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Biodegradation in water: screening tests

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biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999-02-05 to 1999-03-05
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD 301 F test guideline and in compliance with GLP
according to guideline
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
GLP compliance:
Oxygen conditions:
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): The activated sludge was obtained from the municipal sewage treatment plant in Reutlingen.

- Preparation of inoculum for exposure: The innoculum was washed 2 times with dechlorinated tap water and centrifuged at 3000 rpm for 1 minute, 1 g of the wet centrifugate corresponded to approx. 60 mg suspended solids. 0.1 g of the centrifugate were introduced per flask of volume 250 mL.

- Concentration of sludge: 24 mg/L suspended solids

- Water filtered: no
Duration of test (contact time):
28 d
Initial conc.:
102.2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
- Composition of medium: The following stock solution were prepared, see table 1

- Solubilising agent (type and concentration if used): No

- Test temperature: 22 ± 2 °C

- pH: 7.7

- pH adjusted: no

- Aeration of dilution water: Demineralised water was aerated for 1 day with oil-free compressed air. Before usage, the water was allowed to stand for 0.5 hours at 20 °C.

- Suspended solids concentration: 24 mg/L

- Continuous darkness: no

- Culturing apparatus: Closed bottles

- Number of culture flasks/concentration: 6 Flasks: 1. Test article in mineral medium + inoculum; 2: Test article in mineral medium + inoculum; 3: Mineral medium + inoculum (blank); 4: Mineral medium + inoculum (blank); 5: Reference article in mineral medium + inoculum; 6: Test article in dilution water, sterilised with 6 mL HgCl2/L medium (C HgCl2 = 10 g/L; abiotic control).

- Method used to create aerobic conditions: Electrolytic oxygen supply and automatic data registration

- Measuring equipment: BOD-digi (J. Otto-GmbH), electrolytic oxygen supply with automatic data registration

- Test performed in closed vessels: Yes

- Details of trap for CO2 and volatile organics if used: Respirometric BOD-determination with CO2 absorption on soda lime

- Sampling frequency: Continuous BOD measurement

- Sampling method: Automatic data registration

- Inoculum blank: Yes: mineral medium + inoculum.

- Abiotic sterile control: Yes: test article in dilution water, sterilised with 6 mL HgCl2/L medium (C HgCl2 = 10 g/L; abiotic control).

- Toxicity control: No

Reference substance:
benzoic acid, sodium salt
Test performance:
The measurements concerning the innoculum blank, the reference substance and the test article comply with the criteria for validity in the OECD Guideline 301 F.
Key result
% degradation (O2 consumption)
Sampling time:
28 d
Results with reference substance:
Test concentration: 102.2 mg/L

- Theoretical Oxygen demand (ThOD): 1,67 mg O2/mg substance
- ThOD in flask 5: 165 mg O2/L

- BOD in flask 5 after 28 days: 1.691 mgO2/mg substance or 166.6 mg O2/L (including blank)

- Biodegradation after 14 days: 91.5 %

- Biodegradation after 28 days: 97.2 %

Table 2: BOD removal from the test flasks during the 28 day exposure


O2 uptake (mg O2/L)


ThODTest substance








BOD Test substance








BOD Blank















BOD Reference article








BOD Removal








 BOD removal = BODtestsubstance -BODblank-BODabiotic= 193.8 mg O2/L or 1.896 mg O2/mg test substance. 


Validity criteria fulfilled:
Interpretation of results:
other: Readily biodegradable; no information on degradation in 10 day window
A biodegradation value of 73 % after 28 days in a Manometric Respirometry test was determined in a reliable study conducted according to OECD 301 F, and in compliance with GLP. The test substance was considered readily biodegradable.

Description of key information

A Final Decision letter (CCH-D-2114412576-50-01-F) was received for this substance on 09 July 2018 which requested further information on the PBT properties of the Block 10 Aldehyde consituents in this substance. As a first step Abietal was chosen to represent the Aldehyde Block 10 consituents and a ready biodegradation study was comissioned and depending on the outcome of that study the requirement for further testing for Persistence, Bioaccumulation and Toxicity would not be required.

