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Analysis of Biochemical Oxygen Demand, Phosphate and Nitrate

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  • Introduction:

    The purpose of this lab experiment is to introduce students to research-based labs through active learning; that is, the lab experiment will be conducted more like actual research rather than a traditional laboratory experiment. Additionally, what sets this experiment apart from other traditional experiments is that we do not know what the outcome will be. The data that is generated from this experiment will be collected and displayed for the world to see and could possibly drive further research into the water quality of a particular location of the Saginaw Bay Watershed.

    Students will be collecting water samples and performing real-time analysis to collect data for several water quality parameters, including 5-day biochemical oxygen demand, pH, turbidity, total phosphorus, soluble reactive phosphorus, conductivity, and dissolved oxygen.

    In this particular lab, students from different lab sections will collect and prepare samples for students in other lab sections. Monday lab students will be collecting water samples in the Saginaw Bay Watershed and preparing them for phosphate analysis for the Wednesday lab sections the following week, while Wednesday lab sections will be collecting and preparing water samples for a five-day biochemical oxygen demand (BOD5) test for the Monday lab sections. It is very important that you use caution when sampling and preparing the samples for analysis. The other water quality parameters will be obtained on-site through an NSF-funded Hydrolab, which is a multiparameter probe, as well as in the lab.

    Note on Items That Will Be Graded:

    Unlike prior lab experiments, this experiment will involve multiple graded assignments. 1) You will be performing a literature review for prior Saginaw Bay Watershed water quality reports. This background research will be organized and turned in in the form of an annotated bibliography. More information on this literature search will be described later. 2) You will be developing a sampling plan and turning it in for a grade. 3) You will develop a chain-of-custody form and turn it in for a grade. 4) You will turn your report in for a grade. 5) You will fill out a peer evaluation form for your lab group of 3 or 4 students. This evaluation form will impact your overall lab grade in the course and will serve as a layer of accountability.


    Water quality measurements are vital to understand the health of an aquatic ecosystem. One of the most important water quality parameters relates to the nutrient phosphorus. In a water system, phosphorus generally exists in the chemical form of phosphate. Phosphorus is vital for plant growth; however, too much phosphorus can be detrimental to a body of water. As nutrient levels increase, plant growth can increase as well. In some cases, harmful algal blooms (HABs) result, which release toxins into the environment (microcystin being the most harmful). In reality, HABs are not actually algae; rather, they are cyanobacteria. Another effect of heightened nutrient levels and algal growth is that of decreased dissolved oxygen levels. As organic matter decompose, oxygen is used up from the surrounding water, depleting the water of its dissolved oxygen. This can result in anoxic zones in which organisms cannot survive.

    Other indicators of water quality include turbidity and conductivity, which we will be measuring in the laboratory experiment. Turbidity is a measurement of the suspended matter in a liquid. The more suspended matter, the greater the light scattering. Therefore, heightened turbidity can be a marker for poor water quality. As soil is discharged into a water system, this will result in heightened turbidity. As more nutrients adsorb to soil, once the soil is discharged into the water system, these nutrients can desorb and become nutrient sources for plant growth. Conductivity measures the relative amounts of ions in a solution. Therefore, higher conductivities can be correlated with heightened nutrients. These parameters, however, are not always related. It is possible to have heightened turbidity without heightened nutrient levels or heightened conductivity. As always, there are multiple variables that influence the water quality parameters that we measure.

    We will also be measuring the 5-day biochemical oxygen demand (BOD) The BOD measures the amount of dissolved oxygen needed by aerobic biological organisms in a body of water to break down organic material over a 5-day time period.

    In this laboratory, we will be measuring the relationships between the collected data and draw an overall conclusion as to the health of the Kawkawlin River at the sampling sites.

    Step 1: Developing a Sampling Plan

    (To be completed in class on week 1 of this experiment). Due the following week at the beginning of the lab period.

    Based on our in-class exercise involving sampling, each group will need to develop a sampling plan under the following practical guidelines:

    • Each group must choose one sampling site
    • The sampling site must be on the Kawkawlin River
    • The sampling site must be within a 25-30 minute drive from SVSU
    • The sampling site must be accessible by bridge or easily accessible on the bank
    • Each sampling plan must be approved by the instructor

    The sampling plan should be typed, include the group members’ names and signatures, and include the following information:

    • GPS Location with directions from SVSU to the sampling sites
    • Volume and container that will be used for samples
    • What type of sample (i.e. grab, composite)
    • How many samples per sampling site

    Step 2: Chain of Custody Form

    (To be completed in lab on week 1 of this experiment and filled out and turned in at the end of week 2)

    Here, we will brainstorm as a group and determine what the key components of the chain of custody form entail, and we will create a google spreadsheet for us to fill out our data.

    Step 3: Sample Collection and Analysis, Monday

    (To be completed in lab during Week 2):

    The Monday lab students will collect samples from 2 separate locations as defined in your sampling plan. On site, you will determine the temperature, conductivity, dissolved oxygen, pH, and turbidity. Each group of students per lab section will be responsible for properly labeling and documenting their sample preservation on their chain of custody form for the other lab section.

