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To do this lab: 1. Read the Lab 7 Instructions document below. 2. Open the Lab 7 Report document, and answer the Pre-Lab questions. 3. Choose 2 different surface water sites to sample. Ideas: Upstream and then downstream from a wastewater discharge, before and after 2 rivers meet, a lake and a river that feeds it, two different lakes or ponds, etc. You cannot sample tap water (from a faucet). Extra credit to anyone that can sample the Minnesota River and Mississippi River, and compare the two. Wherever you sample, be safe! 4. Following the Lab 7 Instructions, sample the first site, perform the tests, and record the data. (take a photo of the sample site) Repeat this for the second site. 5. Answer the rest of the Lab 7 Questions.

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LAB 7 – Water Quality: Biological Characteristics of Water Pre-laboratory Questions – Answers can be found in the Lab Instructions Background, or in your text. 1. Why do we test water for ph, DO, BOD, turbidity and coliform? 2. Discuss human activities that could affect the quality of the natural surface water at your test site. Specifically address temperature, dissolved oxygen, coliform bacteria, BOD and turbidity. 3. Briefly describe how each of the following parameters interacts with at least one other factor/parameter (for example, how does temperature affect DO?). a. Temperature b. Dissolved Oxygen c. Coliform Count d. Biochemical Oxygen Demand e. Total Suspended Solids or Turbidity Data Table: Water Quality: Biological Data and Site Photos Test Site Site Name or GPS location Elev. (ft) Temp (°C) Dissolved Oxygen (ppm) DO % Saturation BOD (ppm) pH 1 Coliform Bacteria (positive or negative ) Water Vol. for Turbidity Turbidity (NTU) Notes on Site 1 - insert a picture of the body of water you sampled 2 Notes on Site 2 - insert a picture of the body of water you sampled Observation Questions 4. Describe your sampling location. Was the water still or running? If it was moving, how fast (generally)? Was the water clear or cloudy? Was there any algal growth? What about the surrounding land? Was it urban, suburban, or rural? Grassy or forested? 5. How could your sampling location affect the quality of the water in your sample? Discussion Questions 6. RESULTS - Using your data above, How healthy are the water bodies you tested? (Hint, discuss your data results and use them to come to a conclusion). 7. Do all “healthy” bodies of water have the same biological data? Explain. 8. Residents in a riverfront community have complained about a recent decrease in water clarity. Local ire has fallen on a local pig farm that has recently expanded its operations upstream of the town, but the pig farm claims that it is taking actions to protect the waterway and is pointing to the new shopping center across the river as a source of increased sedimentation. What water quality tests could you perform to determine the source of the increased turbidity? How would you expect the samples to differ if the pig farm is responsible? 9. If you could do a biological water quality test on surface water anywhere in the world, where would you test and why? Quality of Surface Waters: Biological Carolina Distance Learning Investigation Manual ©2015,Carolina Biological Supply Company Table of Contents Overview ....................................................................................................... 4 Outcomes ..................................................................................................... 4 Time Requirements ...................................................................................... 4 Background .................................................................................................. 5 Materials ........................................................................................................ 9 Safety ........................................................................................................... 10 Preparation ................................................................................................. 10 Activity 1: Temperature Measurement .................................................. 11 Activity 2: Sample Collection .................................................................. 12 Activity 3: Dissolved Oxygen and Biological Oxygen Demand Test 12 Activity 4: Turbidity Test ............................................................................. 15 Activity 5: Coliform Bacteria Test ............................................................ 15 Disposal and Cleanup .............................................................................. 16 Data Table: Biological Water Quality .................................................... 