Dissolved Oxygen: Rivers Method

Learn more about DO (leaving MassWWP pages)

*For the Mass. Department of Environmental Protection-approved SOP, get the pdf file here

Background Information

Dissolved oxygen is the oxygen dissolved in the river water. It is an important indicator since most aquatic plants and animals need it to survive. The river system both produces and consumes oxygen. The river gains oxygen from the atmosphere through the aerating action of wind or turbulence (cascading water) and from plants through photosynthesis. Respiration by aquatic animals, decomposition, and various chemical reactions consume oxygen. Decomposition of organic matter discharged in wastewater consumes oxygen. If more oxygen is consumed than is produced, dissolved oxygen levels decline and some sensitive animals may disappear. DO levels fluctuate daily and seasonally. They also vary with water temperature - cold water holds more oxygen than warm water. The most critical time for many aquatic animals are early mornings on hot summer days, when river flows are low, water temperatures are high, and plants have not been producing oxygen since sunset.

We measure dissolved oxygen by collecting a water sample in a special bottle, "fixing" or stabilizing the amount of oxygen in the sample by adding certain chemicals, and then measuring the concentration in the lab. DO is measured in either milligrams per liter or "percent saturation." Milligrams per liter (mg/l) is the amount of oxygen in a liter of water and is the same as "parts per million" or ppm.

Percent saturation is the measured mg/l of oxygen in the water sample relative to the mg/l of oxygen that the water sample is capable of holding at a particular temperature. The amount of oxygen that water can hold varies with temperature. Cold water can hold more oxygen than warm water. At less than 100% saturation, the water will tend to take on oxygen from the air. At greater than 100% saturation, it will give off oxygen to the air. At 100% saturation, the oxygen of the water is at equilibrium with the oxygen in the air and no oxygen will be exchanged. Suppose the measured DO of a water sample is 5 mg/l. If you measured a temperature of 20° C, your water sample is capable of holding about 9.0 mg/l (see table below). Dividing the measured DO by the expected DO gives the % saturation, in this case 55%

Massachusetts Water Quality Standards Class A Class B
Concentration 6 mg/l cold water: 6 mg/l warm water: 5 mg/l
Percent Saturation 75% cold water:75% warm water: 60%

Sampling Method for Rivers


  • Alcohol Thermometer
  • BOD bottles, 300 ml (1 per sample, take an extra in case of breakage)
  • Fresh Manganous Sulfate Powder pillows (pillow # 1) (1 per sample)
  • Fresh Alkaline Iodide-Azide Powder Pillows (pillow # 2) (1 per sample)
  • Fresh Sulfamic Acid Powder Pillows (pillow # 3) (1 per sample)
  • Fingernail clippers or scissors for cutting powder pillows
  • Eye dropper for topping off BOD bottle, if necessary
  • Marble (optional)
  • Field Sheet pencils
  • Rubber gloves and safety goggles (to avoid harmful contact with chemicals)
  • Cooler and ice
  • Zip-loc bags (1 gallon size)

Sampling Protocol

ALWAYS TAKE TEMPERATURE AT SAME TIME YOU COLLECT DO SAMPLE -for calculating % saturation later. See Temperature SOP.