    Samples will be placed on ice immediately after collection. Once reaching the lab, samples will be tested for soluble reactive phosphorus (SRP) and total phosphorus (TP) using the Hach Ultra-Low method with a Hach DR6000 spectrophotometer. In order to measure the SRP, the sample must be filtered through a 0.45 µm filter. Approximately 10 mL of the sample should be filtered into a secondary container for use during the SRP analysis to be completed during the normal lecture time on Wednesday. For TP, the sample must not be filtered. Therefore, the TP must be prepared immediately when returning to the lab, followed by filtering and SRP analysis.

    In addition, samples will be prepared for a 5-day Biochemical Oxygen Demand. In order to prepare the samples, the following must be done:

    • Place 0 mL (Blank), 40 mL, 60 mL, 80 mL, 100 mL and 120 mL of sample into 300 mL BOD plastic bottles, add one BOD growth medium packet (Hach product number 1416066, and dilute to volume.
    • Stopper each BOD bottle and mix by inversion
    • Measure the dissolved oxygen concentration (in ppm) in each of the six bottles with the dissolved oxygen probe
    • Place the stopper in each bottle, being careful to not trap air bubbles
    • Add drops of dilution water to the stopper to create a water seal
    • Snap the cap on each BOD bottle and incubate for 5 days at room temperature

    Sample Preparation and Analysis, Monday

    (To be Completed in the Lab During Week 3):

    During the Monday lab of week 3 for lab experiment 5, samples that have been incubating since Wednesday in the analytical lab will be pulled out of refrigeration and the 5-day biochemical oxygen demand (BOD5) test performed. To perform this analysis, a dissolved oxygen probe will be used. Remove the plastic cap and the acrylic stopper, pour a small amount of water out of the bottle, and place the dissolved oxygen probe in the water to record the reading. After performing your BOD tests, set the caps and stoppers aside (They will be reused) and pour the contents of your BOD bottle down the drain and dispose of your bottles.

    Note on 5-day BOD Calculation:

    In order to calculate the 5-day BOD, you will need the initial and final dissolved oxygen readings (in mg/L), as well as the decimal fraction of sample used. For example, for the bottle that included 40 mL of sample will have a decimal fraction of (40/300 = 0.133). The calculation is as follows, where D0 is the initial dissolved oxygen reading on the day of preparation in units of mg/L; Df is the final dissolved oxygen reading after 5 days of preparation; and P is the volumetric fraction of the sample compared to the total volume of the BOD bottle (300 mL):

    \[\textrm{5-day BOD} = \mathrm{\dfrac{D_0 – D_f}{P}} \tag{Equation 5-1}\]

    It is expected that the dilution water blank (zero mL of river water sample) will have an insignificant change in dissolved oxygen, while the five samples containing river water will have a greater BOD than the dilution water blank. It is expected that each of the five samples will have BOD levels that are similar, which is why an average BOD will be reported as well.

    Lab Report Instructions:

    One lab report per group will be turned in for this lab. Your lab report will contain the following:

    • Chain of custody form (Already turned in at the end of week 2)
    • Excel spreadsheet consisting of the following information for each sampling site for each lab. Be sure to differentiate dates and lab meeting time (9:30AM or 2:30PM). Note, even if you did not personally collect these parameters, each group should include all in their report.
      • Hydrolab data
        • Temperature (degrees C)
        • Dissolved Oxygen (mg/L reading only)
        • Turbidity (NTU)
        • Conductivity (µS/cm)
        • pH
      • Total Phosphorus (PO43- - P, ppb)
      • Soluble Reactive Phosphorus(PO43- - P, ppb)
    • Another excel spreadsheet consisting of the 5-day Biochemical Oxygen Demand data for each set of six bottles for each group at each site. Be sure to differentiate lab meeting time (9:30AM or 2:30PM). The BOD data will include the following:
      • Initial reading for each sample (day of preparation)
      • Final reading for each sample (5 days after preparation)
      • 5-day BOD level based on calculation described above (Equation 6-1)
      • Average 5-day BOD level for each group’s set of 5 samples (Blank not included).

    In addition, answer the following questions:

    • Is there a significant difference between the SRP levels the day of sampling and after refrigerating several days? Show your work to receive full credit.
    • Does the BOD level for that particular site correlate with the phosphorus level? Support your answer with logical reasoning.
    • Does the BOD level for that particular site correlate with the hydrolab data? Support your answer with logical reasoning.
    • In general, is the Kawkawlin River a healthy body of water at the time of sampling? This will require some digging on your part. You can look up past Saginaw Bay Watershed water quality reports. Be sure to include appropriate references.

    Completion of a Peer Evaluation Form

    • Each individual per group should fill out a peer review form evaluating each group member. The instructor will maintain these and they will only be seen by the instructor. They will not be released to any other group members. The evaluation forms will be used to inform the instructor about each group members’ level of participation.

    Timeline of Items Due and Due Dates

    • Literature Review of Prior Water Quality Data Due Monday, October 21
    • Sampling Plan Due Monday, October 15
    • Chain of Custody Form Due Monday, October 15
    • Final Report Due Monday, November 6
    • Peer Evaluation Form Due Monday, November 6

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