18 ©2015,Carolina Biological Supply Company Overview Students will collect water samples from local waterways and test them for biological factors that governments and scientists use to monitor the health of natural waterways. Outcomes • Practice proper water sample collection techniques. • Conduct chemical and biological analyses of field water samples. • Compare water test results to environmental standards. Time Requirements This lab requires the collection of water samples at two sites, travel time is in addition to any times listed below. Preparation.............................................................................................40 minutes Activity 1: Temperature Measurement ...............................................10 minutes Activity 2: Sample Collection ...............................................................10 minutes Activity 3: Dissolved Oxygen and BOD Test .......................................20 minutes + 5 days Activity 4: Turbidity Test .........................................................................10 minutes Activity 5: Coliform Test .........................................................................10 minutes + 48 hours ©2015,Carolina Biological Supply Company Background Water is one of the most common compounds on earth, and one of the most precious. Water covers approximately 75% of the earth’s surface, but only 2.5% of this is fresh water, and only a small fraction of that 2.5% is accessible for use and consumption. We depend on a clean water supply for drinking, cleaning, sanitation, recreation, and manufacturing. Public health officials and biologists have developed tests that can be used to monitor the quality of natural waters and to locate sources (and potential sources) of water pollution. In this activity, you will collect samples from local waterways and test and evaluate water quality on the basis of several chemical and physical characteristics used by scientists to analyze natural waters. Nitrogen and Phosphorous Water is an important agent for dissolving and transporting many nutrients (essential compounds for life) through the environment. The growth of primary producers (and, by extension, all organisms in the food chain) in any environment is limited by the scarcity of a particular resource. That resource can be physical, such as light or space, or it can be a nutrient. In terrestrial and freshwater environments, nitrogen or phosphorous often is limiting. Most fertilizers contain nitrogen and phosphorus as watersoluble nitrates (NO3–) and phosphates (PO43–). Rain washes these water-soluble nitrates and phosphates from cultivated land into surface waters where they promote the rapid growth of plants and algae, which speeds up the rate of eutrophication. Eutrophication is a natural, usually gradual process that increases sediment and nutrient availability in water bodies. Human activities lead to cultural eutrophication. Fertilizer runoff from lawns and farms, inadequately treated municipal wastewater, and poorly maintained septic systems all contribute to cultural eutrophication. In agricultural areas with dense animal populations, animal waste adds nitrogen and phosphorus to surface waters. Heavy rain can cause a sudden influx of nutrients, which can lead to rapid and uncontrolled growth in aquatic algae and plant populations. When these organisms die, bacterial-mediated decay depletes water oxygen levels, kills sensitive aquatic organisms, and contributes to sediment buildup. If nitrogen is the limiting nutrient, such as in most brackish or marine waters, nitrogen levels ≥ 0.3 parts per million (ppm) can accelerate natural eutrophication. Phosphorous is usually the limiting nutrient in freshwaters. Phosphorus levels ≥ 0.1 ppm can accelerate natural eutrophication in freshwaters. Temperature Temperature affects many chemical and biological processes. It modifies the solubility of oxygen in water, the rate of photosynthesis in aquatic plants, the metabolic rate of aquatic organisms, and the susceptibility of aquatic species to toxins and disease. Water temperature varies naturally with season and depth, but human activity also can affect temperature in significant ways. For example, heated water from surfaces such as sunlit parking lots, roads, sidewalks, and from industrial processes that use water as a coolant can flow into nearby creeks, streams, or rivers. This thermal pollution can destroy some types of aquatic life. Water loses heat very slowly, so thermally polluted water should be given significant dilution and time to cool before discharge into natural surface waters. Heated water also facilitates the rapid growth ©2015,Carolina Biological Supply Company of some aquatic plants and algae, so thermal pollution can accelerate the rate of cultural eutrophication. Dissolved Oxygen Dissolved oxygen (DO) is essential for biotic diversity in a body of water. The amount of oxygen in water is usually expressed in ppm. Much of the DO in water comes from the atmosphere. Atmospheric oxygen dissolves via diffusion into the water at the surface. Wind and waves stir the water, which dilutes the DO concentration at the surface by mixing, and subsequently increases the amount of oxygen that can dissolve at the surface. Another important source of oxygen in water is photosynthesis performed by aquatic plants and algae. Some of these aquatic photosynthetic organisms are rooted or attached to the streambed, whereas others are planktonic (live in the water column). The amount of photosynthetically generated DO in the water typically peaks in the afternoon when the sun reaches zenith and the most light penetrates the water. Turbidity, or murkiness resulting from a large number of particles in the water, can lower DO levels by reducing photosynthetic rates due to limited light penetration. Many factors influence the amount of DO water can hold. Water with lower temperatures can contain higher oxygen concentrations. Freshwater can hold more oxygen than saltwater. Another factor is altitude and atmospheric pressure; higher gas pressures result in higher gas concentrations dissolving into a liquid. Atmospheric pressure is greatest at sea