Recent Update: Limited time only! Get 30% off for samples that fail the test with bulk orders (≥ 4 samples)! Learn more.

Biodegradability Testing

Biodegradability testing is designed to determine the biodegradability of materials under a given or intended use condition. Although the biodegradation process is complex, the measurement is usually based on simple, nonspecific parameters such as CO2 evolution, oxygen consumption, or the removal of dissolved organic carbon or chemical oxygen demand.

CONCEPT

Biodegradability testing usually contains a blank control group, a reference group, and the test group. Blank control usually contains inoculum only without organic medium, and is normally used to check the CO2 formation or oxygen consumption by the inoculum itself. A reference group usually contains a material that is known to be biodegradable, such as sodium benzoate, sodium acetate, and aniline. This is used to confirm that the system is working properly.

However, some of these groups may not be necessary for a specific method.

A number of standard methods have been published by various organizations, such as OECD, ISO, ASTM, EU, US-EPA, and NITE

Below is a list of methods we currently provide at Aropha:

▶ OECD:
     ➢ OECD 301B (CO2 evolution);
     ➢ OECD 301D (closed bottle);
     ➢ OECD 301F (oxygen consumption);
     ➢ OECD 302B (DOC die away);
     ➢ OECD 303A (activated sludge simulation);
     ➢ OECD 306 (seawater - DOC die away or closed bottle);
▶ ISO:
     ➢ ISO 9408 (oxygen consumption);
     ➢ ISO 9439 (CO2 evolution);
     ➢ ISO 9888 (Zahn-Wellens);
     ➢ ISO 10707 (closed bottle);
     ➢ ISO 11733 (simulation of activated sludge);
     ➢ ISO 14851 (plastics - oxygen consumption);
     ➢ ISO 14852 (plastics - CO2 evolution);
     ➢ ISO 16221 (seawater - various methods);
▶ ASTM:
     ➢ ASTM D5864 (lubricant - CO2 evolution);
     ➢ ASTM D6731 (lubricant - closed respirometer).

OECD 301 CO2 evolution and closed bottle test overview

Some documented methods (sorted in alphabetical order)

