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OUR Level 3 Testing

Updated: Nov 21, 2020

Level 3: Treatability OUR Testing

This level of oxygen uptake rate (OUR) testing is used to evaluate the impact of different chemical compounds or waste streams on a biological treatment system. In particular, Level 3 OUR testing allows you to determine if a waste stream or chemical is desirable, inhibitory, or toxic to the microorganisms in a bioreactor (aeration tank, oxidation ditch, etc.).

Aeration tanks with diffused air.

I've had people ask me what the difference is between inhibition and toxicity, with their thought being these two words describe the same thing, and I can see how this thinking occurs. The best description I've come across is what Michael H. Gerardi provides in his excellent book "Nitrification and Denitrification in the Activated Sludge Process." Though what follows is specifically in reference to the nitrification process I think it is fairly obvious that Mr. Gerardi's defintions apply well to all bacteria in a biological treatment system. I quote as follows:

"Inhibition is a temporary, short-term (acute) or long-term (chronic) loss of enzymatic activity.Toxicity is the permanent loss of enzymatic activity or irreversible damage to cellular structure." He goes on to state: "Nitrifying bacteria cannot acclimate as often to an inhibitory event as can organotrophs. This is due to the fact that organotrophs obtain a relatively large amount of energy from the oxidation of cBOD, and it is energy that permits them to repair damaged enzyme systems. Therefore nitrifying bacteria are more "sensitive" to inhibitory wastes than organotrophs. Thus significant loss of nitrification occurs before significant loss in efficiency of cBOD removal."

The key to doing this testing is that you must first be sure the microorganisms have reached a state of endogenous respiration, wherein all of the oxidizable organics have been consumed. So please review my blog page OUR Level 2 Testing: Time to Endogenous Respiration for details on how to determine if your mixed liquor suspended solids sample has reached, or is near to, endogenous. Level 3 OUR testing should only begin after the completion of Level 2 OUR testing, but, as you will read below, there will be exceptions to this.

Your goal with Level 3 OUR testing is to evaluate the impact of one or more waste streams (or chemicals) independent of any background "noise" from the original organics in the wastewater. When testing different waste streams I like to create a simple fishbone diagram, as shown below, laying out the streams to be tested. In order to begin Level 3 testing you start when the bacteria are hungry, well-oxygenated, and ready to start consuming a new supply of organic material (the sample or stream to be tested), described in detail in the sections that follow.

Fishbone Diagram Showing Various Waste Streams

How Do I Know I Have Reached Endogenous Respiration?

I wish I had a simple, unequivocal answer for you, but the truth is, I do not. But I can give you some guidance from my experience in doing OUR testing. Based on having run many, many OUR tests on both municipal and industrial biological treatment systems, I offer the following:

You need, ideally, for the oxygen uptake rate in the MLSS sample to be ≤9.0 mg/L oxygen/L/hr. If you start your Level 3 OUR testing with the initial OUR at ≤6.0 mg/L oxygen/L/hr, that will be even better. What do I mean by "better?" With a low starting OUR value the influence of the remaining organics in the MLSS sample will have a minimal impact on the new OUR when you start adding your chemical or waste stream of interest to the BOD bottle for testing. But be prepared for the fact that sometimes you will never get close to an OUR of ≤9.0 mg/L oxygen/L/hr.

Every wastewater treatment plant is different and every mixed liquor suspended solids sample is different which means the time it takes to reach endogenous is going to be different for each system you test. Over time I have slowly come to the conclusion that there is no specific point or value below which endogenous respiration can be definitively established. Perhaps for that reason OUR guidelines recommend a minimum period of time for aerating your MLSS sample before beginning treatability testing. The ASTM International organization recommends two hours of aeration before testing and the OECD recommends three hours of aeration. Quotes from both organizations are provided below.


ASTM International

In section 4.4 of the ASTM standard D5120-90 (2009), the following is stated:

“The prepared cell suspension is aerated for a 2-h period. At the end of the period, the respiration rate is determined using a respirometric or an oxygen uptake technique.”

From this quote you can see that a minimum of two hours of aeration for the mixed liquor sample is recommended before treatability testing begins.

The Organization for Economic Co-operation and Development (OECD)

Below I've quoted a fairly large amount of material from the document shown in the graphic below. You can download this document by clicking on the graphic.

OECD Guidelines Graphic


5. The respiration rates of samples of activated sludge fed with synthetic sewage are measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. Under consideration of the realistic exposure scenario, longer contact times could be appropriate. If the test substance is rapidly degraded e.g. abiotically via hydrolysis, or is volatile and the concentration cannot be adequately maintained, additionally a shorter exposure period e.g. 30 minutes can be used. The sensitivity of each batch of activated sludge should be checked with a suitable reference substance on the day of exposure. The test is typically used to determine the ECx (e.g. EC50) of the test substance and/or the no-observed effect concentration (NOEC).

General procedure and measurements

36. Test mixtures, reference mixtures and the blank and abiotic controls are incubated at the test temperature under conditions of forced aeration (0.5 to 1 L/min) to keep the dissolved oxygen concentration above 60 – 70% saturation and to maintain the sludge flocs in suspension. Stirring the cultures is also necessary to maintain sludge flocs in suspension. The incubation is considered to begin with the initial contact of the activated sludge inoculum with the other constituents of the final mixture. At the end of incubation, after the specified exposure times of usually 3 hours, samples are withdrawn to measure the rate of decrease of the concentration of dissolved oxygen in the cell designed for the purpose (Fig.2 of Annex 3) or in a completely filled BOD bottle. The manner in which the incubations begin also depends on the capacity of the equipment used to measure oxygen consumption rates. For example, if it comprises a single oxygen probe, the measurements are made individually. In this case, the various mixtures needed for the test in synthetic sewage should be prepared but the inoculum should be withheld, and the requisite portions of sludge should be added to each vessel of the series. Each incubation should be started in turn, at conveniently timed intervals of e.g. 10 to 15 minutes. Alternatively, the measuring system may comprise multiple probes that facilitate multiple simultaneous measurements; in this case, inoculum may be added at the same time to appropriate groups of vessels.