level, so more oxygen dissolves into water at sea level than at higher elevations. The amount of DO in a body of water can vary greatly due to all these factors. After a DO measurement is taken from a water sample, a percent saturation value can be calculated by comparing the amount of oxygen the water contains at that moment with the maximum amount of oxygen that it could contain at that temperature. Respiring organisms such as animals and bacteria significantly reduce DO levels. Bacteria in animal waste are a major concern in waterways that receive runoff from fields and pastures where livestock are kept. When oxygen becomes depleted in the water, it is said to be anoxic. Anoxic conditions cause marine organisms to suffocate. Coliform Bacteria Coliform bacteria populate the intestinal tract of warm-blooded animals, and disperse throughout the environment along with fecal material. When an animal defecates, large numbers of coliform bacteria (called fecal coliforms) are present in the feces. Coliforms also can be released to the environment from a decomposing body. The presence of large numbers of coliforms in a water sample is a good indication that animal or human waste has polluted the water. Most coliforms do not cause disease, except for a few Escherichia coli strains. However, many pathogenic bacteria and parasites are spread through feces (e.g., those causing typhoid fever, hepatitis, gastroenteritis, and dysentery), and the presence of coliforms indicates that harmful pathogens might be present. Determining the presence of coliforms in drinking water and in swimming areas is a very important tool for protecting public health. ©2015,Carolina Biological Supply Company This is accomplished by collecting a water sample, and inoculating some of the sample on nutrient rich medium that supports coliform growth. After several days, the culture will show bacterial growth. This is the first step in determining the risk of animal or human waste contamination of a body of water. Additional tests can be performed to distinguish between total coliforms, which can have a number of sources, and fecal coliforms, which originate in animal and human intestines. There are potential risks associated with culturing pathogenic bacteria. Therefore, this activity will only examine total coliforms. Total Suspended Solids Total suspended solids (TSS) is a term used to describe and measure the material left behind after a water sample has been filtered and evaporated. Dissolved materials and nutrients in natural water come from a variety of sources. Large concentrations of dissolved solids affect cellular nutrient transport, alter the taste of water, and interfere with industrial processes by fouling pipes and filters. Calcium and magnesium are two minerals commonly dissolved in water. A water-insoluble carbonate forms when aqueous calcium or magnesium hydrogen carbonate is heated. Industrial boilers reach high temperatures and generate solid deposits of magnesium carbonate and calcium carbonate, commonly called scale. These water-insoluble deposits reduce the efficiency of machines and are expensive to remove. TSS measurement requires the use of an analytical balance, which is not practical in the current setting due to cost. Turbidity The turbidity test measures water clarity. Fine particles such as eroded soil, and microscopic organisms such as phytoplankton, contribute to water turbidity. Turbid waters clog fish gills, hinder disinfection processes, and carry entrapped pollutants downstream. Turbidity also blocks sunlight, which prevents photosynthesis below the surface and increases surface water temperature. Turbidity is measured in Nephelometric Turbidity Units (NTU). In this exercise, a modified turbidity tube will be used. A water sample is drained from the tube slowly, and the black and white pattern of a Secchi disk at the bottom of the tube becomes visible. At this point when the disk becomes visible, the sample volume is recorded and translated into NTU. If the pattern is visible when the tube is full, turbidity will be estimated using a saturation chart. Biochemical Oxygen Demand Aerobic bacteria that decompose organic material require oxygen. In general, the more organic material there is in the water the oxygen demand will increase. When biochemical oxygen demand (BOD), sometimes called biological oxygen demand, surpasses the amount of oxygen that can be added to the water, the water becomes anoxic. High BOD coupled with low DO input causes water quality deterioration, which can result in the death of many fish and animals. An increase in nitrates and phosphates in a body of water often increases the growth ©2015,Carolina Biological Supply Company rate of plants and algae. These aquatic organisms subsequently die and are decomposed by aerobic bacteria, which consume oxygen during the process. Another cause of increased BOD is a consequence of fertilizer runoff. An influx of fertilizers and sudden growth of aquatic producers may produce a brief spike in photosynthetically generated DO levels, but BOD rises as the aquatic producers begin to die. pH Water contains hydrogen ions (H+) and hydroxyl ions (OH–). The concentration of H+ ions determines the pH. If the concentration of H+ ions is higher than that of OH– ions, the pH is less than 7.0 and is considered as acidic. Thus the greater the concentration of H+ ions, the lower the pH will be. If the concentration of H+ ions is lower than that of OH– ions, the pH is