Guideline Note Principle Endpoint
ASTM D5864 Biodegradation of Lubricants or Their Components CO2 Evolution Test CO2 Evolution -
ASTM D6139 Biodegradation of Lubricants or Their Components Using the Gledhill Shake Flask CO2 Evolution -
ASTM D6731 Lubricants or Lubricant Components in a Closed Respirometer Closed Respirometer -
EPA OPPTS 835.3100 CO2 Evolution CO2 Evolution -
EPA OPPTS 835.3140 CO2 Headspace Test Closed Respirometer Ready
EPA OPPTS 835.3170 Shake Flask Die-away Test - -
EPA OPPTS 835.3200 Zahn-Wellens / EMPA Test DOC Die Away Inherent
EPA OPPTS 835.3210 Modified SCAS Test DOC Die Away Inherent
EPA OPPTS 835.3215 Concawe Test - Inherent
EPA OPPTS 835.5045 Modified SCAS Test for Insoluble and Volatile Chemicals - Inherent
EPA OTS 795.45 Modified SCAS Test DOC Die Away Inherent
EPA OTS 796.3100 - - -
EPA OTS 796.3180 Modified AFNOR Test - Ready
EPA OTS 796.3200 Closed Bottle Test Closed Bottle Test Ready
EPA OTS 796.3220 Modified MITI Test (I) Closed Bottle Test Ready
EPA OTS 796.3240 Modified OECD Screening Test - Ready
EPA OTS 796.3260 Modified Sturm Test CO2 Evolution Ready
EPA OTS 796.3340 Modified SCAS Test DOC Die Away Inherent
EPA OTS 796.3360 Modified Zahn-Wellens Test DOC Die Away Inherent
EU Method C.4-A DOC Die Away DOC Die Away Ready
EU Method C.4-B Modified OECD Screening Test DOC Die Away Ready
EU Method C.4-C CO2 Evolution CO2 Evolution Ready
EU Method C.4-D Manometric Respirometry Test Closed Respirometer Ready
EU Method C.4-E Closed Bottle Test Closed Bottle Test Ready
EU Method C.4-F MITI Test Closed Bottle Test Ready
EU Method C.5 Biochemical Oxygen Demand Closed Bottle Test Ready
EU Method C.6 Chemical Oxygen Demand COD Ready
EU Method C.9 Zahn-Wellens Test DOC Die Away Inherent
EU Method C.12 Modified SCAS Test DOC Die Away Inherent
EU Method C.29 CO2 Headspace Test Closed Respirometer Ready
ISO 7827 DOC Die Away DOC Die Away Ready
ISO 9408 Manometric Respirometry Test Closed Respirometer Ready
ISO 9439 CO2 Evolution CO2 Evolution Ready
ISO 9887 SCAS Test DOC Die Away Inherent
ISO 9888 Zahn-Wellens Test DOC Die Away Inherent
ISO 10707 Closed Bottle Test Closed Bottle Test Ready
ISO 10708 Two-Phase Closed Bottle Test Closed Respirometer Ready
ISO 11733 Simulation Test - Activated Sludge Unit Simulation (DOC/COD removal) Simulation
ISO 14593 CO2 Headspace Test Closed Respirometer Ready
ISO 14851 Plastics Oxygen Consumption Closed Respirometer Closed Respirometer -
ISO 14852 Plastics CO2 Evolution CO2 Evolution -
ISO 16221 Seawater Various methods -
OECD 301 A DOC Die Away DOC Die Away Ready
OECD 301 B CO2 Evolution CO2 Evolution Ready
OECD 301 C Modified MITI Test (I) Closed Respirometer Ready
OECD 301 D Closed Bottle Test Closed Bottle Test Ready
OECD 301 E Modified OECD Screening Test DOC Die Away Ready
OECD 301 F Manometric Respirometry Test Closed Respirometer Ready
OECD 302 A Modified SCAS Test DOC Die Away Inherent
OECD 302 B Zahn-Wellens / EMPA Test DOC Die Away Inherent
OECD 302 C Modified MITI Test (II) Closed Respirometer Inherent
OECD 302 D (proposed) Closed Respirometer Closed Respirometer Inherent
OECD 303A Simulation Test - Activated Sludge Unit Simulation (DOC/COD removal) Simulation
OECD 303B Simulation Test - Biofilms Simulation Simulation
OECD 306 (I) Shake Flask Test in Seawater DOC Die Away in Seawater -
OECD 306 (II) Closed Bottle Test in Seawater Closed Bottle Test in Seawater -
OECD 310 CO2 Headspace Test Closed Respirometer Ready
Learn More at Aropha Resource Center

Why is Biodegradability Testing?


A large amount of chemicals are produced and released into the environment every year, such as pesticides, herbicides, dyes, and antibiotics, a large portion of which could potentially pose threat to the environment. Chemicals such as chlordane and dieldrin have been removed from the market due to their persistence, bioaccumulation, and toxicity (PBT) properties; in the meantime, regulations have been made to restrict the introduction of PBT substances from entering the market in the first place.

Among the PBT properties, persistence is usually the leading reason resulting in slow degradation in the environment. The accumulation of certain chemicals could potentially be harmful due to continuous exposure and increasing concentrations in the surroundings.

Despite many other transportation processes for the removal of these chemicals such as adsorption, photodegradation, and redox reaction, the biodegradation has been considered as the most fundamental process for the ultimate elimination of PBT chemicals from the environment.

Therefore, biodegradation tests before the introduction of these chemicals to the market has been required by a number of organizations, including Organization for Economic Co-operation and Development (OECD), International Organization for Standardization (ISO), Japanese Ministry of International Trade and Industry (MITI), National Institute of Technology and Evaluation (NITE), European Union (EU), and United States Environmental Protection Agency (US-EPA).

