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Constructed Wetlands Project

Student Lab Book

Pasadena City College Wetlands Research Team

High School Edition

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Written by:

Ibrahim Hajjali, Steven Cardenas, Marisa Robles, William Liang, Thai Vuong, Alieenthea Lam,

J. Elliot Krayer, Rushil Shah, L. Alejandro Del Valle and James Liu.

Funding Partners:

Metropolitan Water District of Southern California

United States Bureau of Reclamation

Los Angeles County Sanitation Districts

Friends of the United Nations

Water for People

American Society of Civil Engineers

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Table of Contents Topic Page

Key Concepts and Suggested Research Topics 2 Introduction 3 What is a Wetland? 3 Why is this Study Important? 4 What you will Investigate 4 Materials for Analysis 5 Methods for Analysis 6 Data Charts 10 Questions 12 Critical Thinking 13 Appendix A 15 Construction of Wetlands 15 Recommendations before you begin 15 Building Materials 15 Wetland Drawings 16 Procedure for Construction 18 Properties of the Pollution 20 Suggested Vegetation 20 Glossary 21

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Key Concepts:

• Function of a Wetland • Common Wetland Flora and Fauna • Carbon Cycle and the Process of Decomposition • Nitrogen Cycle and Nitrification Suggested Research Topics:

• Protists and Protist Diversity • Bacteria and Bacterial Diversity • Plants: Growth, Important Nutrients, and Symbiosis

with Bacteria. • Ecology of Aquatic Environments • Nitrogen Cycle • Carbon Cycle

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Introduction:

What is a Wetland?

Wetlands are areas of land where the soil is saturated by water. They have characteristics of both terrestrial and aquatic systems. Generally there are two categories of wetlands: coastal wetlands and inland wetlands. Coastal wetlands provide the bridge that connects fresh water and salt water, allowing for the exportation of nutrients and organic materials to the ocean. Inland wetlands are found along the edges of rivers and streams. Wetlands can be further classified into four subcategories: swamps, marshes, bogs, and fens. These classifications are based on the plant types present, as well as biotic and abiotic characteristics. Wetlands provide habitats for many hydrophytic plants and animals. Wetlands also have the capability of slowing flood waters and absorbing excess nutrients which could pollute rivers and oceans.

Constructed wetlands are treatment systems engineered to mimic the natural wetlands’ ability to purify water, and aid in the removal of pollution. Some ultimate goals of constructing a wetland include being able to treat waste water and having more control over the flow of storm water.

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Why is this study important?

Fueled by convenience and ignorance, large amounts of waste and pollutants have been disposed of into water supplies all over the world. As a result, potable water is slowly becoming a scarce commodity. The gravity of this matter has only recently been recognized, therefore we must begin to devise methods for remediation. One possible method for water purification is through the use of wetlands. Constructed wetlands may prove to be an economical, efficient and natural methods of reducing the pollutants that are present in our waterways. Research is being done to investigate the rejuvenating effects of wetlands on polluted water.

What you will investigate

In this project, you will observe how the plants and micro-organisms living in your constructed wetland play a role in purifying the water. You will assess the wetland’s ability to purify polluted water by conducting chemical and biological tests. In the chemical analysis, you will measure the concentrations of Ammonium, Nitrates, Phosphates, and Iron. The pH, Biological Oxygen Demand (BOD), and Dissolved Oxygen (DO) will also measured. If available, light microscopes can be used to observe the active micro-organisms in the wetlands, such as bacteria and protists. Your instructor might also chose for you to perform an analysis of the bacteria population diversity and density.

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Materials for Analysis:

10 mL Pipettes* Test Tubes* Chemical Test Kits (i.e. LaMotte AMD-12) for:

Ammonia Nitrate Phosphate Iron Biological Oxygen Demand (BOD) Dissolved Oxygen (DO) pH

Pollution (refer to Appendix)

* One per student/ group Optional Materials Microscopes Slides and covers 15 mL plastic large test tubes* Petri plates with bacteria growth medium (i.e. LB agar)** Spreading tool, (i.e. “L” shaped plastic or glass rods) Pipettes (Micro pipettes and tips if available). Distilled water

** Three plates per student/ group

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Methods for Analysis:

For construction of the wetland refer to Appendix A.

Several chemical tests will be conducted to observe the effects of pollution on the constructed wetland. These tests will be conducted using the chemical test kits. Follow the instructions included in the chemical test kit for proper procedures.

