ISO Standard Methods for Aqueous Aerobic Biodegradability Test
In addition to OECD methods, there are also a number of ISO standard methods for aqueous aerobic biodegradability tests. Here we will introduce eight of them, i.e., ISO 9408, ISO 9439, ISO 9888, ISO 10707, ISO 11733, ISO 14851, ISO 14852, and ISO 16221. Some of them are equivalent to some of the OECD methods. Please note that ISO 14851 and 14852 are designed specifically for plastics.
ISO 9408- Ultimate Biodegradability Test - Oxygen Consumption
Definition
ISO 9408 is an aqueous aerobic biodegradability test that determines the ultimate aerobic biodegradability of organic compounds in aqueous medium by measuring oxygen demand during biodegradation in a closed respirometer. The test normally lasts for 28 days.
Principles
Similar to the OECD 301F Ready Biodegradability Test, ISO 9408 determines the biodegradability of a material by measuring oxygen consumption during the biodegradation process in a period of 28 days in a closed bottle setup (with head space). The degradation percentage is calculated as the ratio of oxygen consumption to the theoretical oxygen demand (ThOD).
The oxygen consumption is usually determined by measuring the quantity of oxygen (produced electrolytically) required to compensate the consumed one to maintain a constant gas volume/pressure in the respirometer flask, or from the change in the volume or pressure in the flask without compensating the consumed oxygen. The evolved CO2 is absorbed by sodium hydroxide solution present in a tube in the flask.
The formula of the test substance and its purity may be known to calculate the ThOD. If the ThOD cannot be obtained, the chemical oxygen demand (COD) may be experimentally determined and used as a less satisfactory alternative.
Information on the toxicity of the test substance is helpful in determining the appropriate dosage so that the material does not inhibit bacteria at the concentration tested.
This method is capable of testing samples that are highly soluble, poorly soluble, insoluble, volatile, involatile, and/or adsorbing.
ISO 9439- Ultimate Biodegradability Test - CO2 Evolution
Definition
ISO 9439 is an aqueous aerobic biodegradability test that determines the ultimate aerobic biodegradability of organic compounds in aqueous medium by measuring CO2 evolution during biodegradation in continuously aerated bottles. The test normally lasts for 28 days.
Principles
The ISO 9439 CO2 evolution method uses respirometry to determine the biodegradability by measuring the CO2 formation during the biodegradation process over 28 days. As the chemical decomposition process primarily result in the formation of CO2 and H2O, the measurement of generated CO2 can well reflect the biodegradation of the test compounds. With the measured and the theoretical CO2 amount, one can easily calculate the degradation percentages over a specified incubation time.
The aeration of the working solution requires the use of CO2-free air, which can be easily obtained by using a few NaOH washing bottles to remove all the CO2 present in the air. After that, CO2-free air can be sent to aerate the working solution.
During the aeration, CO2 is generated as a result of test substance biodegradation. Such CO2 can be easily stripped off the working solution during the aeration process. To capture it, three absorption bottles are usually used containing known amount of Ba(OH)2 to convert CO2 to BaCO3 precipitation. An afterwards acid-base titration using HCl as the titrant can determine the amount of the residual Ba(OH)2, which can be used to back calculate the amount of CO2 generated during the biodegradation.
Information on the toxicity of the test substance is helpful in determining the appropriate dosage so that the material does not inhibit bacteria at the concentration tested.
This method is appropriate for highly soluble, poorly soluble (or even insoluble) and/or absorbing materials. However, since aeration is performed throughout the incubation process, the test substance should not be volatile. ISO 9439 is similar to OECD 301B and ASTM D5864, and the common materials tested by these two methods include lubricants, grease, oil, fuels, surfactants, and personal care products.
ISO 9888- Ultimate Biodegradability Test - Static Test (Zahn-Wellens Method)
Definition
ISO 9888 is an aqueous aerobic biodegradability test that determines the ultimate aerobic biodegradability of organic compounds in aqueous medium by measuring dissolved organic carbon or chemical oxygen demand. The conditions with a high inoculum concentration is optimal for allowing the maximum value of biodegradation. The test normally lasts for 28 days.
