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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 Feb - 29 Mar 2018
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
17 Jul 1992
according to
other: ISO standard 10634
Version / remarks:
"Water quality - Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium", 1995
GLP compliance:

Test material

Test material form:

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Municipal Sewage Treatment Plant, 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands
- Storage length: The freshly obtained sludge was used immediately.
- Pretreatment: Before use, the sludge was washed with mineral medium.
- Preparation of inoculum for exposure: Magnetic stirring
- Concentration of sludge (susended solids): 3.2 g/L
- Initial cell/biomass concentration: 3 mL/L equal to 9.6 mg/L suspended solids
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
18 mg/L
Based on:
test mat.
Initial conc.:
12 mg/L
Based on:
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium: Mineral medium (consisting of solutions A to D according to the guideline) prepared with tap-water purified by reverse osmosis (Milli-RO) and activated carbon
- Test temperature: 22 - 23 °C
- pH: 7.6 (test start), 7.8 (Day 14), 7.4 - 7.5 (Day 28)
- pH adjusted: No. Exception: 1 blank control replicate was adjusted at test start from 7.7 to 7.6 using 1 M HCl.
- Aeration of dilution water: Yes, the test media were aerated with synthetic air during the test.
- Suspended solids concentration at test start: 9.6 mg/L
- Continuous darkness: Yes

- Culturing apparatus: 2 L brown glass bottles
- Preparation of bottles: At the start of the test (Day 0), test and reference item were added to the bottles containing the microbial organisms and mineral components. The volumes of suspensions were made up to 2 L with Milli-RO water.
- Pre-incubation medium: The day before test start (Day -1) the mineral components, Milli-RO water (ca. 80% of final volume) and the inoculum were added to each bottle. This mixture was then aerated over night to purge the system of CO2.
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Aeration with synthetic air (CO2 < 1 ppm)
- Measuring equipment: Titration
- Details of trap for CO2 and volatile organics if used: Three CO2-absorber bottles filled with 100 mL 0.0125 M Barium hydroxide (Ba(OH)2) were connected in series to the exit air line of each test bottle. Synthetic air was passed through the scrubbing solutions at a rate of approximately 1 - 2 bubbles per second (30 - 100 mL/min).
- Other: The test media were aerated and stirred continuously.
- Other: At the start of the test (Day 0), the test and reference items were added to the bottles containing the microbial organisms and mineral components. The volumes of the suspensions were made up to 2 L with Milli-RO water, resulting in the mineral medium described before.

- Sampling frequency: Day 2, 5, 8, 12, 15, 19, 23, and 29
- Sampling method: The CO2-absorber nearest to the test bottle was removed for titration. Each of the remaining two absorbers were moved on position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series.
- Other: The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titration of the remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl). On the penultimate day, the pH of the respective test suspensions was measured and 1 mL concentrated HCl (37%, Merck) was added to the bottles of the inoculum blank and test suspension. The bottles were aerated over night to drive off CO2 present in the test suspension.

- Inoculum blank: 2 replicates (no test item)
- Procedure control: 1 replicate (reference item sodium acetate)
- Toxicity control: 1 replicate (test item + reference item)
Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

% Degradationopen allclose all
% degradation (CO2 evolution)
Sampling time:
28 d
Remarks on result:
Replicate 1
% degradation (CO2 evolution)
Sampling time:
28 d
Remarks on result:
Replicate 2

BOD5 / COD results

Results with reference substance:
The reference item was biodegraded by 85% within 14 d (pass-level: at least 60% within 14 d), thus confirming the suitability of the inoculum and test system.

Any other information on results incl. tables


The study met all the criteria (Table 1) and was thus considered valid.

Table 1: Validity criteria for OECD 301.

Criterion from the guideline


Validity criterion fulfilled

Difference of extremes of replicate values of the removal of the test chemical at the plateau, at the end of the test or at the end of the 10-d window, as appropriate, is less than 20%.

The difference of duplicate values for % degradation of the test item was ≤ 16% (< 20%).


Percentage degradation of the reference compound has reached the pass levels by day 14.

The reference item was biodegraded by 84% within 14 d (> 60%).


The toxicity control should degrade to at least 25% (based on ThOD or ThCO2) within 14 d.

After 14 d, 32% biodegradation was reached in the toxicity control.


The IC content of the test substance suspension in the mineral medium at the beginning of the test must be less than 5% of the TC.

The IC content of the test item suspension was < 5% of the Total Carbon content.


The total CO2 evolution in the inoculum blank at the end of the test should not normally exceed 40 mg/L medium.

The total CO2 release in the blank at the end of the test was 31.8 mg CO2/L (63.6 mg CO2 per 2 L of medium).




The relative biodegradation values calculated from the measurents performed during the test period revealed 2 and 18% biodegradation of the test item based on ThCO2, for the duplicates tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10 -d window) was not met.

In the toxicity control, degradation was 32% after 14 d, which is above the pass level of > 25% in 14 d. Thus, the test item was found not to inhibit microbial activity.

Applicant's summary and conclusion

Validity criteria fulfilled:
For further details please refer to “Any other information on results incl. tables”.
Interpretation of results:
not readily biodegradable