  1. Use a 300 ml BOD sample bottle. The water sample must be collected in such a way that you can stopper the bottle while it is still submerged. That means that you must be able to reach into the water with both arms. For DO samples, an extension pole is not feasible. The water must be deeper than the sample bottle and free of surface scum and debris.
  2. Carefully wade into the stream, walking upstream, and avoid stirring up bottom sediments. Samples should be collected from relatively calm but flowing water, not in nor immediately downstream from a riffle. Stand so that you are facing one of the banks. If you are in a canoe, have your partner steady it and face one of the banks.
  3. Collect the sample so that you are not standing or floating upstream of the bottle. Remove the stopper of the BOD bottle. Point BOD bottle downstream and slowly lower it into the water until the lip is just submerged. Allow the water to fill the bottle very gradually, avoiding any turbulence or air bubbles (this will add oxygen to the sample and skew your results). Submerge completely and allow to overflow to ensure that air bubbles are not trapped in the sample or gently tap the bottle to allow bubbles to escape.
  4. Holding the bottle vertically, remove it from the river, leaving water around the cap at the flared mouth of the bottle. Cap the bottle by dropping the stopper directly into the bottle neck from 1/4 inch above. Check to make sure there are no air bubbles or space left at the top. If there are, you need to start over. Take your eyedropper and fill it with water from the stream. Go to the stream bank for step 5.
  5. If there are no air bubbles present in the bottle, "fix" the sample immediately as described below:
    1. Remove the stopper and add the contents of one Manganous Sulfate Powder Pillow (#1) and one Alkaline Iodide-Azide Powder Pillow (#2) to the 300 ml sample, using scissors or clippers to open packages. HINT: You may need to "roll" the pillow gently between your fingers to ensure delivery of all the powdered reagent into the sample bottle. Residual reagent powder around bottle neck can be washed into the bottle by swirling bottle gently.
    2. Immediately insert the stopper so air is not trapped in the bottle. Holding the stopper in place, invert the bottle several times to dissolve the powder. An orange-brown flocculent precipitate will form if oxygen is present.
    3. Allow sample to sit undisturbed and wait until the flocculent in the solution has settled to the bottom half of the bottle. Again invert the stoppered bottle several times and wait until the flocculent has settled. This insures complete reaction of the sample and reagents. HINT: Don't be afraid to invert vigorously several times for proper mixing and dissolution of reagents.
    4. Remove the stopper and the contents of pillow #3 (Sulfamic Acid). Immediately insert the stopper so air is not trapped and invert several times to mix. The floc will dissolve and leave a yellow color if oxygen is present. HINT: If you have trouble avoiding introducing an air bubble in the sample at this step, put a marble in the sample after adding pillow 3.
      The oxygen in the sample is now "fixed" and ready to be analyzed in the lab.
  6. Cap the bottle and seal by pouring a small amount of water into the flared lip area with the eyedropper of river water you collected in step 4.
  7. Fill out the river field sheet completely. Write the site # on the bottle, if it was not already done for you.
  8. Put all trash you have generated (pillow cases, broken bottles if any) in a zip-loc bag and take it back with you.

Troubleshooting - Lab analysis troubleshooting

Air bubbles may get trapped in the bottle under the glass stopper. This is most likely to happen after pillows # 1 and 2 have been added and the bottle has been inverted. Inverting the bottle may spill any water residing in the neck of the bottle.

TO FIX THIS: Ideally, learn how to drop the stopper from 1/4 inch above the bottle directly into the neck. This will form an airless seal. An alternative is to use an eyedropper filled with stream water from your site. After adding Reagent # 3 (sulfamic acid), dribble a bit of water into the bottle. This has the added advantage of rinsing into the solution any particles clinging to the bottle neck. Alternatively, drop a marble in the sample when you add pillow #3.

Transporting the Sample:
Store the bottle upright in your cooler. If you cannot put ice directly in your cooler because you store other materials in there, use a gallon-size zip-loc bag filled with ice. Put your sample in that zip-loc bag, zip shut and place in cooler with frozen koolit.

The sample must be analyzed in the lab within 8 hours!

Tip: Some groups maintain a complete extra set of bottles, so volunteers can collect next month's bottles as they drop off their samples at the lab. If you follow this procedure, make sure your bottles are kept in Zip-loc bags and boxed, or in some other clean and secure container.


Laboratory Equipment For field sample and QC standard analysis:

  • Hach Digital Titrator
  • Hach Sodium Thiosulfate (0.2 N) Titration Cartridge (with clean delivery tube)
  • Starch Indicator Solution
  • Drop dispenser (for starch solution)
  • Fixed Water Samples in 300 ml BOD Bottles
  • Graduated Cylinder, 250 ml
  • Erlenmeyer Flask, 250 ml
  • Magnetic stirrer and stirring bar (optional)
  • Lab Sheet
  • Safety goggles and gloves

Additional Equipment For Quality Control tests:

  • 500 ml graduated cylinder
  • 500 ml beaker

Quality Control Procedure

- using QC test standard received from EAL:

  1. Remove QC standard from refrigerator, warm to room temperature before testing.
  2. Rinse out a 500 milliliter graduated cylinder by pouring a few mls of the test standard into it, swirling it around the cylinder, then pouring it down the drain.
  3. Carefully measure exactly 300 mls of the test standard into the 500 ml graduated cylinder. When you measure a liquid quantity in a cylindrical container, a "meniscus", (a shallow U shape) forms on the liquid's surface. The bottom of the U should rest on the 300 ml line.
  4. Pour the standard into a 300 ml DO bottle.
  5. Slowly empty the contents of a alkaline iodide-azide powder pillow (Pillow # 2) into the bottle. (You don't use powder pillow # 1 in this QC test). Cap bottle so there is no air bubble, then invert several times. Let the solution settle, then invert several times again.
  6. Empty the contents of a sulfamic acid powder pillow (Pillow # 3) into the bottle. Swirl this around until the reagent is dissolved. The solution should turn yellow.
  7. Measure 100 mls of this "fixed" solution in a graduated cylinder. Transfer to a 250 ml erlenmeyer flask or beaker.