Principles
Similar to OECD 302B, The ISO 9888 Zahn-Wellens test measures the removal of dissolved organic carbon (DOC) or chemical oxygen demand (COD) during biodegradation and the degradation percentage is calculated as the ratio of DOC removal to original DOC loaded (or COD removal to original COD dosed).
The carbon content of the test substance and its purity may be known to calculate the DOC, or otherwise the COD may be measured. Information on the toxicity of the test substance is helpful in determining the appropriate dosage so that the material does not inhibit bacteria at the concentration tested.
This method is capable of testing samples that are highly soluble (> 50 mg DOC/L), non-volatile, and non-adsorbing.
ISO 10707 - Ultimate Biodegradability Test - Closed Bottle
Definition
ISO 10707 is an aqueous aerobic biodegradability test that determines the ultimate aerobic biodegradability of organic compounds in aqueous medium by measuring dissolved oxygen consumption during biodegradation in closed bottles. The test normally lasts for 28 days.
Principles
Similar to OECD 301D, ISO 10707 measures the dissolved oxygen (DO) consumption during aerobic biodegradation in a closed bottle setup without head space. The degradation percentage is calculated as the ratio of DO consumption to the theoretical oxygen demand (ThOD).
Compared to some other methods, ISO 10707 has the top levels of simplicity and applicability.
The formula of the test substance and its purity may be known to calculate the ThOD. If the ThOD cannot be obtained, the chemical oxygen demand (COD) may be experimentally determined and used as an alternative.
Information on the toxicity of the test substance is helpful in determining the appropriate dosage so that the material does not inhibit bacteria at the concentration tested.
This method is capable of testing samples that are highly soluble, volatile, and/or adsorbing.
ISO 11733 - Biodegradability Test - Activated Sludge Simulation
Definition
ISO 11733 is an aqueous aerobic biodegradability test that determines the biodegradability of a material by measuring dissolved organic carbon or chemical oxygen demand under a simulated activated sludge condition. The test normally lasts for 12 weeks.
Principles
Similar to OECD 303A, ISO 11733 is designed to evaluate the primary and/or ultimate biodegradation of water soluble organic materials in a continuously operated activated sludge system simulating the realistic wastewater treatment process. The dosage of the test compound is generally between 10 and 20 mg DOC/L, and the biodegradation percentage is calculated based on the DOC/COD removal.
The ISO 11733 Activated Sludge Simulation test measures the DOC/COD removal during biodegradation. Two parallel units are generally used, one containing organic medium only as a blank control, while the other containing both organic medium and the test compound. The difference of the DOC removal between the two units is believed to be the contribution of the test compound. This method cannot be used for testing insoluble or volatile materials.
ISO 14851- Ultimate Biodegradability of Plastics - Oxygen Consumption
Definition
ISO 14851 is an aqueous aerobic biodegradability test that determines the ultimate aerobic biodegradability of plastic materials in aqueous medium by measuring oxygen demand during biodegradation in a closed respirometer. The test normally lasts for 2 months, but can be extended up to 6 months.
Principles
ISO 14851 determines the biodegradability of a plastic material by measuring oxygen consumption during the biodegradation process in a period of 2-6 months in a closed bottle setup (with head space). The degradation percentage is calculated as the ratio of oxygen consumption to the theoretical oxygen demand (ThOD).
The oxygen consumption is usually determined by measuring the quantity of oxygen (produced electrolytically) required to compensate the consumed one to maintain a constant gas volume/pressure in the respirometer flask, or from the change in the volume or pressure in the flask without compensating the consumed oxygen. The evolved CO2 is absorbed by sodium hydroxide solution present in a tube in the flask.
The formula of the test substance and its purity may be known to calculate the ThOD. The material is preferably in powder form with a maximum size of 250 um in diameter to provide better bioavailability for faster biodegradation.
Information on the toxicity of the test substance is helpful in determining the appropriate dosage so that the material does not inhibit bacteria at the concentration tested.
