# 2.2: Day 2 Procedures - Collection of Water Samples

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Day 2 - Collect (DO) Water Samples from the Charles River--Titrate the Samples with Standardized Sodium Thiosulfate Solution

### TAs Prepare Manganous Sulfate Solution and Alkali-Iodide-Azide Reagent22

For the Manganous Sulfate solution, dissolve 364 g of MnSO4 x H2O into distilled water, filter and dilute to a volume of 1 Liter. The MnSO4 solution should not give a color with starch when added to an acidified potassium iodide (KI) solution.

For the alkaline iodide-azide reagent dissolve 700 g of KOH and 150 g KI in distilled water and dilute to 1 Liter. Then, add a solution consisting of 10 g NaN3 dissolved in 40 mL of distilled water.

#### Collection of Samples from the Charles River23

Follow the instructions of the TA for the time to meet at the pre-determined collection site on the Charles River. Collect the samples to be tested into special 300 mL BOD bottles taking precautions not to introduce air bubbles into the sample collection bottles. Hold the special designed water sampling device snapping the bottle in place then holding the device with BOD bottle approximately one arm length under water and allow the collection bottle to fill slowly with no air bubbles. Once filled, carefully raise the device and insert the glass stopper making sure that no air bubbles are present in your sample especially below the glass stoppered neck area. If you see any air bubbles you should discard the sample and start over again. Once you have collected your samples, carry the samples back to the laboratory for the workup and titration procedure, which follows.

#### Azide – Winkler Method Workup and Titration Procedure

1. Carefully remove the stopper from the 300 mL BOD collection bottle avoiding aeration of the sample. Using a calibrated pipette, add just below the surface of the liquid 2 mL of 2.15M manganous sulfate solution, which has been prepared by the TAs. Pipette the solution in slowly to avoid any introduction of air into your collection bottle.
2. Carefully repeat the above procedure again adding just below the surface of the liquid 2 mL of alkaline-iodide-azide reagent, which has been previously prepared by the TAs.
3. Stopper the collection bottle being careful not to introduce any air into the collection flask and noting that the collection flask now contains an excess of liquid. Holding the stopper securely, invert the bottle several times to mix the sample. Check for air bubbles discarding the sample and starting over if any is seen. If oxygen is present in your collection flask you will see a milky precipitate form initially which quickly turns a yellowish brown color. When the precipitate has settled invert the sample container again allowing the precipitate to thoroughly mix with the sample and then settle out to the bottom again. Do this approximately three times.
4. Carefully remove the stopper and add 2 mL of concentrated sulfuric acid (or about 28 drops from a Pasteur pipette) to the surface of the sample, just letting it gently run down the inside mouth of the collection flask. Carefully stopper and wipe off the top of the flask to remove any trace of acid then invert the bottle and continue mixing thoroughly until the precipitate has dissolved. This may take about 30 minutes or so. If it does not dissolve add another 1-2 mL of acid. The sample is now technically fixed and can be stored in a cool dark place for several hours. After addition of H2SO4, you may see an air bubble in your sample, which is fine at this point.
5. Titrate a volume representing 200 mL original sample after correcting for sample loss by displacement with reagents. Since we have added 4 mL (2 mL each) of MnSO4 solution and alkali-iodide-azide reagents into the 300 mL collection bottle, titrate 200 x 300/(300-4) = 203 mL. Pour 203 mL of the sample from the collection bottle into a 250 mL Erlenmeyer flask. Use a volumetric flasks to measure out 200 mL of the solution for the titration. Use a 10 mL graduated cylinder to measure out the final 3 mL of volume until the 203 mL volume is achieved. Pour the 203 mL into a 250 mL Erlenmeyer flask, insert a stir bar into the flask, and get a good stir rate creating a vortex in the liquid then immediately start the titration. An easier option here is to titrate exactly 200 mL then multiply your result by a correction factor 203/200. Titrate the sample with the standardized thiosulfate solution with constant stirring until a pale yellowish color develops record the amount of titrant used. Add 1 mL approximately 20 drops of 1% starch indicator solution and continue the titration until the solution turns colorless for the first time. Approach the endpoint carefully: as it only takes one drop of titrant to change the color from blue to colorless. Ignore the return of the blue color with time after the first colorless endpoint has been reached. Record the volume of titrant used. Each pair of students should do three titrations.

Calculate the dissolved oxygen content of your samples in mg/L and in ppm.

For the dissolved oxygen (DO) determination error analysis: Calculate the error propagation for the DO concentrations for each trial. For the error in the thiosulfate concentration, use the standard deviation provided by your TA associated with the class average if that is not available use your own thiosulfate data.

For the DO concentrations, calculate the average, standard deviation, and the 95% confidence interval.