greater than 7.0 and is considered as alkaline (or basic). The pH scale is from 0 to 14. The pH scale is logarithmic; as the pH increases by one whole number, the H+ ion concentration decreases 10 times. For example, a pH change from 5 to 6 indicates a tenfold decrease in H+ concentration, and a pH change from 4 to 6 indicates a hundredfold decrease in H+ concentration. Water rarely exists at the neutral pH of 7. Saltwater typically is slightly basic, with pH of 8 or higher. Freshwater usually has pH of 6.5, but this varies greatly between bodies of water. Organic materials can affect the pH of water. Fallen leaves often contain tannic acid, which stains the water brown and reduces pH. Some bacteria create acidic waste products that lower the pH of water. Acid rain has a widespread effect on natural waterways in the eastern United States. Oxides of nitrogen and sulfur are released into the atmosphere by burning fossil fuels. They mix with atmospheric water and form nitric and sulfuric acids. These acids are mixed with rainwater and delivered to natural waterways and soils. Normal rain has pH of 5.6 (due to dissolved carbon dioxide from the atmosphere forming carbonic acid). Rain is considered to be acid if the pH is lower than 5.6. Many freshwater organisms have adapted to a specific pH range. For some organisms, the optimum pH range is quite narrow. A sudden or drastic pH change will kill some of these organisms or reduce survival and reproduction, which disrupts the food chain and the aquatic community. Lower pH levels also can release heavy metals from the surrounding bedrock into the water. Once dissolved, these metals can interfere with the uptake of dissolved oxygen by aquatic organisms. ©2015,Carolina Biological Supply Company Materials Included in the materials kit for Biological Water Quality Factors LaMotte Dissolved Oxygen TesTabs™ (in the small pouches) LaMotte Coliform TesTabs™ (in the large glass vials wrapped in bubble wrap) 125 mL collection bottle Small glass sample vial Sample comparison chart Needed from the equipment kit 100 mL graduated cylinder Thermometer Test tube rack Plastic pipettes Permanent marker Needed, but not supplied Aluminum foil Timer or stopwatch GPS or GPS-enabled phone (recommended) Household bleach solution ©2015,Carolina Biological Supply Company 2 packs of 4 2 3 Safety Gloves and safety glasses should be worn any time you are dealing with chemicals, including the TesTabs™. Natural water samples can contain harmful contaminates and organisms. Wear your gloves and safety glasses during sample collection as well. Read all the instructions for this laboratory activity before beginning. Follow the instructions closely and observe established laboratory safety practices, including the use of appropriate personal protective equipment (PPE) described in the Safety and Procedure section. Safety while performing field work is of greatest importance. Never conduct field work on your own, especially in remote areas; always take a responsible partner with you. Prepare and leave a field safety plan, complete with planned survey locations and expected return time, with a responsible party. Wear appropriate clothing (long pants and close-toed shoes), sunblock, and insect repellant if necessary. You may be working in waterways; therefore, rubber boots or waterproof shoes would be optimum if available. Be familiar with potential hazards in your chosen survey areas. Never enter a wetland area if there is a flood risk. Please review the Field Work portion of the Lab Safety Manual or your school’s specific safety guidelines for more information. Make sure that you are healthy enough to participate in field work. If in doubt, consult your medical practitioner. Do not eat, drink, or chew gum while performing this activity. Wash your hands with soap and water before and after performing the activity. Clean up the work area with soap and water after completing the investigation. Keep pets and children away from lab materials and equipment. Preparation 1. Select your field sites for the following activities. This kit has enough test materials for two locations. You will perform activities 1, 2 and 4 for your first sample in their entirety before collecting a second sample at a second site and performing the activities again. Activities 3 and 5 must be started before collecting your second sample, but the activites can run concurrently for both samples once started. 2. Ideal water sources include shallow creeks, rivers, springs, or ponds. Locate ©2015,Carolina Biological Supply Company several possible test sites on a map. Make sure these areas are accessible to you. Sites located in county or state parks are accessible, but you will need to acquire permission from landowners before visiting privately owned sites. 3. Activities 1 and 2 will be performed at the field site. Activity 3 will be started at the field site and completed after 5 days of incubation. Activities 4 and 5 can be started at the field site or within 23 hours of sample collection. It is common practice to use a cooler to transport samples because they need to be kept cool and dark to prevent post-sampling photosynthesis from skewing test results. 