ISO 14852- Ultimate Biodegradability of Plastics - CO2 Evolution
Definition
ISO 14852 is an aqueous aerobic biodegradability test that determines the ultimate aerobic biodegradability of plastic materials in aqueous medium by measuring CO2 evolution during biodegradation in continuously aerated bottles. The test normally lasts up to 6 months.
Principles
The ISO 14852 uses respirometry to determine the biodegradability by measuring the CO2 formation during the biodegradation process up to 6 months. As the plastic material decomposition process primarily result in the formation of CO2 and H2O, the measurement of generated CO2 can well reflect the biodegradation of the test material. With the measured and the theoretical CO2 amount, one can easily calculate the degradation percentages over a specified incubation time.
The aeration of the working solution requires the use of CO2-free air, which can be easily obtained by using a few NaOH washing bottles to remove all the CO2 present in the air. After that, CO2-free air can be sent to aerate the working solution.
During the aeration, CO2 is generated as a result of test substance biodegradation. Such CO2 can be easily stripped off the working solution during the aeration process. To capture it, three absorption bottles are usually used containing known amount of Ba(OH)2 to convert CO2 to BaCO3 precipitation. An afterwards acid-base titration using HCl as the titrant can determine the amount of the residual Ba(OH)2, which can be used to back calculate the amount of CO2 generated during the biodegradation.
Information on the toxicity of the test substance is helpful in determining the appropriate dosage so that the material does not inhibit bacteria at the concentration tested.
This method is appropriate for highly soluble, poorly soluble (or even insoluble) and/or absorbing materials. However, since aeration is performed throughout the incubation process, the test substance should not be volatile.
ISO 16221 - Biodegradability Test - Seawater
Definition
ISO 16221 is an aerobic biodegradation test that determines the ultimate biodegradability of an organic material in seawater relying on the microorganisms originally present in seawater without the addition of a specific inoculum. When artificial seawater is used, however, inoculum may come from other materials of marine origin. Five methods are specified, i.e., (I) DOC die-away, (II) closed bottle, (III) two-phase closed bottle, (IV) CO2 evolution, and (V) CO2 headspace tests. They are directly modified and adapted from the fresh water methods ISO 7827, 10707, 10708, 9439, and 14593, respectively. A maximum test duration of 60 days is allowed.
Principles
Similar to OECD 306, ISO 16221 targets the aerobic biodegradability of organic materials in marine environment. Directly adapted from five fresh water ISO methods mentioned above, the ISO 16221 provides several different analytical methods and system setups to accommodate materials with a wide range of physico-chemical properties.
Since nutrients (mineral medium) are added and the concentration of test substance is much higher than would be present in the sea, the tests do not simulate the marine environment.
If a sample shows low biodegradability results, it does not necessarily mean it cannot be degraded, but indicates further study is necessary, for example, using as low a concentration of the test compound as possible.
Because the test conditions of marine environments are generally less favorable than those of limnic test systems, the test duration of ISO 16221 can be up to 60 days compared to the usual time of 28 days for fresh water systems.
Applicabilities
| Test | Analytical method | Sample info required | Poorly soluble | Volatile | Adsorbing |
|---|---|---|---|---|---|
| ISO 9408 (Oxygen consumption) | Oxygen consumption | ThOD or COD | + | +/- | + |
| ISO 9439 (CO2 evolution) | CO2 evolution | Carbon content | + | - | + |
| ISO 9888 (Zahn-Wellens) | DOC or COD | Carbon content or COD | - | - | - |
| ISO 10707 (Closed bottle) | Dissolved oxygen | ThOD or COD | - | + | + |
| ISO 11733 (Activated sludge simulation) | DOC and/or COD | Carbon content and/or COD | - | - | - |
| ISO 14851 (Plastics - oxygen consumption) | Oxygen consumption | ThOD | + | +/- | + |
| ISO 14852 (Plastics - CO2 evolution) | CO2 evolution | Carbon content | + | - | + |
| ISO 16221 (Seawater) | Various methods | Carbon content or ThOD | + | + | + |