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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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
Adopted July 17, 1992
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium diisopropylnaphthalenesulphonate
EC Number:
EC Name:
Sodium diisopropylnaphthalenesulphonate
Cas Number:
Molecular formula:
sodium diisopropylnaphthalene-sulfonate
Test material form:
solid: particulate/powder
Details on test material:
white to off-white lumps

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): Effluent freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage
- Laboratory culture: /
- Method of cultivation: /
- Storage conditions: The filtrate was kept aerated until inoculation.
- Storage length: /
- Preparation of inoculum for exposure: /
- Pretreatment: /
- Concentration of sludge: 4 mL filtrate of secondary effluent per litre of final volume.
- Initial cell/biomass concentration: /
- Water filtered: yes
- Type and size of filter used, if any: Secondary effluent was filtered through a coarse filter paper, the first 200 mL were discarded.
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
2 mg/L
Based on:
test mat.
Initial conc.:
4 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Test duration 28 days for the inoculum blank and test suspension
14 days for the procedure and toxicity control
Test bottles 250-300 mL BOD bottles with glass stoppers.
Milli-RO water Tap-water purified by reverse osmosis (Millipore Corp., Bedford, Mass., USA).

Stock solutions of mineral components
A) 8.50 g KH2PO4
21.75 g K2HPO4
67.20 g Na2HPO4.12H2O
0.50 g NH4Cl
dissolved in 1 litre Milli-RO water, pH 7.4 + 0.2
B) 22.50 g MgSO4.7H2O dissolved in 1 litre Milli-RO water.
C) 36.40 g CaCl2.2H2O dissolved in 1 litre Milli-RO water.
D) 0.25 g FeCl3.6H2O dissolved in 1 litre Milli-RO water.

Mineral medium 1 mL aliquots of solution (A) to (D) were mixed and made up to 1 litre with Milli-RO water. The concentration of dissolved oxygen was measured for control purposes. The mineral medium was left at test temperature to obtain an oxygen-saturated solution at the start of the test. Care was taken to ensure the solutions were not supersaturated with oxygen.

Test set up Inoculum blank: Containing only inoculum (no test item)
Procedure control: Containing reference item and inoculum
Test suspension: Containing test item and inoculum (applicable for both test concentrations)
Toxicity control: Containing reference item, test item at the lowest concentration and inoculum.
Number and groups of test bottles Individual BOD bottles were prepared for each measuring point, except that the bottles measured at the start were also
measured on day 7. Parallel groups were prepared to allow duplicate measurements of oxygen consumption at the test intervals.
Illumination The test bottles were protected from light.
Reference substance
Reference substance:
other: sodium acetate

Results and discussion

Test performance:
The pH values at the start of the test were between 7.4 and 7.5.
The temperature recorded in a vessel with Milli-RO water in the same room varied between 21 and 23°C, and complied with the requirements as laid down in the study plan.
% Degradationopen allclose all
% degradation (O2 consumption)
for the low test item concentration
St. dev.:
Sampling time:
21 d
% degradation (O2 consumption)
for the high test item concentration
St. dev.:
Sampling time:
21 d
Details on results:
The relative biodegradation values calculated from the O2 measurements performed during the test period revealed no biologically relevant biodegradation of CH04207 at the low and high concentration of CH04207 (mean value of 3% and 2%, based on ThOD, respectively).
In the toxicity control, more than 25% biodegradation occurred within 14 days (mean value of 25%, based on ThOD). Therefore, the test item was assumed not to inhibit microbial activity.

BOD5 / COD results

Results with reference substance:
In the procedure control, sodium acetate was degraded by at least 60% (82%) within 14 days

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
In conclusion, CH04207 was not readily biodegradable under the conditions of the closed bottle test performed presently.
Executive summary:

The objectiveofthe studywasto evaluate the organic test item CH04207 for its ready biodegradability in an aerobic, aqueous medium with microbial activity introduced by inoculation of secondary effluent during a test period of 28 days.

The study procedure described in this report was based onthe OECD guideline No. 301 D, 1992.

CH04207 consisted of white to off-white lumps with a purity of >97%. The Theoretical Oxygen Demand (ThOD) of CH04207 was calculated to be 2.14 mg O2/mg based on the molecular formula. Nominal concentrations of 2 and 4 mg/L were tested. No correction was made for the purity or composition of the test item.

The following test set up was applied, with duplicate vessels for each time point:

·     Inoculum blank (only inoculum, no test item);

·     Procedure control (sodium acetate at 2 mg/L and inoculum);

·      Test item (CH04207 at 2 mg/L and inoculum);

·      Test item (CH04207 at 4 mg/L and inoculum);

·      Toxicity control (sodium acetate at 2 mg/L, CH04207 at 2 mg/L and inoculum).

A clear and colourless stock solution was prepared by dissolving 50 mg test item in mineral medium. Magnetic stirring was used to accelerate dissolution of the test item in mineral medium and to obtain a homogeneous stock solution. Volumes of stock solution corresponding with the test concentrations were then combined with inoculum and mineral medium.

Measurements of the oxygen concentrations were performed at the start of the experiment (day 0) and on day 7, 14, 21 and 28.

The relative biodegradation values calculated from the O2measurements performed during the test period revealed no biologically relevant biodegradation of CH04207 at the low and high concentration of CH04207 (mean value of 3% and 2%, based on ThOD, respectively). The toxicity control showed that CH04207 did not inhibit microbial activity.

Since all criteria for acceptability of the test were met, this study was considered to be valid.

In conclusion, CH04207 is designated as not readily biodegradable.