Algal Response to Chlorine Effluence Frank Wolf Pittsburgh Central Catholic High School PJAS 2009

Purpose The purpose of this project is to discern the effect(s) of the addition of various concentrations of chlorine, a common disinfectant and means of sterilization, on the growth of chlamydomonas and euglena, two species of alga.

Chlorine Exists as a toxic, yellow-green, diatomic, gas in its elemental state (Cl2) Generally found in nature as the chloride ion (Cl-) Because of its highly reactive nature, it is usually found in a combined state with other substance(s), generally as a salt  i.e., sodium chloride in seawater and in minerals deposited in the earth The chlorine used for various industrial purposes is produced through the electrolysis of sodium chloride

Chlorine’s Use in Sanitation Chlorine - the most common substance used in water sanitation because of its natural toxicity Forms of chlorine used for water sanitation: solid, liquid, and gaseous forms  Granular (solid) form of chlorine was used in this study In sanitation, most specifically water sanitation, an important reaction occurs that allows it to sanitize  When chlorine (C1 2 ) is added to water (H 2 0), a chemical reaction occurs that produces hypochlorous acid (HOCl) and hypochloric acid (HCI)  Hypochlorous acid is the active, killing form of chlorine that actually does the sanitizing Hypochloric acid is not directly involved in the sanitation reaction(s) from this point onward  Hypochlorous acid sanitizes the water by killing the microorganisms present  Hypochlorous acid continues to kill microorganisms in this way until it encounters a microorganism containing nitrogen or ammonia In the process of destroying the microorganism, the hypochlorous acid, after combining itself with the nitrogen or ammonia, is broken down to its component atoms and becomes a chloramine, a combined chlorine substance A chloramine is not capable of sanitation

Algae in Pool/Spa Water One of the microorganisms that chlorine is frequently used to kill in water sanitation is algae One of three types of algae grow in pool water  Green algae  Mustard algae  Black algae Both algae observed are classified as green algae When a green alga grows in pool water, it forms a film on the various surfaces of the pool and/or floats atop the water, making it appear as a light green Green algae will grow in pool water when nutrients are present and there is not a sufficient level of free chlorine (uncombined chlorine available for sanitation) maintained Most common method of dealing with algal growth in pool/spa water is a strong dosage of chlorine (super chlorination) Green algae are very susceptible to this type of chemical treatment  Growth is stopped and the algae are killed.

Chlamydomonas A unicellular, photosynthetic alga Egg-shaped cells that are approximately 10 um in length and are capable of propelling themselves through water using their two, 12-um flagellum Employ the most accurate form of steering known among microbes  Cells use a beam of light as an origin of sorts and swim toward it or away from it in almost a straight line Cells possess their green color from the chlorophyll contained in the large chloroplast in each cell. Can be found in freshwater as well as in moist soil Characteristics of chlamydomonas that lend to its frequent use as a scientific model:  Can be grown under various conditions, including on plates or in test tubes  Normal room temperature permits its growth  Standard fluorescent lights support its photosynthetic processes  Do not require any organic food material to thrive, only CO 2 and light

Euglena Uses its flagella as its means of movement Uses a beam of light to assist in its steering ( same method as chlamydomonas) Cells vary in length from 20 to 300 um Cells possess both animal-like and plant-like characteristics  Contains chloroplasts and is therefore photosynthetic  Can also take in pre-formed food sources like a heterotrophic cell Rely most heavily on photosynthesis to sustain life Because of these features, botanists’ and zoologists’ opinions conflict on whether euglena should be classified as a plant or a more animal- like classification Can be found in nutrient-rich freshwater or in sewage systems because of its ability to absorb nutrients from decaying materials When it is cultured in the dark, it loses its chloroplast  Even when it is reintroduced to a lit environment, it does not regain its chloroplast  Once the chloroplast is lost, the cell resembles its protozoan relative, astasia

Null Hypothesis Chlorine will not have a significant impact on the growth of either species of alga.

Materials Spectrophotometer 49 glass test tubes Test tube racks Distilled water 0.15 grams granular chlorine Wax paper Pipettes (micro and macro) Pipette tips 48mL Chlamydomonas 48mL Euglena Container for creation of 10x stock solution Pens/pencils Calculator Data collection sheet Test tube labels Desk lamp

Procedure (1) Place desk lamp in the experimental site so that is approximately 45 cm away from the test tube racks. This light will be on a 12 hr on/12 hr off cycle to create the suggested environment for the algae. The experimental site will be kept at room temperature throughout the duration of the study. Create the 10x stock solution using 0.15 grams of chlorine and 1 liter of distilled water. Add 2mL of Chlamydomonas to 24 of the tubes and 2ml of Euglena to an additional 24 tubes. In order to create a 0x concentration of chlorine in four of the Chlamydomonas tubes, add 3mLs of distilled water to each tube. Repeat the prior step for four of the Euglena tubes.

