Glyphosate Effects on algal population Patrick Keating Pittsburgh Central Catholic High School PJAS 2019 Grade 9

Pesticide Runoff One of the fastest growing and largest threats to global water resources Rainwater collects the excess chemicals, flows into rivers and lakes, causes toxicity and/or eutrophication Eutrophication results in oxygen crash Kills many organisms

Herbicide Used to eliminate unwanted plants COULD HARM NON-TARGET SPECIES Could harm aquatic organisms Could affect algal growth GLYPHOSATE

ROundup 2% Glyphosate and isoproplyamine salt 2% pelagonic acid and related fatty acid 96% Other ingredients

Glyphosate Common Herbicide Main ingredient in Roundup Kills broad leafed plants and grasses

Chlamydomonas reinhardtii Round, flat, green algae Photosynthetic Found in many places Can thrive in complete darkness, carbon source Often used in biological research

Euglena gracilis Cylinder-shaped, unicellular, green Algae Found worldwide, typically in fresh water or sewage systems Photosynthetic, Hetrotroph Also commonly used in biological research

Purpose To determine the effects of Roundup on algal population growth

Past Studies “Combination Effects of Herbicides on Plants and Algae” states that most of the herbicides used were harmful to plants and algae “Herbicides and Insecticides Effects on Green Algae and Cyanobacteria Strain” stated that the herbicides that were able to be tested were “extremely toxic” to green algae

Hypothesis Null: Roundup will not have a significant effect on growth of Chlamydomonas reinhardtii or Euglena gracilis Alternative: Roundup will have an effect on growth of Chlamydomonas reinhardtii or Euglena gracilis

Materials Soil water Spring water Spectrophotometer Hemocytometer 23 Watt Lightbulb 13x100 Culture Tubes Micropipettes (p1000, p200, p20) Chlamydomonas reinhardtii Euglena gracilis Glyphosate Test Tube rack

Procedure Made test tube concentrations with both types of Algae, (see Slide 13) Placed 13x100 culture tubes beneath 23 watt lightbulb for 12 hours a day, complete darkness for other 12 hours Mix tubes around before measuring in the spectrometer Measure all thirty tubes in the blanked spectrometer at 430nm (Carolina Science Spectrophotometer) Cell counts performed from days 11-26-18 to 12-5-18 On day 10, take Hemocytometer cell counts performed

Test Tube Concentration Control (0%) 0.05% 0.005% Spring Water 1.5ml 1 ml 1.45 ml Soil Water Algae 2.5ml (Euglena) 2.5 ml (Euglena) 2.5 ml (chlamyd) Glyphosate (10% stock) 0 ml 0.5 ml 0.05 ml

Glyphosate Effects on Euglena

Glyphosate Effects on Chlamydomonas 0.727451332

Hemocytometer Readings: Euglena 0% 0.005% 0.05% P Value: 0.00225

Hemocytometer Readings: is Chlamydomonas 0% 0.005% 0.05% P Value: 6.925 E-8

Conclusion The ANOVA allows null rejection Different concentrations of Glyphosate significantly effected algal growth for both species of algae Significant effects on 0.05% concentrations of both algae

Limitations and Extensions Limited range of exposures Unnatural environment Optimal Lighting? Spectrophotometer evidence not completely reflective of population size Extensions: Wider ranges of exposure More herbicides Use natural pond Optimize lighting and care Synergistic Effects with other chemicals

Bibliography Britannica, The Editors of Encyclopaedia. “Euglena.” Encyclopædia Britannica, Encyclopædia Britannica, Inc., 27 Dec. 2017, www.britannica.com/science/Euglena. “Chlamydomonas Reinhardtii.” Science360 News Service | National Science Foundation, news.science360.gov/obj/pic-day/49c9d5f2-c84a-4e13-9b0b-5b8cecbd29df/chlamydomonas-reinhardtii. “Chlamydomonas Reinhardtii v4.0.” Home - Pseudomonas Fluorescens Pf0-1, genome.jgi.doe.gov/Chlre4/Chlre4.home.html. Piri, M., and V. Ördog. “Herbicides and Insecticides Effects on Green Algae and Cyanobacteria Strain.” Iranian Journal of Fisheries Sciences, Iranian Journal of Fisheries Sciences, “Runoff, Factors Affecting.” Water Encyclopedia, www.waterencyclopedia.com/Re-St/Runoff-Factors- Affecting.html. https://www.researchgate.net/publication/6500177_Combination_effects_of_herbicides_on_plants_and_ algae_Do_species_and_test_systems_matter

ANOVA: Euglena (day 6) Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column 1 5 0.66 0.132 7E-05 Column 2 2.33 0.466 0.00128 Column 3 0.86 0.172 0.00052 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 0.332653333 2 0.166326667 266.8342246 1.13E-10 3.885293835 Within Groups 0.00748 12 0.000623333 Total 0.340133333 14

ANOVA Chlamydomonas (Day 6) Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column 1 5 0.89 0.178 0.00032 Column 2 3.58 0.716 0.00453 Column 3 1.23 0.246 0.00038 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 0.85828 2 0.42914 246.1606119 1.81486E-10 3.885293835 Within Groups 0.02092 12 0.001743333 Total 0.8792 14