The results show that Abietal meets the criteria in the OECD 301B to be labelled readily biodegradable e.g. at least 60% biodegradation in 28 days. Meeting the 10-day window was not a requirement according to both the ECHA Final Decision Letter and OECD guidance documents.

The test item is non-persistent, and satisfies the criteria to be labelled readily biodegradable based on the results obtained in the second experiment.

Abietal is representative of the Block 10 consituents and is therefore as it is readily biodegradable it is not considered PBT or vPvB.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Biodegradation studies for Abietal.

First, Abietal was put through a feasibility study in which 3 different inoculum were tested. The objective of this study was to evaluate the feasibility of the proposed adaptations, and to determine the optimal experimental conditions to assess biodegradability of Abietal (test item) in aerobic aqueous medium, with inoculation using activated sludge, standardized soil, or a mixture of these two. Adaptations are in accordance with OECD 301 B and/or enhancements described in ECHA Guidance. The duration of the test was extended to 42 days, which makes this an enhanced ready biodegradability study, as described in ECHA Guidance.

The test item did not meet the criteria to be classed as readily biodegradable under the conditions of the test whilst using any of the tested inocula. However, the study was conducted under the strict conditions of an OECD 301B ready biodegradation test, while Abietal achieved significant ultimate biodegradation across all inocula (>50 – 86% degradation) in less than 60 days. These results indicate that there is a strong potential for Abietal to ultimately biodegrade rapidly in the environment and therefore it may not have to be classified as persistent.

A full OECD 301B study under GLP, with potential extension to 60 days, is proposed to determine whether the substance can be classified as readily biodegradable or if it can be classified as not persistent according to the ECHA Guidance Chapter R.7b and R.11.

Definitive OECD 301B study

The objective of the study was to evaluate test item Abietal for its ready or ultimate biodegradability in an aerobic aqueous medium with microbial activity introduced by inoculation with activated sludge.

In total, two experiments were performed. The first experiment showed high test item variability. Therefore, the experiment was repeated. The variability was confirmed to be test item related in the second experiment. Since both experiments provide valuable information to support the overall conclusion, both experiments are reported in this report. With the exception of the difference between replicate values, the study met the validity criteria prescribed by the Study Plan and was considered to be valid.



Biodegradation (1stexperiment)

47.9 % (19 % to 79 %) after 42 days. (Repeated due to high variability between replicates, significant degradation observed)

Biodegradation (2ndexperiment)

66.8 % (42 % to 104 %) after 48 days (non-persistent criteria met)

60.4 % (45 % to 83 %) after 28 days, with exclusion of replicate B (readily biodegradable criteria met)


No inhibition of microbial activity.

Abiotic degradation

Formation of CO2was not observed in the abiotic controls


Abietal met the criteria to be classified as readily biodegradable.

The high rate of degradation observed in both experiments supports the conclusion that Abietal can be classified as non-persistent in accordance with the ECHA information requirements guidance R.11.

DTO biodegradation as a whole substance

A ready biodegradation value of 73.2% (measured as percentage of theoretical oxygen demand) was achieved in 28 days using a method consistent with OECD Guideline 301 F (manometric respirometer) for Distilled Tall Oil (DTO). Although, information about the 10-day window was not available, the degradation rate is very rapid for a very poorly soluble substance. The reference substance attained 92% degradation by Day 14 and percentage degradation between replicate test samples was ± 20%, satisfying the validity criteria of the test.

In other supporting studies, DTO was readily biodegradable in a GLP test conducted according to OECD 301D (Closed Bottle Test) using an equal volume mixture of three commercially available products. The substance achieved significant levels of biodegradation (60% in 28 days) but failed to meet the 10-day window. The biodegradability of a commercial grade of DTO was measured in a GLP test according to OECD Guideline 301E (Modified OECD Screening Test). The test medium comprised an aqueous extract of the product, prepared by loading culture medium with 10 g/l test substance and stirring for 23 hours, standing for 1 hours and removing the aqueous extract by siphoning. The aqueous extract attained 64% degradation in 28 days.

However, the assessment of biodegradability of the test substance is based on the test conducted in accordance to OECD 301F because this is more applicable to a poorly soluble substance like DTO. As a whole, it can be concluded that DTO is readily biodegradable for purposes of classification and labelling.