The kits will be used to determine the relative concentrations of the following key chemicals: Ammonia, Nitrates, Phosphates, Iron, BOD, DO and pH. The chemical tests should be run daily for at least one week before the pollution is introduced. Data should be collected at approximately the same time each day for the increased accuracy. Test samples should be taken from both the input and output regions. This will give a better idea of the transition between the input and output. Water samples should be taken from all the troughs and the concentrations of each chemical should be averaged to obtain daily totals.

Biological tests can be performed using a light microscope, if available. Take 1 drop of water from the input, output, and middle regions and prepare a wet mount slide. Do not focus on one trough but collect samples from all the troughs to reduce error. If there is insufficient time and supplies, samples may be omitted from the middle of the wetland. Observe the micro-organisms and count the number of species. Draw basic pictures of individual species to help differentiate them from each other, or for a challenge, identify them by name.

To calculate the protist population, count the number of individuals from each species that are in your field of vision. Then determine the number of fields on the slide. To do this, use the ruler in the scope or measure the scope by placing a ruler beneath it; use this measurement to find the area of the scope. Then, find the area of the cover slip. After obtaining both areas, divide the area of the cover slip by the area of field of view. Multiply the number of individuals by the number of fields on the slide. This method will only give an

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average, but it is more time efficient than counting individual micro-organisms.

The analysis of the bacteria population diversity and density can be preformed at your instructor’s discretion. All of the materials used for this portion of the lab should be sterile to ensure that any growth on the Petri dishes is from the wetland and not outside contamination.

The bacterial count can be obtained by means of serial dilutions conducted before and after the addition of pollution to the wetland. Take a 10 mL water sample from the input and

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Daphne

Paramecian

Spirogyra

Diatoms Anabaena

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output regions of the wetland. These will be considered your original samples. To perform the serial dilutions add 1 mL of the original sample using your pipette to the first test tube and dilute with 9 mL of distilled water. Next, hold the test tube firmly with one hand and gently flick the bottom of the test tube with your finger to ensure the original sample and the distilled water has been mix thoroughly. Repeat this process for all of the dilutions. The original sample is now diluted 100 fold. Label this test tube “1/100”. To perform the next dilution, transfer 1 mL from the 1/100 tube to the second test tube and mix with 9 mL of distilled water. Label this test tube “1/10,000”. Then using the 1/10,000 test tube as a base, repeat the dilution process once more in the third test tube. This test tube should be labeled “1/1,000,000”.

Next label three LB agar petri dishes 1/100, 1/10,000 and 1/1,000,000. Do not open the cover and expose the agar to open air unnecessarily. Opening the covers unnecessarily will contaminate the plates. Use your micropipetter to draw 5 µL from the tube containing the 1/100 dilution. If micropitters are not available find an instrument that will deliver close to 5 µL of solution, approximately one small drop. Use one hand to lift the cover of the LB agar petri dish labeled 1/100 open it just enough to allow you to insert you pipette tip and keep it over the plate to prevent anything from falling in and deliver the solution onto the middle of the plate.

Gently spread the solution evenly over the surface of the agar, using your sterile spreading tool. Then close the lid and tape the cover and plate together. Repeat this procedure with the other test tubes and LB plates. Allow the cultures to grow for one week; this should give the microorganisms enough time to divide to yield visible colonies. The progress should be tracked to ensure that over-growth does not occur. If an incubator is used the time allowed can be reduced to one day.

After one week observe the number of different colored colonies on each plate and the total number of colonies. Assume each colony originated from one bacteria cell.

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Differences in the color of the bacteria colonies display the diversity of life forms in the water. Identify the plate that contains the most separate colonies, meaning the colonies look like individual disks. Calculate the density of bacterial life per mL in the original sample taken from the wetland. Divide the number of colonies by the amount of solution streaked on each plate.

If possible the bacterial diversity and count should be performed both before and after the pollution event. The data may then be combined to extrapolate trends.

Once the pre-pollution data has been collected, your instructor will introduce 5-10 gallons of pollution. Allow the pollution to run through the wetland for approximately one hour, before recording the chemical readings. Do not take any biological readings. Resume biological readings on the day following the introduction of pollution. Continue testing for a minimum of two weeks after the introduction of pollution. Record data in a table similar to the one provided, for each chemical tested.

