OECD 301B Test: 60 % ThCO2 in 28 Days Explained

In short

OECD 301B is a ready-biodegradability test that measures the carbon dioxide a substance releases as micro-organisms break it down. A substance passes if it reaches 60 % of its theoretical CO2 within 28 days, with 60 % achieved inside a 10-day window that starts when 10 % degradation is reached.

OECD 301B, also called the Modified Sturm test, is the CO2 evolution member of the OECD 301 ready-biodegradability series. It is the method most often requested for surfactants and detergent ingredients in Europe, because carbon dioxide is a direct measure of mineralisation rather than a proxy. This page covers what the test measures, how it runs, what a pass really requires, how it compares with 301F and 301D, and how to read a signed report.

Method: CO2 evolution (Modified Sturm)
Duration: 28 days
Pass criterion: 60 % ThCO2, inside a 10-day window
PureSurf result: NEXOVANT F Eco: 74.7 % ThCO2 (GLP)

The worked example on this page is our own GLP study. See NEXOVANT F Eco's GLP 301B result

What OECD 301B measures

In a 301B test the substance is the only carbon source offered to a small, unadapted microbial inoculum in a mineral medium. Air scrubbed of carbon dioxide is bubbled through the vessels, and the carbon dioxide the culture produces is trapped and quantified, usually in barium or sodium hydroxide traps or by an inorganic-carbon analyser.

The measured carbon dioxide is expressed as a percentage of the theoretical carbon dioxide, ThCO2, which is the amount that would be produced if every carbon atom in the dose were oxidised. Because ThCO2 counts only carbon that has actually left the molecule as CO2, a 301B percentage is a statement about ultimate biodegradation, not about the parent structure merely losing its surface activity.

Parameter measured	Carbon dioxide evolved, as % of theoretical CO2 (ThCO2)
Test duration	28 days, with degradation plotted at least every few days
Inoculum	Activated sludge, secondary effluent or surface water, unadapted, low cell density
Test concentration	Typically 10 to 20 mg organic carbon per litre
Controls	Inoculum blank, reference substance (for example sodium benzoate), toxicity control
Suitable substances	Non-volatile, water-soluble or stably dispersible, including most surfactants

The 60 % pass criterion

A substance is classified as readily biodegradable under 301B when it reaches 60 % ThCO2 within the 28-day test. The 60 % figure is lower than 100 % by design: part of the carbon is assimilated into new microbial biomass instead of being respired, so full mineralisation of the dissolved fraction still leaves the CO2 yield short of the theoretical maximum.

The reference substance has to degrade normally, the inoculum blank has to stay low, and the toxicity control has to show that the test substance is not inhibiting the very organisms meant to degrade it. If any of those fail, the percentage on the front page of the report means nothing.

A result below 60 % is not proof of persistence. It means the substance did not meet the strict ready criterion under these conditions, and an inherent biodegradability test (OECD 302 series) or a simulation test may be the appropriate next step.

What the 10-day window means

The pass is not only about the final number. The 60 % must be reached inside a 10-day window that opens the moment degradation passes 10 %. The window exists to separate substances that degrade rapidly once micro-organisms encounter them from substances that need a long acclimation before anything happens.

A curve that crawls to 12 % by day 6, sits flat, and then climbs to 68 % by day 26 fails the ready criterion even though 68 % is well above 60 %. For multi-component substances such as some surfactant blends, the window may be waived if the components are assessed individually, and a report that relies on that allowance should say so explicitly.

OECD 301B compared with 301F and 301D

The three methods answer the same question with different instruments, and they do not always give the same answer for the same substance. Oxygen-based methods can read low when nitrification consumes oxygen, and CO2-based methods can read low when carbonate chemistry traps inorganic carbon.

Ready-biodegradability methods most often used for surfactants.
Method	Principle	Parameter measured	Pass threshold	Best suited to
OECD 301B	CO2 evolution (Modified Sturm)	Carbon dioxide, % ThCO2	60 % in 28 days	Non-volatile organics, surfactants, poorly soluble but dispersible substances
OECD 301F	Manometric respirometry	Oxygen demand, % ThOD	60 % in 28 days	Surfactants, coloured or turbid samples, substances needing a continuous curve
OECD 301D	Closed bottle	Dissolved oxygen depletion, % ThOD	60 % in 28 days	Low-solubility and low-dose substances, volatile substances

When two data sets disagree, check the method before the number. A 301B and a 301F result on the same substance are not interchangeable, and the EU Detergents Regulation accepts several of these methods for the same claim.

Where 301A and the DOC methods fit

OECD 301A (DOC Die-Away) and OECD 301E (Modified OECD Screening) track dissolved organic carbon removal rather than a respiration parameter, and their pass threshold is 70 % DOC removal rather than 60 %. That difference exists because carbon can leave the solution by adsorption or precipitation without being mineralised, so the DOC methods need the higher bar.

For surfactants the DOC family is used with care: surface-active molecules adsorb onto biomass and vessel walls, which can remove carbon from solution without degrading it and inflate the apparent result. That is one of the reasons 301B and 301F dominate surfactant dossiers.

Typical failure modes

Most 301B failures are not chemistry failures. They are dosing, solubility or inhibition problems that a pre-test would have caught.

Inhibition: the substance is dosed above its toxicity threshold for the inoculum, so degradation stalls. The toxicity control reveals it.
Poor dispersion: an undissolved substance offers little surface to the organisms and reads low.
Weak or wrong inoculum: sludge from a plant that never sees comparable chemistry degrades slowly, and the reference substance control will look sluggish too.
Carbonate interference: alkaline media or carbonate-containing test items distort the CO2 balance.
Nitrogen release: amine-containing substances can release ammonium, which consumes oxygen in 301F, so a 301B run avoids that particular artefact.
Slow start: the substance degrades well but only after acclimation, so it misses the 10-day window while exceeding 60 % overall.