4. Select an area to test based on the way the watershed flows. Choosing sites above and below the convergence of two waterways helps pinpoint the source of any pollution found in the samples. Prepare a map that marks the chosen sites. Each site can be identified with latitude and longitude using a GPS or GPSenabled phone. For accurate measurement of dissolved oxygen (Biological Water Quality), determine the approximate altitude of the sample location. There are several web sites that can help you identify the altitude of a pinned location (e.g., http://www.freemaptools.com/elevation-finder.htm and http://www.daftlogic.com/sandbox-google-maps-find-altitude.htm). There are also free apps for smart phones, such as “My Altitude” for iOS and “Get Altitude” for Android. 5. Gather all materials that you need before starting the field trip. Make sure that you have the LaMotte TesTabs™, the water-resistant sample comparison sheets, Table 1 and Figure 1, the data table, a thermometer, sterile water sample collection bottles, aluminum foil, gloves, goggles, a marking pen, and a cooler with ice if you will be transporting samples. 6. At the test location, collect each sample from a location that is representative of the waterway per the activity instructions. Best results will be obtained if samples are collected from locations that are not close to storm water drains and drainage ditches. For the most reliable results, water samples should be collected midstream. Water near the shore generally is not representative of an entire stream or river, because some runoff is highly concentrated and has not yet mixed with the main body of water. In some situations, (e.g., if the water is too deep), it may not be possible, feasible, or safe to obtain an ideal sample. In this case, collect a water sample from a safe location. Activity 1: Temperature Measurement 1. While wearing gloves, submerge the thermometer 4 inches below the surface of the water. 2. Keep the thermometer in the water for 2 minutes. Remove it from the water and ©2015,Carolina Biological Supply Company immediately record the water temperature at this site in the Data Table. Activity 2: Sample Collection 1. Remove the lid from the 125 mL sampling container. 2. Rinse the container two or three times with water from the sample site. 3. Replace the lid. 4. Plunge the sample bottle several inches under the surface of the water. 5. Remove the lid. 6. Turn the bottle into the current, if there is one, and allow the water to fill the container. Hold the sample container underwater for at least 30 seconds. 7. Replace the cap while the bottle is still submerged. There should be no air left in the bottle. 8. This sample will be used for Activities 45. Activity 3: Dissolved Oxygen and Biological Oxygen Demand Test 1. This test should be performed at the test site. Wear gloves when working with test site water. 2. Measure and record the temperature of the water site as described in Activity 1. 3. Submerge a small glass tube several inches under the water surface. Make sure that the water completely fills the tube. Remove it from the water. 4. Drop two Dissolved Oxygen TesTabs™ into the tube. Water will overflow the tube. 5. Place the cap on the tube and tighten it without allowing any air bubbles into the tube. 6. Mix the sample by repeatedly turning the tube upside down and righting it until the tablets are dissolved. This will take approximately 4 minutes. 7. Incubate the sample for 5 more minutes. 8. While sample 1 is incubating, repeat step 3. Cap the tube and tighten it without allowing any air bubbles into the tube. Wrap the tube in aluminum foil and set aside at room temperature for 5 days. This is sample 2. 9. After sample 1 has incubated for 5 minutes, compare the sample color with the color chart. Record the result as ppm dissolved oxygen in the data table ©2015,Carolina Biological Supply Company The remainder of this procedure can be completed at home. 10. Calculate dissolved oxygen percent saturation as follows: a. To obtain the corrected DO value, multiply the DO (ppm) value by the correction factor that corresponds to the altitude of the test site. Refer to the Altitude Correction Factor Table (Table 1). b. Print out Figure 1. Locate the corrected DO value on the “Oxygen (ppm)” axis, and mark the point. c. Mark the temperature of the sample on the “Water temperature (°C)” axis of Figure 1. d. Using a straightedge, connect the two points. The intersection of the line with the “% Saturation” value is the corrected dissolved oxygen percent saturation level for the sample. e. Record the DO % Saturation value in the data table. 11. After 5 days, unwrap sample 2 and repeat steps 410. 12. Calculate the BOD by taking the difference between the two samples. BOD is reported in ppm. Record the BOD in the data table. ©2015,Carolina Biological Supply Company Table 1: Altitude Correction Factors Equivalent Altitude (ft.) 0 542 1,094 1,688 2,274 2,864 3,466 4,082 4,756 5,403 6,065 6,744 7,440 8,204 8,939 9,694 10,472 Figure 1: Dissolved Oxygen Saturation ©2015,Carolina Biological Supply Company Correction Factor 1 0.98 0.96 0.94 0.92 0.90 0.88 0.86 0.84 0.82 0.80 0.78 0.76 0.74 0.72 0.70 0.68 Activity 4: Turbidity Test 1. Wear gloves when working with test site water. 2. Fill the graduated cylinder from your equipment kit to the 100 mL mark with water from your collection bottle. 3. Remove the cylinder from its base and place it over the black and white pattern on the turbidity chart. 4. Look straight down through the water sample, and observe the pattern through the water column. If you cannot see the pattern, proceed to step 4a. If you can, proceed to step 4b. a. Pour off a small amount of water (
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