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Data Charts: Chemical

Test Day Concentration (ppm)

Before Input Output

Day 1 Day 2 Day 3 After Input Output Day 6 Day 7 Day 8

Micro-organisms Input Middle

Species 1

Species 2

Species 3

Output

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Bacterial Fauna Before

Input 1/100 1/10,000 1/1,000,000 Types Diversity Mean

Output 1/100 1/10,000 1/1,000,000 Types Diversity Mean

After Input 1/100 1/10,000 1/1,000,000 Types Diversity

Mean

Output 1/100 1/10,000 1/1,000,000 Types Diversity Mean

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Questions:

1. Discuss your observations of the following. Be as descriptive as possible:

• Ammonia: • Nitrate: • Phosphate: • Iron: • pH: • Biological Oxygen Demand: • Dissolved Oxygen:

2. What effect does the chemical concentration have on the micro-organisms?

3. What effect does the chemical concentration have on the plants? 4. Explain any connections between the concentrations of different chemicals. 5. Explain the changes in bacterial population density and diversity with relation to time. 6. Identify the key chemical cycles present in the bio-filtration processes. 7. Does the wetland display the ability to filter graywater? Use specific examples from your data to support your answer. 8. How can the system be made more efficient? 9. What role do the micro-organisms play in the bio-filtration process?

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Critical Thinking:

1. Describe the changes in the nitrate and ammonium levels with respect to every stage of the nitrogen cycle. Include the key micro-organisms involved and the role they play in the cycle. 2. Use the data you collected to analyze and explain how pH changes are related to the chemical changes and explain how these changes effected the protist population. 3. Discuss the transferability of wetlands including their

economic feasibility and how practical they are in different climates and regions.

4. Wetlands provide a method by which wastewater can be

purified by natural means allowing for its reuse. What consequences can be associated with mishandling waste water?

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Appendix A

Construction of Wetland

Recommendations before you begin:

The wetland requires an area of 15 feet by 5 feet. Construction on a table is preferable because the wetland outflow needs to be a sufficient height off the ground. The designated space for the wetland should remain relatively undisturbed for the course of the experiment. The wetlands should be constructed near a water source (i.e. faucet or garden hose) to provide the water running through the wetlands. Also ensure adequate drainage is available and that the table, floor, etc beneath the wetland will not be damaged in the event of leaks.

Building Materials: 1 10-15 gallon Plastic Storage Container (Input Bucket)

2 10 ft Plastic Rain Gutters (Troughs) 1 5 ft PVC piping (½ inch diameter) 4 PVC elbows 4 PVC ball valves 1 Tube of caulking 1 Caulking gun 1 Hand saw 1 Drill 1 Hole Saw (⅝ of an inch) 2 2’’x 8’’x 24’’ wood boards* 4 4’’x 4’’x 28’’ wood boards* PVC sealer / cement Measuring tape Air Dust Nylon Filter (1 inch thick)** Sediment (sandy soil) Native wetland plants (discussed later)

* The length of the boards should determined by the length of the bucket. **Enough to cover what is mentioned in the procedures.

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The quantities on the previous page are the minimum requirements necessary for the construction of two troughs. These quantities may be increased if you wish to perform the experiment on a larger scale. If possible this is suggested since it allows for the collection of more data which will increase your accuracy. Wetland Drawings:

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Procedure for Construction:

Set the input bucket at one end of the designated space. Drill two holes, ⅝ of an inch in diameter, into the side of the input bucket. These holes should be positioned 1 to 1 ½ inches from the bottom, spaced far enough apart so that, when inserted, the PVC pipes will easily drain into the troughs. Next cut eight 7-inch pieces of pipes using the hand saw. Connect one PVC pipe to either side of each ball valve. Insert one end of the assembled pipe structures approximately 1 inch into the bucket. Seal the gap between the bucket and the pipes using the caulking. A proper seal is essential to prevent leaks. Glue an elbow to the other side of the pipe structure using PVC cement, face the elbow so it will drain down.

Prop the input bucket 7 inches off the table using two 4’’x 4’’x 28’’ wood boards stacked on top of each other. Once the bucket is in position use the 2’’x 8’’x 24’’ wood board as a support beneath the pipes.

Place the troughs directly beneath the pipes that are connected to the input bucket. Next drill holes, ⅝ of an inch in diameter, at one end of each of the troughs. Cut two 2 ½-inch pieces of PVC pipe and insert them roughly 1 inch into the end of each trough. Use caulking to close the gaps between the pipes and the trough. Using the PVC cement, glue an elbow on both of these pipes. Glue the last two pipe structures to the elbows; face the elbows so they will drain down.