Procedure (2) In order to create a 0.01x concentration of chlorine in four of the Chlamydomonas tubes, add 2.995mL of distilled water and 0.005mL of the 10x stock solution to each tube. Repeat the prior step for four of the Euglena tubes. In order to create a 0.1x concentration of chlorine in four of the Chlamydomonas tubes, add 2.95mL of distilled water and 0.05mL of the 10x stock solution to each tube. Repeat the prior step for four of the Euglena tubes. In order to create a 1x concentration of chlorine in four of the Chlamydomonas tubes, add 2.5mL of distilled water and 0.5mL of the 10x stock solution to each tube. Repeat the prior step for four of the Euglena tubes.

Procedure (3) In order to create a 1.5x concentration of chlorine in four of the Chlamydomonas tubes, add 2.25mL of distilled water and 0.75mL of the 10x stock solution to each tube. Repeat the prior step for four of the Euglena tubes. In order to create a 2x concentration of chlorine in four of the Chlamydomonas tubes, add 2mL of distilled water and 1mL of the 10x stock solution to each tube. Repeat the prior step for four of the Euglena tubes. Observing that the day that the tubes are prepared is Day 1, absorbance readings will be taken using a spectrophotometer set to 430 wavelengths once a day on Days Please note that the tubes are to be mixed by inversion ten times just prior to taking the readings using the wax paper as a temporary lid.

Chlamydomonas Data Analyses Comparisont-valueVariation 0x and 0.01x2.93Insignificant 0x and 0.1x1.60Insignificant 0x and 1x5.27Significant 0x and 1.5x5.32Significant 0x and 2x5.43Significant Day 7 p-value: 2.3E-7 Comparisont-valueVariation 0x and 0.01x3.72Significant 0x and 0.1x2.43Insignificant 0x and 1x7.82Significant 0x and 1.5x7.90Significant 0x and 2x7.95Significant Day 14 p-value: 6.55E-10 *If the t-value is greater than the critical t, then the variation is Significant. *0.05 critical t value = 3.62

Chlamydomonas Analysis Day 7 Data Analyses  ANOVA showed significant variation between the groups with a p-value of 2.3E-7 Reject the null hypothesis with a high confidence level  Dunnett’s Test Experimental groups that varied insignificantly from the control were the 0.01x and 0.1x groups Experimental groups that varied significantly from the control were the 1x, 1.5x, and 2x groups Day 14 Data Analyses  ANOVA showed even greater variation between the groups with a p-value of 6.55E-10 Reject the null hypothesis with an even higher confidence level  Dunnett’s test Experimental group that varied insignificantly from the control was the 0.1x group Experimental groups that varied significantly from the control were the 0.01x, 1x, 1.5x, and 2x groups All but one of the experimental groups demonstrated the same degree of variation as Day 7 (meaning that the type of variation, either ‘Significant’ or ‘Insignificant’, remained the same for each group but one, not the actual t Values for the groups)

Chlamydomonas Analysis (continued) Why were the algae in the tubes with a 2x concentration of chlorine not annihilated by this super-dose of chlorine?  Environmental Differences: Suggested environment for using chlorine (pools or spas) vs. Experimental environment in which chlorine was used (test tube environment) Water Motion  Pool or spa – constant motion and filtration of the water  More difficult conditions for the algae to establish itself  Test tubes – water was only disturbed just before spectrophotometer readings were taken  More hospitable conditions for the algae to establish itself and thrive Lighting and temperature  Pool or spa – amount of light supplied to the algae and the temperature of its environment vary  More difficult conditions for the algae to establish itself  Test tubes – night/day cycle (12 hour dark/12 hour light) and temperature (room temperature) were established to ensure optimal growing conditions for the algae  More hospitable conditions for the algae to establish itself and thrive