What interferences could have affected your DO calculations using the modified Winkler titration procedure? Do an error analysis on your sample results. Calculate the saturation level (SL) for your water samples. Do your results indicate that the Charles River water will support aquatic life?

### Dissolved Oxygen % Saturation and Measuring Temperature of Water

Not only pollutants that enter the river effect dissolved Oxygen levels in river water; they are also affected by Temperature and atmospheric Pressure. For example, the lower the temperature, the more oxygen that can dissolve in the water. As the water warms up, the saturation level of DO will drop. You will need to measure the temperature and pH of the water at the collection site. The best way to measure temperature is to simply insert the thermometer directly into the Charles River. This should be done immediately at the time and place you collect the sample. Simply lower the thermometer tip a few inches below the water surface, or place the thermometer into the sample container and allow the thermometer time to equilibrate with the collected water in your container. For the pH we will have a calibrated pH meter on hand and will read the pH directly from the meter. The meters will be calibrated in the lab using two buffers pH 7 and pH 10.

#### Calculate the temperature and pH of your water sample and discuss why they are important in terms of their variation and impact on pollution. Relate the values to your measured DO level.

The actual dissolved oxygen that we calculate in our experiment is in units of mg / L and represents the amount of oxygen gas dissolved in one liter of river water. Dissolved oxygen concentrations can range from 0 to upwards of 15 mg/L. As we look at the water quality of the Charles River, it might be useful to have another way to express it other than in the units of mg / L. Frequently, when talking about DO concentrations, the term % saturation is used. The saturation level of DO (SLDO) represents the theoretical amount that the river could potentially hold based on conditions of temperature, atmospheric pressure and altitude. As a general rule % saturation levels less than 60% are not good and represent unacceptable DO levels.24,25 Levels between 60 to 70% are considered to be Satisfactory, and those between 70% and 90% Very Good, % saturations of 90% to 100% are generally viewed as being Excellent. Levels above 100% indicate supersaturation.26,27 After determining your measured DO concentration you will calculate the % saturation of your sample.

There are several methods for determining the saturation level of the dissolved oxygen (SLDO) in the Charles River. Knowing the DO and the SLDO we can calculate the actual % Saturation Level of DO that is the ratio of the measured DO in ppm divided by the SLDO in ppm.

$\rm % SL = \dfrac{\text{Actual DO in ppm}}{\text{SLDO in ppm}} \times 100$

Since the % saturation depends on both the temperature and pressure (elevation) a pressure correction factor should be included. In Appendix I there is a DO Pressure correction chart. Simply find the correct barometric Pressure and take the pressure correction factor and multiply it times the DO concentration that you have measured. This becomes your pressure corrected DO concentration. Because the Charles River is at sea level we do not have to worry about a major elevation Pressure correction. With the corrected DO measurement in hand; you can use the nomograph chart in Appendix II as a quick solution to determine the % saturation level for the Charles River. Simply find the corrected DO measurement on the bottom scale, mark off the corresponding temperature of the river water in degrees Celsius on the top scale, and connect the two marks with a straight line. The point where the line crosses the % saturation axis for your water sample is known as the % saturation level.

An even better way would be to calculate the saturation level of dissolved oxygen (SLDO) directly taking vapor pressure and temperature into account making use of a simple empirical formula derived from Henry’s Law28. This formula has been reported to work well for temperatures between 00 C and 500 C, and allows us to calculate the amount of oxygen that theoretically could be present in oxygen-saturated water. The formulas apply to oxygen in distilled water:29

$\rm \text{ppm dissolved oxygen} = \dfrac{(P - p) \times 0.678}{35 + T} = SLDO$

0°C < T < 30°C

$\rm \text{ppm dissolved oxygen} = \dfrac{(P - p) \times 0.827}{49 + T} = SLDO$

30°C < T < 50°C

where P is the barometric pressure at the collection site in mm Hg, T is temperature of water in °C, and p is the aqueous vapor pressure in mm Hg. To calculate p the vapor pressure of water in the air you can use the following equation:

$\rm p_{\text{water vapor}} = e^{(20.386 - \dfrac{5132}{T})}$

where P= vapor pressure in (mm Hg) and T= temperature of air in Kelvin (K).

You can now take your corrected DO concentration and divide it by the SLDO, then, multiplying this by 100 gives you your % saturation.

Calculate the % saturation for your sample and from the % oxygen saturation level determine if there is a deficit or surplus of oxygen present. Explain your reasoning behind the deficit or surplus in the context of what it means in terms of respiration and aquatic life. Comment on any errors that could have caused any discrepancies in your calculated % saturation. No error propagation necessary for the SLDO and %SL results.

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