Biodegradation mechanisms

Aerobic biodegradation is a natural process where complex organic substances are broken down into smaller and simpler compounds by the enzymes produced by the microorganisms when oxygen is present. It usually involves the metabolic and enzymatic processes with the final products of carbon dioxide and water. Although complicated, the biodegradation tests usually measure relatively simple and non-specific parameters such as oxygen uptake, CO2 production, or dissolved organic carbon (DOC).

The organic compounds can be either electron donors or acceptors depending on their oxidation states, while oxygen usually acts as an electron acceptor or being involved in the activation of the substrate via oxygenation reactions.

The fundamental units of biodegradation are functional groups, as these are the ‘elements’ that undergo the transformation during the course of microbial catabolism. Each functional group may undergo several types of enzymatic transformations. Therefore, different compounds may show substantially different biodegradation behaviors. However, with about 40 most relevant functional groups found in the environmentally important compounds, and perhaps three to four transformation reactions for each, the University of Minnesota Biocatalysis/Biodegradation Database has been successfully collecting such information for two decades.

On the other hand, the functional groups/substructures that are beneficial for biodegradation generally follow the order: ester, amide, anhydride > hydroxyl > carboxyl, epoxide, site of unsaturation > benzene ring, methyl, methylene, while the negative influences were molecular mass, branching, halogenation, and nitrogen heterocycles.

For example, the initial attack on the aromatic ring is mostly assumed to be electrophilic in nature. Nitrogen ([-CN, -NO2, -NH3+, -CONH2]) and halogenic substituents strongly deplete the electron density of the aromatic ring and decrease the rate of biodegradation, while in the presence of hydroxyl and carboxyl groups was found to increase the biodegradation.

In addition, compounds that are already partially oxidized are generally considered to be more easily attacked than those which are not, and the initial degradation step is considered to be rate limiting step.

Learn More at Aropha Resource Center

Requirement and Applicability of Different Methods

Despite all other facts about the material compositions, local or regional regulatory requirements, and customers' own needs, the most important factors that determine which method to use for the aqueous biodegradation tests are the related physicochemical properties of the materials including solubility, volatility, and adsorptivity

As the adsorbing substances are usually hard to deal with and can easily lead to falsely high results, it is a good practice to avoid methods that are not able to handle adsorbing substances (e.g., OECD 301A).

The table below summarizes the applicability of different methods for test substances with different physicochemical properties.

Test Analytical method Sample info required * Poorly soluble Volatile Adsorbing
OECD 301B (CO2 evolution)
CO2 evolution Organic carbon content + - +
OECD 301D (Closed bottle)
Dissolved oxygen
ThOD or COD
+/- + +
OECD 301F (Oxygen consumption)
Oxygen consumption
ThOD or COD
+ +/- +
OECD 302B (Zahn-Wellens/EMPA Test)
DOC or COD Organic carbon content or COD +/- - -
OECD 303A (Activated sludge unit)
DOC and/or COD
Organic carbon content and/or COD
+/- - -
OECD 306 (Seawater)
DOC or dissolved oxygen Organic carbon content or ThOD
+/- + +
ISO 9408 (Oxygen consumption)
Oxygen consumption
ThOD or COD
+ +/- +
ISO 9439 (CO2 evolution)
CO2 evolution Organic carbon content + - +
ISO 9888 (Zahn-Wellens)
DOC or COD Organic carbon content or COD +/- - -
ISO 10707 (Closed bottle)
Dissolved oxygen
ThOD or COD
+/- + +
ISO 11733 (Activated sludge simulation)
DOC and/or COD
Organic carbon content and/or COD
+/- - -
ISO 14851 (Plastics - oxygen consumption)
Oxygen consumption
ThOD
+ +/- +
ISO 14852 (Plastics - CO2 evolution)
CO2 evolution Organic carbon content + - +
ISO 16221 (Seawater)
Various methods Organic carbon content or ThOD + + +
ASTM D5864 (Lubricant - CO2 evolution)
CO2 evolution Organic carbon content + - +
ASTM D6731 (Lubricant - Closed respirometer)
Oxygen consumption ThOD or COD + +/- +