GLP, cost and turnaround

For any regulatory use, run the study under Good Laboratory Practice at a monitored facility. GLP is a data-integrity framework, not a higher pass threshold: it governs the study plan, raw data retention, equipment calibration, personnel records and an independent quality-assurance audit. A non-GLP screening result is fine for internal ranking and useless for a dossier.

Plan on roughly 28 days of test time plus preparation and reporting. In practice a signed GLP 301B report usually lands eight to twelve weeks after the substance and its analytical documentation arrive at the laboratory, and contract prices in Europe typically sit in the low four figures per substance. Rush handling shortens queueing, never the 28-day test itself.

Send substance identity, purity, carbon content and, where relevant, the ThCO2 calculation basis with the sample.
Agree in advance whether the result will be reported on a total or an active-content basis.
Budget for a repeat: a first failure caused by inhibition is common and a lower dose often resolves it.

How to read a 301B report

Read a report in this order and the headline percentage becomes much easier to trust or discard.

Study identity: guideline version, GLP statement, test facility and signature date.
Test item: name, batch, purity and whether results are on an active or total basis.
Validity criteria: reference substance above 60 % by day 14, low inoculum blank, replicate agreement.
Toxicity control: degradation in the mixed vessel, which shows the substance did not poison the inoculum.
The degradation curve, not just the endpoint: check where 10 % was passed and whether 60 % arrived inside the following 10 days.
Final classification wording: "readily biodegradable" versus "not readily biodegradable under the conditions of the test".

If a supplier quotes a percentage without the method, the duration, the basis and the 10-day window verdict, treat it as marketing rather than data and ask for the study reference.

Worked example: NEXOVANT F Eco

NEXOVANT F Eco, our bio-based amine oxide grade optimised for the environmental profile, reached 74.7 % ThCO2 in 28 days under OECD 301B in a GLP study, which meets the ready-biodegradability criterion. The same GLP programme characterised aquatic toxicity under OECD 201 and OECD 202 and confirmed the material is non-mutagenic under OECD 471.

The performance grade, NEXOVANT F Pro, is a separate case and shows why grade-level reporting matters. A manometric respirometry study (OECD 301F) gave 78.9 % ThOD on an ammonium basis, but that report is not yet signed, and an earlier OECD 301B study on the same substance reached 47.4 % ThCO2, below the threshold. We publish both numbers and will publish the final classification when the report is signed.

NEXOVANT F Eco, OECD 301BFinal GLP	74.7 % ThCO2 in 28 days, readily biodegradable
NEXOVANT F Pro, OECD 301BGLP, in finalisation	47.4 % ThCO2 in 28 days, below the 60 % criterion
NEXOVANT F Pro, OECD 301FGLP, in finalisation	78.9 % ThOD on an ammonium basis, report not yet signed

Frequently asked questions

What is the OECD 301B test?

OECD 301B is a ready-biodegradability test, also called the Modified Sturm test, in which micro-organisms are given the test substance as their only carbon source and the carbon dioxide they release is measured over 28 days and expressed as a percentage of the theoretical carbon dioxide.

What is the difference between OECD 301B and 301F?

301B measures the carbon dioxide produced (% ThCO2) and 301F measures the oxygen consumed in a manometric respirometer (% ThOD). Both run for 28 days with a 60 % threshold. 301F suits coloured or turbid samples and gives a continuous curve; 301B avoids the oxygen artefact caused by nitrogen release from amine-containing substances.

How long does an OECD 301 test take?

The test itself runs 28 days. Including sample preparation, analytics, reporting and GLP quality assurance, a signed report usually takes eight to twelve weeks from the day the substance reaches the laboratory.

What does the 10-day window mean?

The 10-day window is the period that starts when degradation first exceeds 10 %. To be classified readily biodegradable, the substance must reach 60 % within those 10 days, not just at some point before day 28. It filters out substances that need a long acclimation before they degrade.

Is 60 % biodegradation good?

Yes, 60 % ThCO2 or ThOD inside the window is the formal pass for ready biodegradability. It is not a partial result: part of the carbon becomes microbial biomass rather than carbon dioxide, so 60 % already corresponds to essentially complete degradation of the available fraction.

Which OECD 301 test is best for surfactants?

301B and 301F are the two workhorses. 301B is preferred when carbon dioxide is the cleaner signal, for example with amine-containing surfactants that release ammonium. 301F is preferred when a continuous oxygen curve or a turbid sample makes respirometry easier. The DOC-based methods are used with care because surfactants adsorb onto biomass.

Does ready biodegradable mean the same as biodegradable?

No. Ready biodegradability is a strict, defined classification from a 28-day test with an unadapted inoculum. "Biodegradable" on its own has no test behind it. Between the two sits inherent biodegradability, which shows a substance can degrade under favourable conditions but not that it degrades quickly in the environment.

What is ThCO2?

ThCO2 is the theoretical carbon dioxide: the mass of carbon dioxide that would be produced if every carbon atom in the dosed substance were fully oxidised. Measured carbon dioxide divided by ThCO2 gives the degradation percentage reported in a 301B study.

Does a 301B pass mean the substance is safe for aquatic life?

No. Biodegradability and aquatic toxicity are separate endpoints. A substance can degrade quickly and still be acutely toxic while it is present, which is why OECD 201 and OECD 202 results are reported alongside 301B data.

Content last reviewed: 2 August 2026 by Prof. Katalin Barta Weissert

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