37. The activated sludge concentration in all test, reference and blank (but not abiotic control) mixtures is nominally 1.5 g/L of suspended solids. The oxygen consumption should be measured after 3 hours of exposure. Additional 30-minute exposure measurements should be performed as appropriate and previously described in paragraph 5.


Unfortunately, aerating a mixed liquor sample for two to three hours is often not sufficient to reach endogenous respiration, which sometimes makes treatability testing an all-day activity. The graph below shows the results from a municipal wastewater treatment plant's MLSS where, after 6.3 hours of aeration, endogenous respiration still had not been reached, though we appeared to be getting close. Keep in mind this is a mixed liquor sample that has been sitting in a bucket being aerated, without any additional food being added, with just the remaining organics in the sample supplying food to the bacteria, elevating the oxygen uptake rate.

Time to Endogenous Respiration

In the graph above, showing the extended oxygen uptake rate in the MLSS from a municipal plant, we do see a steady, but slow reduction in the OUR. To be noted, this MLSS is from a well-designed wastewater plant that is not operating at full capacity. In comparison, let's look at the graph below, from a marginally-designed industrial wastewater treatment plant with a heavily loaded (overloaded) sequencing batch reactor. The OUR of the mixed liquor starts out very high (32.0 and then again, an hour later, 29.7 mg Oxygen/L/hr) decreasing only 7.1% from the 29.7 value to 27.6 after nearly five hours of aeration without food being added. When you encounter this situation, where there continues to be a large background demand from complex organics being broken down very slowly, you have to make a decision on whether or not to proceed with your treatability testing. And this is where the minimum aeration period provides guidance. In the graph below it really wouldn't have mattered if we had started the treatability testing after two or three hours of aeration. Personally, I would aerate for three hours when the OUR is high before proceeding to the treatability test.

Industrial Plant Extended OUR

Performing the Treatability Testing

The way you go about doing this testing is to generate OUR data using several different sample volumes. Once you’ve confirmed that endogenous respiration has been reached, or you have aerated your MLSS sample for three hours, you run at least three increasing sample volumes using, for example, 0.3 mL, 0.5 mL, and 0.7 mL of test sample added to the 300 mL BOD bottle which is then filled to just overflowing with MLSS as pictured in the graphic below. A magnetic stir bar needs to be in the BOD bottle because the sample needs to be well-mixed during the 15-minute OUR test. The mixing assures a steady flow of MLSS across the surface of the DO probe.

Increasing sample volume being added to BOD bottles.

Logistical or Time Concerns with Treatability Testing

Since each sample requires a 15 minute time period to complete an individual OUR test, you can see that testing three consecutive samples at increasing sample volume requires a minimum of, practically speaking, at least 50 minutes (dumping first sample, adding new sample, etc). With just one magnetic stirrer, DO probe/meter, etc. combined with multiple streams to be tested, you can also see that treatability testing can take a very long time, all day when added to the three-hour aeration period discussed above. If this is what you are faced with, which I often am, the testing time can only be reduced when you are fortunate enough to have access to three complete OUR setups, as I was recently.

The image below shows three dissolved oxygen probes mated to three handheld meters and three magnetic stirrers. (Actually, the magnetic stirrer shown all the way to the right in the image was capable of stirring the contents of four BOD bottles simultaneously.) With this setup you can complete a series of three iterations using three dosage levels in just 15 minutes. Obviously, this provides a huge reduction in the time you spend waiting the OUR test to run.

Extended OUR Test Setup

After generating OUR data using three or more sample volumes, you produce a simple bar graph such as the one shown below. In this example, the sample being tested was leachate from an active municipal solid waste landfill and there was concern about possible inhibition or toxicity from the leachate. But these are good results and the interpretation of this graph is straightforward. Because the OUR is increasing with each increase in sample volume, converted here to "equivalent" flow rates, we know the leachate sample is not inhibitory. In fact, the more of this sample we feed to the activated sludge (the bacteria), the more the microorganisms like it. So we would want to be careful in introducing this waste stream at too high a flow rate to be certain we don’t exceed the oxygen generation capability of the aeration system.

Leachate addition to activated sludge

Interpretation of Test Results

The graphic below shows how to interpret the results from testing different sample volumes. An increasing OUR is the best result though an OUR that stays constant with increasing sample volume also indicates no inhibition from the waste stream. Where you need to be careful in trying to treat a particular waste stream is when you have a decreasing OUR with increasing sample volume. When you see that negative slope, you know you have an inhibitory waste that will need to be introduced slowly so the microorganisms can acclimate to it. And if the OUR drops to zero you know you’ve got a toxic waste that is going to need some sort of physical or chemical treatment, such as the addition of hydrogen peroxide or potassium (or sodium) permanganate, before it can be introduced to an activated sludge system. (If you click on the graphic you will open a full-page PDF version that is easier to see and is optimized for printing.)

Graphical interpretation of OUR results.


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