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Environmental fate & pathways

Biodegradation in water: screening tests

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biodegradation in water: ready biodegradability
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From September 19 to October 21, 2016
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Reliability of the source study is 1
according to guideline
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
according to guideline
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
Inoculum or test system:
other: Secondary effluent of the waste water treatment plant of Pardubice processing predominantly municipal sewage.
Details on inoculum:
Preparation of the inoculum: The fresh collected waste water was filtered through paper filter. COD was determined and waste water was aerated till using.
Duration of test (contact time):
ca. 28 d
Initial conc.:
ca. 4.89 mg/L
Based on:
test mat.
Initial conc.:
ca. 3.4 mg/L
Based on:
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Test procedure:
Series of bottles for the test
-2 x 9 bottles with the test substance and inoculated medium
-2 x 9 bottles with the reference substance and inoculated medium for the check of inoculum activity
-9 bottles with the test substance and inoculated medium for the determination of oxidized nitrogen forms
-2 x 5 bottles with the test and the reference substance mixture and inoculated medium for the toxicity test
-2 x 9 bottles with inoculated medium only for the blank determination

The bottles were placed into thermostat.
Reference substance:
benzoic acid, sodium salt
% degradation (O2 consumption)
ca. 0
Sampling time:
28 d

Quality criteria

Prescribed criteria of the test validity:

The test should satisfy the following quality criteria given in the EU test method:

1. Oxygen depletion in the inoculum blank should not exceed 1.5 mg O2·L-1after 28 days.

2. The residual concentration of oxygen in the test should not fall below 0.5 mg·L-1in any time.

3. The difference of extremes of replicate values of the removal of the test substance at the end of the test should be less than 20 %.

4. The percentage degradation of the reference substance should reach the level for ready biodegradability (min. 60 %) by 14 days.

5. If in a toxicity test, containing both the test substance and the reference chemical, less than 25 % degradation should occur in 14 days, the test substance could be assumed to be inhibitory. The test series should be repeated, if possible, using a lower concentration of test substance or a higher concentration of inoculum.


The following values were attained in the current study:

1. Oxygen depletion in the inoculum blank reached 0.91 mg·L-1during 28thday of the test.

2. The lowest concentration of dissolved oxygen in bottles was 5.13 mg·L-1.

3. The difference of two determinations of the test substance degradation at the end of test was 2.4 %.

4. The percentage degradation of the reference substance has reached the level for the ready biodegradability (min. 60 %) by the 7thday of the test.

5. In the toxicity test, after 14 days of incubation the degradation of the test and reference substance was 26.5 %. Therefore the test substance is not inhibiting for the used inoculum.

Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
Not readily biodegradable
Executive summary:


The test was performed according to Method C.4E - Closed Bottle Test, Council Regulation (EC) No. 440/2008, published in O.J.L 142, 2008. The results of biological degradation are related to experimentally determined COD values of the test and reference substance at the beginning of the test.

The test substance was sufficiently soluble in used mineral medium so the dosage from the stock solution was carried out.

Sodium benzoate was used as the reference substance. The dosage was carried out from the stock solution.

COD of the test substance in medium at the beginning of the main test:             3.40 mg/l

COD of the reference substance in medium at the beginning of the main test:     3.47 mg/l

In parallel to the main test the toxicity test was performed.

Based on the test substance contains nitrogen in the chemical composition, the oxidized nitrogen forms were determined and the correction for nitrification was carried out.

The test was performed at temperature of 20 ± 1 °C with the pH values of solutions 7 - 8 at the beginning of the test.


In this 28-day study of ready biological degradability the degradation of 0 % of the test substance was attained in the end of study.


Description of key information

Not readly biodegradable

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

No information about the Biodegradability in water of the test substance is available.
Nevertheless, by the nature of their design and use, dyes are not intended to be readily biodegradable as this would assist in the rapid destruction of the dyestuff, rendering it unfit for purpose during its application. As such, it is accepted that these substances are not readily biodegradable under relevant environmental conditions. A published study (Pagga & Brown, 1986) describes the results of the testing of 87 dyestuffs in short-term aerobic biodegradation tests. The authors of this publication concluded that dyestuffs are very unlikely to show any significant biodegradation in such tests and that 'there seems little point in carrying out such test procedures’ on dyestuffs.