - Thanks to the Howard Hughes Medical Institute for sup-

porting the Whitman Summer Institute for Precollege %ache&, where this collaboration began.

Literature Cited 1. McLaughlln.J. L.; Chang, C . 4 . : Smith, D. L. S f d Nat Pmd C k m . 1991.9: m 0 9 . 2. Kendler, B. S.:Koritr. H.G.: Gibaldi,A.Amr Bid. noch 1992,5411). 4650. 3. Sigetist, H. E. The Gmd Doctors: trans., Paul, E. and C.; Norton: New Yor*, 1933. 4. Budavati, S., Ed. The Memk Index, 11th ed.; Me& & Co.: Rahway, NJ, 1989. 5. Bmuk, B. Plants Consumed by Mon;Academic Pleas: Iandon, 1975.

50 Journal of Chemical Education