Monarchs in the Classroom

We wanted to find out if acid rain that comes in contact with monarch larvae affects the weight of the adults. We applied three different concentrations of acid rain (3.5, 4.0, and 5.0 pH) to the larvae using a mist process. Our control was distilled water (7.0 pH). All conditions other than the acid rain concentrations were controlled. Our results showed that larvae that had been misted with the strongest acid rain (lowest pH) had the highest average mass as adults. The average mass of the adults decreased with the weaker acid rain concentrations (higher pH), although the control group was intermediate in size. We are uncertain why the weight increased with stronger levels of acid. Before we draw any conclusions from our experiment we would want to repeat it.

Acid rain has been found to negatively affect invertebrates (Baines 1990). One of the students in our team had done some research with dragonflies and acid rain. He found that with decreased (more strongly acidic) pH levels there was a higher rate of mortality among the dragonfly larvae, and those that survived were small, weak, and deformed as adults. We decided to do a similar test with monarchs to see if they were affected by acid rain.

Our team did some research to find out where acid rain comes from in our area. It is believed that the major cause of acid rain in Minnesota is due to coal burning plants in the Dakotas (EPA 2000, MPCA 1997). Minnesota has no control over those plants, but our team decided to test the rain in our area to see if it is acidic due to these plants or other sources. We tested rainwater for six weeks in our school nature area to find the pH level. The average pH of rain in our area was 5.3. This was consistent with the DNR report of 5.4 (DNR 2000, EPA 2000). Once we had a good understanding of acid rain in our area we set up our experiment. We applied different concentrations of acid rain to larvae every day throughout the larval stage to see if it would affect the weight of the adults. We used a concentration that was close to that found in our area (5.0 pH), as well as concentrations (3.5 pH and 4.0 pH) found in areas that have problems with acid rain.

The final step of our research was to see if the adults had been affected by the acid rain that we applied during the larvae stage. We tested this by observing and comparing coloration and markings to other monarchs raised under normal conditions, as well as by measuring the mass of each adult. We chose the mass measurement because it has been correlated with their life span. Some research suggests that the more mass an adult has the longer they live (Oberhauser 2000). We felt longer life spans would be beneficial to monarchs, so we wanted to find out if there would be a difference in mass measurements among the treatments.

Null: The concentrations of acid rain will have no effect on the adult weight of monarchs.

Alternative 3: Adult mass will decrease until a threshold concentration is reached (4.0 pH), after which additional increases in acid concentration will have an effect on adult mass.

For our experiment we obtained 75 eggs laid by several females on the same day. We used dilute sulfuric acid to make our acid rain recipe. The concentrations of acid rain were 5.0 pH, 4.0 pH, and 3.5 pH. Our control was distilled water, which has a pH of 7.0. After the eggs hatched we distributed 2nd instar larvae into 12 identical plastic (4.5 x 6.5 x 12 inch) containers. The containers are a product made by Sterilite, and are identical to the ones used at the University of Minnesota in the Monarchs in the Classroom Program. We placed 5 larvae in each container. We had three containers for each concentration. The containers were kept in a room where light, temperature, and humidity were all kept constant. Every day we misted each caterpillar with 3 ml of liquid. Each caterpillar was put on a paper towel outside of its container. We also sprayed 5 ml of each treatment on each plant. The liquid was measured in a graduated cylinder, and an atomizer was used to spray the acid rain so that all the liquid would come out of the container. The floral container that we put the milkweed stem into was also filled with 13 ml of the appropriate solution. We fed the larvae common milkweed, and picked all of it from the same field. We tried to make sure the plants were relatively the same size, had the same amount of leaves, and were about the same age. We sprayed the larvae every day until they pupated. When the adults emerged we waited 24 hours and then weighed them in wax envelopes. We subtracted the weight of the envelope from the overall mass, so we could get an accurate mass of the adults. Once the adults were weighed we also checked them for deformities and other detectable abnormalities. When our data collection was complete, we released our monarchs into the wild.

We analyzed our data using a statistical test. In order to determine the significance of the differences in weight among the different concentrations we used a t-test. To test all four concentrations we completed six t-tests. The results of our statistical tests are found in the next section of our paper.

Figure 2. We collected common milkweed plants from the wild and washed them before misting them with the acid rain solutions. Larvae were reared in plastic tubs with 5 larvae per tub and 3 tubs per treatment.

The results of our experiment were quite surprising. Adults that had been misted with the strongest acid rain (3.5 pH) had the highest average mass of 0.65 grams. The second highest average mass was 4.0 pH with 0.60 grams, then the control 0.55, and finally the 5.0 pH with 0.51 grams (Figure 3).

The mortality rate throughout our experiment was much less than anticipated. Only 4 monarchs died throughout the entire experiment, one in each concentration. Therefore, none of the treatments appeared to effect survival rates.

Once we had taken a look at our overall data, we then analyzed individual data of each treatment by doing t-tests to compare the average mass of adults in each pair of treatments. Table 1 shows the p-value for each treatment pair (there were 26 degrees of freedom in each test). We found that the average adult mass in each treatment was significantly different from the others. Our t-test results allowed us to reject the null hypothesis for all treatments because they were all significantly different from the others.

Strength of Acid Rain:	3.5 pH	4.0 pH	5.0 pH	Control
3.5 pH	--	--	--	--
4.0 pH	0.009	--	--	--
5.0 pH	0.000	0.000	--	--
Control	0.000	0.002	0.005	--

Table 1. P-values resulting from t-tests of adult mass for each pair of treatments. P-values are the probability or chance that two groups are NOT different from each other, so a low p-value means that there is a very small chance that two groups are NOT different. This means that there is a large chance that the two groups ARE different.

We collected qualitative data about the appearance of the adult butterflies from each treatment. We looked for discoloration, scarring, extra body parts, missing body parts, different markings, normal markings, approximate wing length, crumpled or not crumpled wings and general flying ability (all butterflies were able to fly away upon release). We could not detect any differences between the treatments. We also had some butterflies caught in the wild and some from other experiments to which we qualitatively compared our acid rain butterflies. Other than the fact that there was a mass difference, there were no other differences that we could detect.

Acid rain has been found to have a negative affect on many components of our environment, including invertebrates (DNR 2000). Research suggests that many invertebrates suffer a retarded growth or shortened life span due to the affects of acid rain (Hare 1990, Stewart 1990). A previous experiment done by one of our team members also found this to be true in dragonflies (Schulke 1999). However, we are still not certain why the mass of our adult butterflies increased with stronger levels of acid rain in this experiment. Although there appeared to be no abnormalities or deformities, the adult monarchs were released within three days of emergence. We did not monitor their life span or any other biological characteristics. Based on the information gained during our experiment we wish we had kept our butterflies to observe the rest of their life cycle. Our team has agreed to replicate our experiment next summer to see if our results are similar.

We would first and foremost like to thank our families, Robin and Bill Schulke, Bonnie and Rod Wentzel, Spencer, Brittany, Chris and Courtney Black for all their patience and support. A huge thanks to Dr. Janet Rith-Najarian for her acid rain recipe and supplies. We would also like to thank Monarchs in the Classroom @ the University of Minnesota for their supply of eggs for the experiment. A special thanks goes to Michelle Solensky and Anja Brunet for their help and guidance. We would also like to thank the National Science Foundation (ESI-9731429) for their support and continued commitment to science.

Schulke, Tye. 1999. The effects of acid rain on dragonfly larvae. Minnesota State Science Fair.