2. Brian Crofoot Mount Union College Department of Biology April 20, 2010

3. Copyright Notice Original content, excluding all photographs, Copyright 2010 Brian Crofoot. This work, excluding all photographs, is licensed under the Creative Commons Attribution-ShareAlike 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by- sa/4.0/. Photographs used for Commentary, Research, and Non-Profit Educational uses.http://creativecommons.org/licenses/by- sa/4.0/

4. Overview 1. Background information a. Copper Sulfate b. Duckweed c. Water types 2. Experiment a. Introduction b. Experimental c. Results d. Conclusion 3. Questions & Answers

5. First things first

6. Copper Sulfate What is Copper Sulfate? A. It’s a fungicide and herbicide B. Copper as an element is essential for plant life C. A substance with many uses Source: Alibaba.com Source: practicalphysics.org

8. Why Copper Sulfate? Why is copper sulfate used? How does it affect plants? Its an oxidant It targets the chloroplasts in plants Why do we need to understand its toxic effects? To better understand how it affects fish, birds, plants, and other organisms We need to know if organisms exposed to it will be affected by it

9. Why Duckweed? What is Duckweed? A member of the Lemna genus A small and very common plant Why is it used in ecotoxicological research? It can be used as a model organism in toxicological research Easy to find and cultivate Source: Bonniesplants Source: Alberto Godoy

11. Water Types Natural Water From a natural environment Treated Waste Water Has lots of nutrients Tap Water It is drinking water and I wanted to learn about how it affects copper sulfate’s toxicity on duckweed Spring Water To redo a previous experiment

12. Results from a Previous Bioassay

14. Hypotheses Copper sulfate is harmful to Duckweed if it is placed in treated waste water, natural water, and tap water, and in spring water in concentrations of more than 1.0g/L. Copper sulfate acts as a nutrient for Duckweed if it is placed in spring water of concentrations of no more than 1g/L CuSO4 Duckweed will survive the best if it is placed in treated waste water

15. Experiment

16. Bioassay What is a Bioassay? Its an experimental method used to study the effects of toxins on organisms It is an EPA approved technique Why is this type of experiment important? Its purpose is to study how chemicals affect organisms

17. Ecotoxicology A field of biology that tries to understand how toxins and pollutants affect organisms and ecosystems. Tries to find ways to help organisms cope with pollutants and toxins Bioassays are just one tool used in ecotoxicology

18. Procedure Summary 1. Acclimate Duckweed for 1 week 2. Amounts of copper sulfate and waters were measured 3. Solutions were mixed 4. 10 duckweed were placed into each beaker Bioassay techniques were followed Each beaker had 200mL of the specific water type Each beaker had a certain concentration of copper sulfate Copper (II) Sulfate pentahydrate was used EPA Guidelines were followed 4 water types -Natural Water from Deer Creek Reservoir -Spring Water -Tap Water -Treated Waste Water Concentrations of Copper Sulfate  0g/L (control groups)  0.1g/L  1.0g/L  10g/L  100g/L

20. Results

21. LC 50 Spring WaterTap Water Natural Water Treated Waste Water Day 1 1 g/L CuSO4100 g/LNone Day 3 1g/L 10 g/L Day 5 1 g/L Day 7 0.1 g/L LC 50 Values

22. ANOVA Analysis Day 1's 2 Way ANOVA Results Source of VariencedfFP-value Concentration 476.230 Water Types 323.620 Interaction of Water types and concentration 125.430

23. Results Summary Initial All duckweed are green and alive Day 1 Many Duckweed died in the higher concentrations of 10g/L & 100g/L, while as they survived in the lower concentrations Day 3 All water types had almost no deaths in the two lowest concentrations of 0.0g/L & 0.1g/L All duckweed in the 100g/L had died in all water types Day 5 All duckweed in the 10g/L solutions had died in all of the water types Started to notice something weird about the duckweed Day 7 Generally more than half of the duckweed in the 0.1g/L had died in all of the water types All duckweed in the 1g/L solutions had died in all water types

24. Results Summary All p-values are significant and are below 0.01 after an ANOVA analysis of the data Only 1 duckweed died out of all of the control groups LC50s increased as time passed Duckweed survived the best in treated waste water Duckweed fared the worst in Spring Water

25. Conclusions The types of water significantly affect Duckweed’s survivability when exposed to copper sulfate The concentration of copper sulfate significantly affects Duckweed There is a relationship between the concentration of copper sulfate and Duckweed’s survival There is a significant interaction between copper sulfate and water types on Duckweed’s mortality rate

26. Duckweed’s Responses Duckweed tries to combat copper sulfate by using antioxidants and proteins Copper sulfate affects duckweed by causing oxidative stress It affects duckweed’s chloroplasts

27. In the Future… Redo this experiment to get a bigger data set Try using other types of water Try using another organism Try using another concentration of copper sulfate

28. Recap Copper sulfate is a toxic chemical Duckweed is a useful organism to study toxins in aquatic environment A bioassay is an EPA approved experimental procedure

29. Did you know? Alliance’s water is obtained from Deer Creek Reservoir. Copper Sulfate is not very toxic to humans unless ingested or inhaled. Waste Water Treatment in the US is regulated by the EPA and State governments The government has set tough regulations on the quality of the water we use everyday

30. Important References Hopkin, Steve P., D.B. Peakall, R.M. Sibly, and C. H. Walker. Principles of Ecotoxicology, Third Edition. 3 ed. Boca Raton: CRC, 2005. Print. Razinger J, Dermastia M, Drinovec L, Drobne D, Zrimec A, Koce JD (2007): Antioxidative Responses of Duckweed (Lemna minor L.) to Short-Term Copper Exposure. Env Sci Pollut Res 14 (3) 194–20 Wu, Lin. Class Lecture. Introduction to Ecotoxicology. Mount Union College, Alliance, Ohio. 2009

31. Special Thanks Alliance Water Treatment Facility and its employees Dr. Lin Wu Mount Union College

32. Any Questions? The End Any Questions?