Biodegradability test (screening test) has been a common practice to investigate the environmental fate of newly developed materials once they are released into the environment.
Traditional experimental tests are conducted to investigate the biodegradability.
However, recent advances in computer science has provided numerous in-silico ways to predict the biodegradability based on machine learning models.
In this section, we will dive into both segments to take a quick look at how they can help us achieve biodegradability estimation (for both ready and inherent biodegradation).
For the experimental test (for ready and inherent biodegradation), the detailed system setups and procedures have been very well documented into different series of standard methods by different organizations, such as OECD, EPA, EU, and ISO.
A glance of different biodegradation test guidelines
Below is a glance of these documented standard methods (sorted in alphabetical order).
|ASTM D5864||CO2 Evolution Test||CO2 Evolution||-|
|ASTM D6731||Closed Respirometer Test||Closed Respirometer||-|
|EPA OPPTS 835.3120||CO2 Headspace Test||Closed Respirometer||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 796.3260||Modified Sturm Test||CO2 Evolution||Ready|
|EPA OTS 796.3340||Modified SCAS 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|
|ISO 5815||Biochemical Oxygen Demand after 5 Days||Closed Bottle Test||Ready|
|ISO 9888||Zahn-Wellens Test||DOC Die Away||Inherent|
|ISO 10707||Closed Bottle Test||Closed Bottle Test||Ready|
|ISO 10708||Closed Bottle Test||Closed Bottle Test||Ready|
|ISO 14593||CO2 Headspace Test||Closed Respirometer||Ready|
|ISO DIS 9408||Oxygen Demand in a Closed Respirometer||Closed Respirometer||Ready|
|ISO DIS 9439||CO2 Evolution||CO2 Evolution||Ready|
|OECD Guideline 301 A||DOC Die Away||DOC Die Away||Ready|
|OECD Guideline 301 B||CO2 Evolution||CO2 Evolution||Ready|
|OECD Guideline 301 C||Modified MITI Test (I)||Closed Respirometer||Ready|
|OECD Guideline 301 D||Closed Bottle Test||Closed Bottle Test||Ready|
|OECD Guideline 301 E||Modified OECD Screening Test||DOC Die Away||Ready|
|OECD Guideline 301 F||Manometric Respirometry Test||Closed Respirometer||Ready|
|OECD Guideline 302 A||Modified SCAS Test||DOC Die Away||Inherent|
|OECD Guideline 302 B||Zahn-Wellens Test||DOC Die Away||Inherent|
|OECD Guideline 302 C||Modified MITI Test (II)||Closed Respirometer||Inherent|
|OECD Guideline 302 D||Closed Respirometer||Closed Respirometer||Inherent|
|OECD Guideline 306 (I)||Shake Flask Test in Seawater||DOC Die Away in Seawater||Seawater|
|OECD Guideline 306 (II)||Closed Bottle Test in Seawater||Closed Bottle Test in Seawater||Seawater|
|OECD Guideline 310||CO2 Headspace Test||Closed Respirometer||Ready|
Summary of different guidelines
The table above indicates that even though there are quite a large number of standard methods, they are mostly similar to each other in terms of principles and endpoints.
Among different principles, the Closed Bottle Test was found to be the most popular with 11 individual guidelines, followed by DOC Die Away with 10 guidelines.
The least popular guidelines were Closed Bottle Test in Seawater and COD, both having only one guideline.
|Number||Principle||Number of guidelines|
|1||DOC Die Away in Seawater||1|
|2||Closed Bottle Test in Seawater||1|
|6||DOC Die Away||10|
|7||Closed Bottle Test||11|
As for the endpoints of the tests, most guidelines are for Ready biodegradability test with a total number of 26, while only 8 are designed for Inherent biodegradability test.
|Number||Endpoint||Number of guidelines|
Among the different series of methods shown above, OECD methods gained the highest popularity in many countries.
In addition to above 301 and 302 series of tests for ready and inherent biodegradability tests, respectively, OECD also has a 303 series of test.
OECD methods were well developed to accommodate most of the biodegradation test requirements.
301 and 302 are designed for ready and inherent biodegradability test, respectively, under relatively stringent conditions (high substance to inoculum ratio), while 303 are for simulated biodegradation under identical conditions compared to the real activated sludge treatment processes.
The 301 and 302 methods are considered acceptance tests instead of rejection tests, meaning that if a sample fails these tests, it does not necessarily mean this compound cannot be degraded once it enters the environment. It could still be potentially biodegradable under more favorable conditions (e.g., higher microorganism concentration, and/or longer exposure time).
