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Environmental fate & pathways

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:
14 Mar - 01 Apr 2016
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 301 A (Ready Biodegradability: DOC Die Away Test)
Version / remarks:
according to guideline
EU Method C.4-A (Determination of the "Ready" Biodegradability - Dissolved Organic Carbon (DOC) Die-Away Test)
Version / remarks:
No 440/2008
according to guideline
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
GLP compliance:
yes (incl. QA statement)
Federal Office for the Environment (FOEN)
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): wastewater treatment plant, ARA Birs (Birsfelden / Switzerland)
- Storage length: 2 d (sludge was aerated at room temperature)
- Preparation of inoculum for exposure: based on the determined dry weight, defined amounts of the activated sludge suspension were added to mineral medium to obtain a final concentration of 30 mg dry material per liter.
- Pretreatment: sludge was washed 3 times by centrifugation, and resuspension of the solid material in tap water and finally in mineral medium. Final sludge suspension were dried to determine the dry weight. Sludge was suspended in mineral medium to obtain 4 g dry material per liter. Prior to use, the dry weight was determined again.
Duration of test (contact time):
16 d
Initial conc.:
60.2 mg/L
Based on:
test mat.
Initial conc.:
34.2 mg/L
Based on:
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
- Composition of medium: according to guideline
- Test temperature: 22.6 °C
- pH: 7.3 - 7.4
- Dissolved oxygen concentration: 8.4 - 8.6 mg O2/L
- Continuous darkness: difusse light
- Other: aggitation: 130 rpm

- Culturing apparatus: 2 L Erlenmeyer glass vessels with a fill volume of 1 L and loosely covered with an aluminum cap
- Number of culture flasks/concentration: 2
- Measuring equipment: see details on analytical methods

- Sampling frequency: test item and inoculum control: 0, 2, 5, 7, 9, 12, 14 and 16 d, reference control: 0, 2, 7, 14 and 16 d, toxicity control: 0, 2, 7, 14 and 16 d
- Sampling method: one sample of about 10 mL was taken from each vessel per sampling timepoint

- Inoculum blank: 2 replicates
- Toxicity control: 1 replicate
- Reference control: 2 replicate
Reference substance:
benzoic acid, sodium salt
51.4 g/L
Preliminary study:
Solubility Pre-Experiment:
Prior to test start, the solubility of the test item in mineral medium was checked, and the test item was found to be soluble at a concentration of 100.1 mg/L. The measured dissolved organic carbon content (DOC) of this solution was 0.540 mg C/mg test item. The measured total organic carbon content (TOC) was 0.546 mg C/mg test item.
Key result
% degradation (DOC removal)
Sampling time:
16 d
Remarks on result:
other: the test was ended after 16 exposure d since the biodegradation curve of the test item had reached a plateau over at least three determinations
Details on results:
10-day window criterion fulfilled: 86% biodegradability on day 7
Results with reference substance:
99% biodegradation by day 14

Toxicity control: 97% degradation within 14 days and 98% degradation within 16 d. Therefore, the test item is considered to be non-inhibitory to microorganisms.

Table 1: Biodegradation of the Test Item and the Reference Item.

% Biodegradation
Time d Test item Reference item Toxicity control
0 0 0 0
2 5 88 44
5 29 n.d. n.d.
7 86 99 80
9 103 n.d. n.d.
12 98 n.d. n.d.
14 98 99 97
16 98 99 98
Validity criteria fulfilled:
Interpretation of results:
readily biodegradable
The substance was found to be completely and readily biodegradable after 16 days of exposure to activated sludge under the conditions of the conducted DOC Die-Away Test (OECD 301 A).
The pass level for ready biodegradability, i.e. at least 70 % removal of DOC in a 10-day window within the 16-day period of the test, was reached at Exposure Day 7 (86 %).
Executive summary:

In accordance with the test guidelines the test was ended after 16 exposure days, i.e. ahead of the normally 28 exposure days, since the biodegradation curve of the test item had reached a plateau over at least three determinations.

In the toxicity control, the initial DOC decreased by 97 % within 14 days of exposure. Thus, the test item had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 60.0 mg/L.

Description of key information

Readily biodegradable: 98% after 16 d (DOC removal) (OECD 301A)

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

One experimental study is available investigating the ready biodegradability of the substance. The study was performed according to OECD 301 A and GLP. Activated sludge from a domestic sewage treatment plant was used as inoculum. According to the guideline the test was ended after 16 d of exposure, since the biodegradation curve of the test item had reached a plateau over at least three determinations. 98% degradation of the substance was recorded based on DOC removal after 16 d. The 10-day window criterion was fulfilled, as 86% biodegradation was reached on day 7. Thus, the substance is readily biodegradable according to the OECD criteria under the chosen test conditions. A toxicity control containing both, reference substance and test substance, did not indicate inhibitory effects of the substance to the inoculum (97% degradation within 14 days and 98% degradation within 16 d).