Place the pieces of the filter about 5 inches in from either end of the troughs, so that the water will flow through it upon entering and exiting the wetland. The filters prevent plant and organisms from entering or leaving the wetland. Also, line the inside of the input bucket with the filter to pre-vent the pipes and control valves from clogging. Place enough sediment in the bottom of the troughs to cover about 1 ½ inch of the trough. Plant the plants in the sediment (refer to the Suggested Vegetation).

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Add water to the system using the valves to regulate the inflow and outflow. Make certain enough water is left in the wetlands to support the hydrophytes. After the wetland is finished allow approximately two weeks time for the plants to adjust to their new environment and become established in the wetland, before beginning the tests.

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Duckweed

Common Cattail

Floating Primrose Willow

Bermuda Grass

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Properties of the Pollution:

The pollution should consist of a biodegradable solution, such as biodegradable laundry detergents or soaps, this will simulate household graywater. About 5 gallons of the pollution should be used for the experiment, or as much as the bucket can hold. Suggested Vegetation:

The plants used in the wetland should be hydrophytes, plants that require constant water. Plants may be purchased at any nursery that sells pond plants. Suggested species are Typha latifolia (common cattail), Ludwigia peploides (floating primrose willow), Lemnaceae sp. (duckweed), and Cynodon dactylon (bermuda grass). They are listed in order of their importance to the function of the wetland. The Typha latifolia is essential to the wetland because it is belived to perform most of the purification of water.

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Glossary:

Abiotic: The non-living component of the environment that affects ecological functions. I.e. rock, water, temperature, climate, pressure, wind, etc. [Latin: a non + bio life] Ammonium: An ion, NH4 +, derived from ammonia by combination with a hydrogen ion, that can be an inorganic source of nitrogen. The primary sources of ammonia in water are bacterial decomposition of organic waste, and excretions of aquatic animals. Ammonium serves as a source of nitrogen, which is used as a nutrient for growth for aquatic plants. Biodegradable: The ability of a substance to be broken down either physically or chemically by micro-organisms. For example, plant matter is biodegradable; however, plastic is non-biodegradable. Bio-filtration: The distribution of settled sewage on a bed of inert granular material through which it is allowed to percolate. In doing so, the runoff is aerated, thus allowing aerobic bacteria to reduce its BOD. Biological Oxygen Demand (BOD): A measure of the quantity of dissolved oxygen used by the bacteria as they break down organic wastes. Biotic: The living component of the environment that affects ecological functions, i.e. trees, animals, and fungi. Dissolved Oxygen (DO): The amount of oxygen dissolved in water and therefore freely available to aquatic organisms such as fish for respiration. Graywater: Household drain water from sinks, tubs, and washing. Requires some degree of treatment before it can be reused because it is likely to contain soap, contaminants from the kitchen, etc. Can be used for irrigation purposes or for flushing toilets.

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Hydrophyte: A plant adapted to growing in water, waterlogged soil, or on a substrate that becomes flooded on a regular basis. [hydro water + phyte plant] hydrophytic, adj. Iron: An important nutrient for many organisms, present in most natural waters. Soil and rock provide the most common sources of iron in water. Micro-organisms: Organisms of microscopic or sub-microscopic size, too small to be seen with the unaided eye, i.e. bacteria, protozoa or protists. Nitrate: Formed when ammonia is degraded by micro-organisms in soil or groundwater. Nitrate occurs in water as Nitrate (NO3

-), Nitrite (NO2-),and Ammonium

(NH3). Organic Materials: Carbon containing substances found in plants, animals, and their remains. pH (Potential of Hydrogen): A measure of the acidity or basicity (alkalinity) of a material when dissolved in water. It is expressed on a scale from 0 (more acidic) to 14 (more basic), with 7 being neutral. Phosphate: A nutrient that acts as a fertilizer for aquatic plants. When nutrient levels are high, excessive plant and algae growth creates water quality problems. Phosphorous occurs in natural waters in the form of phosphates (PO4

3-). Over half of the phosphates in lakes, stream and rivers come from detergents. Potable water: Water that is free from impurities in amounts sufficient to cause disease or harmful physiological effects. Better known as drinking water. Remediation: The treatment process for removing the contaminants in the water.

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