301 ready biodegradation tests: An acceptance test, not a rejection test. It selects chemicals that do not have to be tested further because high biodegradability is expected in sewage treatment plants. Measurement is based on nonspecific parameters like DOC, BOD, or CO2. Developed to devise screening methods to determine whether a chemical is potentially easily biodegradable, rather than to predict the actual rate, of biodegradation in the environment. A readily biodegradable material is assumed to be able to undergo rapid and ultimate biodegradation in the environment. Therefore no further investigation on the biodegradability, toxicity, or other environmental effects is normally required.
302 inherent biodegradability tests: Allow prolonged exposure of the test substance to microorganisms and a low ratio of test substance to biomass, which offers a better chance to obtain a positive result compared to tests for ready biodegradability. Biodegradation percentages above 20% may be regarded as evidence of inherent, primary biodegradability, whereas biodegradation percentages above 70% may be regarded as evidence of inherent, ultimate biodegradability.
303 simulation tests: Based on a simulation of the conditions existing in an activated sludge plant. This test should be used for any chemicals that did not pass the previous tests, either to confirm or disprove the first results obtained.
A test usually starts from the lowest tier of methods (301). If it fails, one can move on to higher tiers until a confident conclusion can be drawn.
Environmental risk assessment can be performed if a compound fails all these tests.
More information can be found in the OECD official document.
ASTM D5864 and ASTM 6731
ASTM D5864 and D6731 are two ASTM methods specifically designed for the biodegradation test of lubricants or their components. ASTM D5864 measures the CO2 evolution, which is equivalent to the OECD 301B method. ASTM D6731 measures the oxygen consumption, which is equivalent to the OECD 301F.
ASTM6731 is our preferred method for lubricant test due to its simple system setup and high applicability.
Prediction using machine learning
Traditional standard methods for biodegradation tests are generally very time-consuming and labor-intensive. To overcome these drawbacks, a number of alternative approaches have been proposed in the literature to decrease the time and labor input (e.g., for BOD tests).1
With the increase of the documented experimental data as well as the development of data science in recent years, machine learning has been encouraged to develop predictive models for easy prioritization of newly developed compounds.2-5
At Aropha, a significant portion of our efforts have been dedicated to develop such predictive models. Please visit the "Machine Learning Prediction" page for more details.
Test methods we provide at Aropha
What we provide
At Aropha, we are dedicated to provide accurate but also cost-effective methods to meet our customers' needs at the lowest prices.
To achieve these goals, we are putting a significant amount of efforts to incorporate automation and other novel technologies for ready, inherent, and simulated biodegradability, and in-silico prediction using machine learning.
Since the closed respirometer and closed bottle tests have the top levels of simplicity and applicability, OECD 301F and OECD 301B are the mostly recommended methods.
In addition to experimental tests, we also provide predictive models developed based on machine learning for cheap, quick, and reliable predictions of target compounds (with specific chemical structures). Please visit the page "Machine Learning Prediction" for more details.
- Jouanneau, S.; Recoules, L.; Durand, M. J.; Boukabache, A.; Picot, V.; Primault, Y.; Lakel, A.; Sengelin, M.; Barillon, B.; Thouand, G. Methods for assessing biochemical oxygen demand (BOD): a review. Water Res. 2014, 49, 62-82.
- Cheng, F.; Ikenaga, Y.; Zhou, Y.; Yu, Y.; Li, W.; Shen, J.; Du, Z.; Chen, L.; Xu, C.; Liu, G.; Lee, P. W.; Tang, Y. In silico assessment of chemical biodegradability. J. Chem. Inf. Model. 2012, 52 (3), 655-69.
- Mansouri, K.; Ringsted, T.; Ballabio, D.; Todeschini, R.; Consonni, V. Quantitative structure-activity relationship models for ready biodegradability of chemicals. J. Chem. Inf. Model. 2013, 53 (4), 867-78.
- Pizzo, F.; Lombardo, A.; Brandt, M.; Manganaro, A.; Benfenati, E. A new integrated in silico strategy for the assessment and prioritization of persistence of chemicals under REACH. Environ. Int. 2016, 88, 250-260.
- Fernandez, A.; Rallo, R.; Giralt, F. Prioritization of in silico models and molecular descriptors for the assessment of ready biodegradability. Environ. Res. 2015, 142, 161-8.