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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:
17 December 2014 - 15 January 2015
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was performed according with OECD Guideline No. 301 B and in accordance with GLP principles.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
according to
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
according to
ISO DIS 9439 (Ultimate Aerobic Biodegradability - Method by Analysis of Released Carbon Dioxide)
Version / remarks:
according to
other: ISO International Standard 10634 "Water Quality - Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium" (1995)
GLP compliance:

Test material

Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): NS-1000
- Appearance: Colorless clear liquid
- Storage condition of test material: Refrigerated (2ºC to 8ºC) in the dark, purged with nitrogen.

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source: The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Treatment: The freshly obtained sludge was used immediately. The concentration of suspended solids was 4.1 g/L in the concentrated sludge (information obtained from the municipal sewage treatment plant and confirmed by determination of the amount of suspended solids). Before use, the sludge was allowed to settle (59 minutes) and the supernatant liquid was used as inoculum at the amount of 10 mL/L of mineral medium.
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
48 mg/L
Based on:
test mat.
Initial conc.:
12 mg/L
Based on:
other: Total Organic Carbon (TOC)
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium: 1 litre mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli-RO water
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 Milli-Q water and made up to 1 litre, pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H2O dissolved in Milli-Q water and made up to 1 litre.
C) 36.40 g CaCl2.2H2O dissolved in Milli-Q water and made up to 1 litre.
D) 0.25 g FeCl3.6H2O dissolved in Milli-Q water and made up to 1 litre.

- Test temperature: varied between 21.4 and 22.2 °C
- pH:
At t=0 d: 7.4-7.5
At t=28 d: 7.4 - 7.7
- pH adjusted:no
- Aeration of test medium: Overnight prior to the start of the test. During the test the medium was aerated and stirred continously.
- Continuous darkness: yes

- Culturing apparatus: 2 litre glass brown coloured bottles
- Number of culture flasks/concentration:
Test suspension: containing test substance and inoculum (2 bottles).
Inoculum blank: containing only inoculum (2 bottles)
Positive control: containing reference substance and inoculum (1 bottle).
Toxicity control: containing test substance, reference substance and inoculum (1 bottle).
- Method used to create aerobic conditions:
Synthetic air (a mixture of oxygen (ca. 20%) and nitrogen (ca. 80%), was passed through a bottle with barium hydroxide solution (0.0125 M) to trap CO2 (which may be present in small amounts and then sparged through the solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).
- Details of trap for CO2:
CO2 was trapped in barium hydroxide solution (0.0125 M) through precipitation of barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampoule).
Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers was moved one position into the direction of the test bottle. A new CO2-absorber was placed at the far end of the series.
Phenolphthalein (1% solution in ethanol, Merck) was used as pH-indicator.
On day 28, the pH of all test suspensions was measured and 1 mL of concentrated HCl (37%, Merck) was added to the bottles of the inoculum blank and test suspension. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 29.

Since NS-1000 was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L, weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components (test substance bottle A: 95.2 mg; test substance bottle B: 95.3 mg and toxicity control bottle: 95.4 mg). To this end, 10 mL of Milli-RO water was added to each weighing bottle containing the test substance. After vigorous mixing (vortex) the resulting emulsion was added quantitatively to the test medium. The test solutions were continuously stirred during the test, to ensure optimal contact between the test substance and the test organisms.

- Sampling frequency: Titrations of the test solution and inoculum blank were made on days 2, 5, 7, 9, 14, 23, 27 and 29. Titrations for the positive and toxicity control were made over a period of at least 14 days.
- Sampling method: Titration of the whole volume of CO2-absorber solution (see above)

- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes
Reference substance
Reference substance:
other: Sodium acetate

Results and discussion

% Degradation
% degradation (CO2 evolution)
Sampling time:
28 d
Remarks on result:
other: Average percentage biodegradation of two replicate bottles (13% and 6% biodegradion in bottle A and B respectively). HCl added on the 28th day (last CO2-measurement on the 29th day)
Details on results:
- Theoretical CO2 production:
The ThCO2 of NS-1000 was calculated to be 0.93 mg CO2/mg.
The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.

- Biodegradation:
The relative biodegradation values calculated from the measurements performed during the test period revealed no significant biodegradation of NS-1000 (10% after 28d).
In the toxicity control more than 25% biodegradation occurred within 14 days (34%, based on ThCO2). Therefore, the test substance was assumed not to inhibit microbial activity.
Functioning of the test system was checked by testing the reference substance sodium acetate, which showed a normal biodegradation curve.

BOD5 / COD results

Results with reference substance:
The positive control substance was biodegraded by at least 60% (76%) within 14 days.

Any other information on results incl. tables

Acceptability of the test

1.    The positive control substance was biodegraded by at least 60% (76%) within 14 days.

2.    The difference of duplicate values for %-degradation of the test substance was always less than 20% (≤ 7%).

3.    The total CO2 release in the blank at the end of the test did not exceed 40 mg/L (52 mg CO2 per 2 litres of medium, corresponding to 26 mg CO2/L).

4.    The Inorganic Carbon content (IC) of the test substance (emulsion) in the mineral medium at the beginning of the test was less than 5% of the Total Carbon content (TC). Since the test medium was prepared in tap-water purified by reverse osmosis (Milli-RO water (Millipore Corp., Bedford, Mass., USA, carbon levels < 500 ppb)), IC was less than 5% of TC (mainly coming from the test substance, 12 mg TOC/L).

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
under test conditions no biodegradation observed
No significant biodegradation of NS-1000 was observed. Therefore it is concluded that the substance was not readily biodegradable under the conditions of the Modified Sturm test.
Executive summary:

The ready biodegradation of NS-1000 in water under the conditions of the carbon dioxide (CO2) evolution test (modified Sturm test) was investigated according to OECD guideline 301B and GLP principles. A single test concentration of 48 mg/L (12 mg/L as TOC) was tested during 28 days.

No significant biodegradation of NS-1000 was observed (10% after 28d). Therefore it is concluded that the substance was not readily biodegradable under the conditions of the modified Sturm test.

In the toxicity control more than 25% biodegradation occurred within 14 days (34%, based on ThCO2), indicating that the test substance does not inhibit microbial activity at a nominal concentration of 48 mg/L.

The study is considered to be reliable without restrictions.