This fixed sample is now ready to titrate. Since this is a 100 ml sample, use the 0.2 N sodium thiosulfate cartridge. This means that you'll also use the 0.02 digit multiplier when you're ready to calculate mg/l, as the digit multiplier changes with the strength of sodium thiosulfate that you use.

River Samples Analysis

  1. Insert a clean delivery tube into the titration cartridge.
  2. Attach the cartridge to the titrator body.
  3. Over a sink, hold the titrator with the cartridge pointing straight up so any bubbles will drift to the top of the cartridge. Turn the delivery knob to eject air and a few drops of titrant - until there are no more bubbles present in the delivery tube or the cartridge. Reset the digit counter to 0.
  4. Use the graduated cylinder to measure 100 ml of "fixed" field sample from the 300 ml BOD bottle.
  5. Transfer the sample into a 250 ml erlenmeyer flask or 250 ml beaker. Place the flask on a white surface because you will need to observe a color change.
  6. Place the delivery tube tip into the solution and swirl gently the flask (or use a magnetic stirrer) while turning the delivery knob. Continue titrating until your sample turns a pale yellow color. If the sample abruptly turns clear, you've gone past the endpoint and selected the wrong sodium thiosulfate or sample size. Re-titrate using a larger sample volume or lower strength sodium thiosulfate.
  7. Add a few drops of Starch Indicator Solution and swirl to mix. This will turn your field sample dark blue.
  8. Continue to titrate and swirl your sample until it turns clear. Record the digits required. If you are unsure that the color change from blue to clear was complete, deliver another few digits, one at a time. If no further color change is noted, use the first recorded number as your value. Otherwise, use the last digit at which a color change was noted.
  9. Calculate mg/l of DO: DO = Digits Required x 0.02
  10. Record your result on the data sheet.

In the Lab: Dissolved Oxygen Troubleshooting

1) Some brown particles may remain when the sample is ready for titration. This can cause variable results because the chemicals in the sample are now unevenly concentrated.

TO AVOID THIS: Carefully observe the BOD bottle after adding all three reagents. If particles are visible, or if there is a deposit on the bottom of the bottle, try shaking the bottle to dissolve any remaining solid matter. If this doesn't work, use a plastic, teflon, stainless steel or glass stirring rod or spatula to stir up the bottom sediments. This should allow the acid in the solution to fully dissolve the particles. You are then ready to titrate.

NOTE: Make sure you rinse the stirring rod well after trying this, to avoid corrosion of your utensil.

2) If your results seem wildly inaccurate, check to see you are using the correct Sodium Thiosulfate cartridge. Some folks have been known to use a sulfuric acid cartridge by mistake (that cartridge is used for pH and alkalinity analysis).

3) If you have titrated a quality control sample from the standard solution received from MassWWP and your value seems very high (remember, DO almost never goes above 14 mg/l in natural settings), it may be because you added powder pillow #1 to the standard before titrating. This QC test only uses pillows # 2 and # 3.

3b) Advance the plunger manually or with the delivery knob until titrant is forced out of the delivery tip and the delivery tube is filled with solution. Do this as you would a hypodermic syringe, with the delivery tip nearly straight up to remove all bubbles.

4) Sometimes an old cartridge can give an inaccurate reading, particularly if it has been left uncapped and allowed to evaporate somewhat. If you suspect the cartridge, try using a new one.

Dissolved Oxygen Calibration Table*

Temperature (°C) Dissolved oxygen (mg/l)
at 100% saturation
0 14.6
1 14.2
2 13.8
3 13.5
4 13.1
5 12.8
6 12.5
7 12.2
8 11.9
9 11.6
10 11.3
11 11.1
12 10.8
13 10.6
14 10.4
15 10.2
16 10.0
17 9.7
18 9.5
19 9.4
20 9.2
21 9.0
22 8.8
23 8.7
24 8.5
25 8.4

Water can hold a limited amount of dissolved oxygen. When it holds the maximum amount it can, a water body is said to be at saturation, or 100% saturated. The dissolved oxygen (in mg/l) of water at saturation changes with temperature: the higher the temperature, the less oxygen water holds.

MA Surface Water Quality Standards express minimum criteria for dissolved oxygen in both mg/l and % saturation.

To calculate % saturation of the sample, you divide the measured dissolved oxygen content of your sample by the maximum oxygen content at the temperature of your sample. (See table at right).

* Note in high altitude areas (none in Massachusetts) or water with high chloride concentrations, an additional correction may be necessary. These values are correct for a barometric pressure of 760 mm or sea level altitude.

We have an Excel workbook that contains a utility to calculate DO % saturation.