*"Sample info required" is the information needed to calculate the biodegradation percentages. This must be available for a selected method.
*"Organic carbon content" is the ratio of the organic carbon weight to the weight of the sample. It can be calculated by the sample formula (e.g., acetic acid C2H4O2, organic carbon content is 12*2/(12*2+1*4+16*2)=40%). Try our Online C% Calculator. If the formula is unknown, we can send the sample out to a third party lab for you for analysis (normally $110 with a 10-day turnaround time).
*"ThOD" can be easily calculated based on the formula of the sample. Try our Online ThOD Calculator.
*"COD": we provide COD analysis at Aropha.

Recommended Methods for Different Physicochemical Properties

1. If the test substances are soluble in water to at least 100 mg/L, non-volatile, and non-adsorbing, they may be assessed by all the methods. The OECD 301D, OECD 301F, ISO 9408, and ISO 10707 are recommended due to their high reliability and simple experimental setup.

2. For soluble and volatile (e.g., Henry's law constant < 50 L·atm/mol) substances, only the closed bottle (OECD 301D and ISO 10707) and closed respirometer (OECD 301F, ISO 9408 and ASTM D6731) methods are suitable.

3. For insoluble and non-volatile substances such as lubricant, oil, and grease, the CO2 evolution (OECD 301B, ISO 9439, and ASTM D5864) or closed respirometer (OECD 301F, ISO 9408 and ASTM D6731) methods can be used.

4. For insoluble and volatile substances, only the closed respirometer methods (OECD 301F, ISO 9408 and ASTM D6731) are applicable.

5. For highly volatile substances (e.g., Henry's law constant > 50 L·atm/mol), the closed respirometer methods (OECD 301F, ISO 9408 and ASTM D6731) may not be applicable. Instead, the closed bottle test (OECD 301D and ISO 10707) is more appropriate.

6. OECD 302B and 303A may only be used if a material has failed the OECD 301 tests.

7. Strongly adsorbing materials may not be tested by OECD 302B, OECD 303A, ISO 9888, or ISO 11733.

Substance properties Example substances Applicable methods Recommended methods (ranked)
Soluble, non-volatile, non-adsorbing
Personal care product... All OECD 301F, ISO 9408, OECD 301D, ISO 10707
Soluble, volatile
Pharmaceuticals, pesticides, herbicides... OECD 301D, OECD 301F, ISO 10707, ISO 9408, ASTM D6731
OECD 301D, ISO 10707, OECD 301F, ISO 9408
Insoluble, non-volatile
Lubricant, oil, grease...
OECD 301B, OECD 301F, ISO 9439, ISO 9408, ASTM D5864, ASTM D6731 OECD 301F, ASTM D6731, ISO 9408
Insoluble, volatile
Petroleum fuels, paints, dry cleaning agents...
OECD 301F, ISO 9408, ASTM D6731 OECD 301F, ASTM D6731, ISO 9408
Highly-volatile
Paints, pharmaceuticals, and refrigerants...
OECD 301D, ISO 10707 OECD 301D, ISO 10707

Note: ASTM D5864 and ASTM D6731 are specified for the biodegradation tests of lubricants or their components. ISO 14851 and 14852 are for plastic materials. They may not be selected if the test substances are not in corresponding categories.

Aropha Resource Center

To learn more about different types of biodegradation tests, their applicability, biodegradation mechanisms, and many other information such as case studies, publications, and blogs, please check our Aropha Resource Center
.

Aropha resource center

CUSTOMERS

Initiate a Project with Us Today

Get StartedTalk to Us