Aquatic Toxicology Toxicology is the study of the effects of pollutants on ecosystem health. General ecosystem health is difficult to precisely measure, so toxicologists often rely on indicator species. Indicator species are species considered representative of a portion of the ecosystem, for example aquatic plants are often used to test water quality. To determine how an indicator species will respond to a particular pollutant (or a mixture of pollutants), a bioassay is conducted. Bioassays are also called biological assays, and are a type of in vitro experiment conducted to measure the effects of a substance on a living organism. In a toxicological bioassay, representatives of the indicator species are exposed to a range of concentrations of the pollutant(s) in question. The response of the indicator species to the pollutant can be measured in terms of growth, reproduction, or survival. Exposure can be acute (high concentration over a short duration) or chronic (long-term exposure). In our bioassay, we’re using duckweed (family Lemnaceae), a small aquatic plant that grows floating on the surface of wetlands, ponds and lakes. There are approximately 20 species of Duckweed in the United States. The most commonly used in bioassays is Lemna minor. Duckweed is a flowering monocot. Each individual is composed of one or more fronds attached to a rootlet that hangs down into the water column. Each frond is actually a combined leaf and stem. Though duckweed is a flowering plant, it rarely flowers and typically reproduces by budding. Flowering L. minor Budding L. minor 1 During budding, a new frond grows from a bud on the parent plant. The new frond eventually grows its own rootlet and breaks free from the parent plant to become an independent individual. Duckweed is an ideal bioassay test organism, as its growth can be easily measured by counting the number of new fronds which develop in test solutions versus control solutions in just a few days. We are going to test the effects of fertilizer runoff on water quality, by measuring Lemna minor growth and survival in differing concentrations of lawn fertilizer. We are also going to test two different fertilizers. Fertilizer runoff is a major problem in areas with manicured lawns such as neighborhoods, business districts, golf courses, as well as agricultural areas. Fertilizers contain nutrients such as nitrogen and phosphorus, and often contain herbicides as well. A great deal of the fertilizer (and other chemicals) applied to lawns and fields is carried away by rain and wind, and ends up in nearby water bodies. This anthropogenic input of nutrients creates an imbalance in the affected aquatic ecosystems. Imbalances lead to eutrophication, a situation where excess nutrients cause increased plant and/or algal growth and decay. Eutrophication often favors fast-growing “weedy” species over others with slower growth rates. Weedy species are then able to out-compete their slow-growing neighbors, and can literally choke a water body. Eutrophication is a significant problem around the world. http://www.botany.wisc.edu/wisflora/pictures/xl_photos/LEMMIN_DW_XL.jpg 2 Experimentation: A Duckweed Bioassay of Lawn Fertilizer Runoff Materials • Fluorescent lights • Lemna minor – 5 per beaker • Beakers (3 per treatment) • Fertilizer solutions • Forceps • Clear plastic film • Spring water Methods 1. Determine which compounds and concentrations your group will be testing. Half of the groups in your class will test Fertilizer A, and half will test Fertilizer B. Form two hypotheses: one which addresses the effect of concentration of the fertilizer your group is testing, and one which addresses the effect of Fertilizer A versus Fertilizer B. Your instructor will provide information on each. 2. Each group will setup three treatments: 1) “high” runoff, 2) “low” runoff, and 3) control (no fertilizer). Each treatment will be composed of three beakers (replicates). Be sure to label! 3. Transfer 100 ml of the appropriate medium to each beaker. Stock solutions for each fertilizer are on the side bench. For a high runoff treatment, take 50 ml of fertilizer solution, add 50 ml of spring water and mix well. For a low runoff treatment, take 20 ml of fertilizer solution, add 80 ml of spring water and mix. Be sure to use the appropriate pipettes, and clean technique to prevent contamination of stocks or treatments! What should you put in your control beakers? 4. Using forceps, transfer five green, healthy L. minor individuals with two fronds each to each beaker, cover with clear plastic film, and place under the lights. 5. Let the beakers sit undisturbed for five days – keep them covered, and do not remove water. 6. At the end of the five-day growth period, count the number of fronds in each beaker. It can be difficult to decide which fronds are large enough to count – make a group decision, and then be consistent with every beaker so that your results will be comparable. 7. Also count the number of yellow fronds, rootless fronds, sinking fronds or otherwise unhealthy individuals in each beaker. 8. Record your results in the table below. 9. Graph the following: 1) the mean number of fronds for each treatment, 2) the mean number of unhealthy individuals in each treatment. 3 10. Bring your results to next week’s lab for comparison with the other groups. We will be looking for differences between fertilizer concentrations as well as differences between the two fertilizers (A and B). Hypotheses: Results Number of Number of Mean # Mean # of Solution and Unhealthy Notes and Fronds per of Unhealthy Concentration Individuals Comments Beaker Fronds Individuals per Beaker Control Discussion What conclusions did you form? What are some ways that fertilizer runoff could be minimized? 4 What are some alternatives to applying fertilizer? In situations like the one pictured above, where a fast-growing species like L. minor begins to choke the surface of a water body, what will happen to the overall health of that ecosystem? Will other aquatic plants survive? What other organisms will be impacted – will fish, birds, amphibians and other species be impacted? How? What will happen as the Lemna dies and sinks to the bottom? References Methods Cunningham, W. P. and B. W. Saigo. 1997. Environmental Science: A Global Concern. Wm. C. Brown Publishers: Dubuque, IA. Environmental Inquiry – Bioassays Using Duckweed http://ei.cornell.edu Keddy, C. J., J. C. Greene, and M. A. Bonnell. 1995. Review of whole-organism bioassays: Soil, freshwater sediment, and freshwater assessment in Canada. Ecotoxicology and Environmental Safety 30: 221-251. United States Environmental Protection Agency www.epa.gov Photos Kuban State Agricultural University: http://www.mobot.org/jwcross/duckweed/Russe/index-e.htm Norwegian Botanical Association: http://www.nhm.uio.no/botanisk/nbf/plantefoto/Lemna_minor.htm University of Wisconsin, Madison. Deparrment of Botany: http://www.botany.wisc.edu/wisflora/pictures/xl_photos/LEMMIN_DW_XL.jpg 5.