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Diss Factsheets

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:
12 Nov - 10 Dec 2015
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 301 E (Ready biodegradability: Modified OECD Screening Test)
according to guideline
EU Method C.4-B (Determination of the "Ready" Biodegradability - Modified OECD Screening Test)
GLP compliance:
Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Germany

Test material

Constituent 1
Details on test material:
- Name of test material: reaction mass of potassium sodium (2R,3R)-2-hydroxy-3-(phosphonatooxy)butanedioate and potassium sodium (2S,3S)-2-hydroxy-3-(phosphonatooxy)butanedioate
- Molecular weight (if other than submission substance): 369.25 g/mole
- Physical state: white to slightly yellow powder of crystalline appearance
- Lot/batch No.: 010-2065
- Expiration date of the lot/batch: September 16, 2019
- Chemical stability: insensitive to exposure to light, considered to be stable in alkaline and neutral solutions (6 < pH <1 2)
- Storage condition of test material: storage at < 40 °C in a tightly closed container under dry conditions (substance very hygroscopic)

Study design

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): Ruhrverband Kläranlage, Schmallenberg, Germany (sampled on 12 Nov 2015)
- Pretreatment: the effluent was allowed to settle for 1 h and filtered through a coarse filter paper. Decanted effluent or filtrate was kept aerobic until use
- Amount of inoculum at test initiation: 0.5 mL effluent per litre of medium
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
25 mg/L
Based on:
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
- Composition of medium: according to guideline (for the test deionized water, free from inhibitory concentrations of toxic substances like Cu2+ ions, was used, containing no more than 10% of the organic carbon content introduced by the test item. This is checked in regular intervals by DOC analysis)
- Test temperature: 21.5 – 23.0 °C during test
- pH: 7.4 ± 0.2 at test initiation
- Light conditions: dark or diffuse light

- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: shaking machine
- Measuring equipment: concentrations of DOC in samples from each flask were determined in best of five measurements of one sample in a TOC analyser (Shimadzu TOC-V-CPH)
- Test performed in open system: yes, the openings of the flasks were covered in such a way as to allow free exchange of air between the flask and the surrounding atmosphere

- Sampling frequency: DOC was determined in each flask at test start, day 1, 3, 7, 11, 14, 18, 21, 27 and 28
- Sampling method: before sampling any evaporation losses were made good from the flasks by adding water in the required amount. Culture medium was mixed thoroughly prior to sampling. Material adhering to the walls of the vessels was redissolved or re-suspended before sampling. The sample was membrane-filtered immediately after it has been taken. Filtered samples were analysed on the same day.

- Inoculum blank:2 vessels
- Toxicity control: 1 vessel
- Procedural control: 1 vessels containing reference item and inoculum
- Adsorption control: 1 vessel containing test item, inoculum, and a sterilising agent

Reference substance
Reference substance:
benzoic acid, sodium salt
25 mg DOC/L

Results and discussion

% Degradation
% degradation (DOC removal)
St. dev.:
Sampling time:
28 d

BOD5 / COD results

Results with reference substance:
The reference substance sodium benzoate was degraded to 98% within the first 14 d

Any other information on results incl. tables

The biodegradation within the 10-day window was 11%. The 10-day window started at day 1. No lag phase / adaptation phase was noticeable. The test item was not toxic to microorganisms under test conditions as biodegradation of the item mixture reached 54% at day 14 (demanded threshold value according to guideline is 35%).

No significant adsorption of the test substance occurred: 0% (actually -4%) decrease of DOC in the adsorption control after 28 d of incubation (a maximum of 5% decrease in the course of the study – at day 18). According to Beek* et al. (2001), adsorption can be neglected when DOC decrease is lower than 20% within the first day. Consequently, the decrease in the test suspension and toxicity control can be assumed to be due to biotic degradation. The DOC Die Away Test fulfills the validity criteria of the guideline:

·        With 5% the difference of extremes of replicate values of the removal of the test item was less than 20% throughout the test

·        The percentage degradation of the reference item has exceeded the pass level of 70% by day 14

Table 1: DOC degradation (% from initial concentration). Mean values of the replicates. TI = Test solution (test item); PC = Procedural control (reference item); TC = Toxicity control (reference and test item); ADC = Adsorption control (inoculum sterilized with HgCl2).

1 9.88 74.91 33.03 3.29
3 12.46 95.49 54.1 3.3
7 11.11 92.38 52.37 0.81
11 11.49 96.93 53.9 3.74
14 10.35 97.55 53.73 1.3
18 13.47 99.68 55.92 5
21 10.17 97.75 54.02 0.39
27 10.11 98.1 53.16 -0.18
28 11.22 98.12 52.34 -4.08


*Beek, B., Böhling, S., Franke, C., Jöhnke, U., Studinger, G., Thumm, E.: The assessment of Biodegradation and Persistence. In: The Handbook of Environmental Chemistry Vol. 2 Part K Biodegradation and Persistence (Ed. Beek, B.) (2001). Springer Verlag, Heidelberg.

Applicant's summary and conclusion

Interpretation of results:
under test conditions no biodegradation observed