Chlamydomonas Analysis (continued) Why did the algae from the 0.01x and 0.1x experimental groups, which had started off and remained similar to the other experimental groups through Day 4, experience such rapid growth on Day 5 and throughout the rest of the study?  The chlorine’s instructions state that the product should be introduced into a spa with a strong dose, followed up by considerably smaller daily doses and weekly shock treatments, or super-doses of chlorine similar to the introductory amount Please note that the working concentration observed in this study (x) was the amount suggested to be added to a new spa (super chlorination)  At Day 4, the chlorine began to lose effectiveness, resulting in the growth of all the groups, most notably in the 0.01x and 0.1x experimental groups  Because these experimental groups had such a little concentration of chlorine to begin with, they were able to recover more quickly than the other groups and, in the long run, achieve overall greater populations  The 1x, 1.5x, and 2x experimental groups, because the higher concentrations of chlorine increased the duration and power of the chlorine’s effect, were not able to recover from the damage to their populations.

Euglena Data Analysis Comparisont-valueVariation 0x and 0.01x1.14Insignificant 0x and 0.1x3.34Insignificant 0x and 1x1.19Insignificant 0x and 1.5x0.21Insignificant 0x and 2x0.17Insignificant Day 7 p-value: Day 14 p-value: Due to the lack of variance between the groups shown by the p-value, a Dunnett's test was not carried out on the Euglena data from Day 7. *If the t-value is greater than the critical t, then the variation is Significant. *0.05 critical t value = 3.62

Euglena Analysis Day 7 Data Analyses  ANOVA Showed insignificant variation between the groups with a p-value of Accept the null hypothesis  Dunnett’s Test Because of the lack of variation between the groups shown by the p- value, no Dunnettt’s test was carried out on the Euglena data from Day 7 Day 14 Data Analyses  ANOVA Showed variation between the groups with a p-value of Reject the null hypothesis with a fair level of confidence  Dunnett’s Test All of the experimental groups varied insignificantly from the control

Euglena Analysis (continued) Why were the algae in the tubes with a 2x concentration of chlorine not annihilated by this super-dose of chlorine?  The same factors that explained the high concentrations of chlorine not completely killing off the chlamydomonas explain why euglena survived two times the suggested weekly dosage of chlorine 1. Lack of constant water movement and/or filtration allowed the euglena to establish itself more easily and effectively 2. Constant environmental conditions (lighting and temperature) removed potential obstacles for the euglena’s growth  Both of these reasons demonstrate why more chlorine would be necessary to achieve the same affect in the test tube environment of the study Why did the readings in all of the experimental groups increases from Day 4 to Day 5?  This, just like the occurrence in the chlamydomonas portion of the study, can be seen as the chlorine’s effectiveness fading because of the lack of the suggested daily treatments

Euglena Analysis (continued) Why did the 0.01x concentration experimental group outgrow the control group?

Limitations/Extensions Limitations:  The significant difference between the range of the chlamydomonas readings and that of the euglena readings, chlamydomonas being far higher  One may think that it is simply showing that the chlorine had a far greater effect on the growth of euglena than it did on the growth of chlamydomonas.  Why is this not the most likely explanation? Upon closer examination, one will notice that it is not only the experimental groups that are lower, but the control as well Scientist concludes that the culture of euglena used to create the tubes was not as healthy as the chlamydomonas culture because the euglena culture was not as fresh Extensions:  Performing a similar study in an environment more like that of a pool or spa, possibly through the use of a water cycling or filtration system  Different forms of chlorine (i.e., liquid, gaseous, etc.) could be tested to determine their effects on the growth of the algae and to see if one form is the most effective in killing algae  Test different types of algae, such as mustard or black algae, in a similar study to determine what level of chlorine-related stress that they could handle

Works Cited “About Chlamydomonas”. 19 Dec < html>. Alcamo, Edward, Ph.D. Fundamentals of Microbiology, Sixth Edition. Sudbury, Massachusetts: Jones and Bartlett Publishers, Blashfield, Jean F. Sparks of Life, Chlorine. Austin: Raintree Steck-Vaughn Publishers, “Chlorine.” 19 Dec “Chlorine Chemistry.” 17 Dec Dusenbery, David B. Life at Small Scale, The Behavior of Microbes. New York: Scientific American Library, “Euglena”. 26 Dec Postgate, John. Microbes and Man, Fourth edition. Cambridge, United Kingdom: Cambridge University Press, “Spectrophotometry”. 27 Dec Stwertka, Albert. A Guide to the Elements: Revised Edition. New York: Oxford University Press, “Water Chemistry for Swimming Pools”. 28 Dec < nc.us/ehs/chem.htm>.