Registration Dossier

Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Qualifier:
according to
Guideline:
ISO DIS 9439 (Ultimate Aerobic Biodegradability - Method by Analysis of Released Carbon Dioxide)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Identification: V173520
Appearance: White powder (determined by WIL Research Europe)
Batch: AN
Purity/Composition: Not indicated
Test substance storage: At room temperature
Stable under storage conditions until: 20 April 2017 (expiry date)

Chemical name (IUPAC), synonym or trade name: 1,1,2,2-tetramethyl-1H-Benz(e)indolium 4- methylbenzenesulfonate
CAS Number: 141914-99-0
Molecular formula: C23H25NO3S
Molecular weight: 395.53
Solubility in water: >10% (NTU >>>)
Stability in water: Unknown

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic (adaptation not specified)
Details on inoculum:
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.
The freshly obtained sludge was kept under continuous aeration until further treatment. The concentration of suspended solids was determined to be 4.5 g/l in the concentrated sludge. Before use, the sludge was allowed to settle (44 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 concentration
Initial conc.:
12 other: mg TOC/l
Based on:
other: TOC
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Test procedure and conditions
Test duration: 28 days (last CO2 measurement on day 29).
During the test period, the test media were aerated and stirred continuously.

Test vessels: 2 litre glass brown coloured bottles.

Milli-RO water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon.

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-RO water and made up to 1 litre, pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H2O dissolved in Milli-RO water and made up to 1 litre.
C) 36.40 g CaCl2.2H2O dissolved in Milli-RO water and made up to 1 litre.
D) 0.25 g FeCl3.6H2O dissolved in Milli-RO water and made up to 1 litre.

Mineral medium: 1 litre mineral medium contains: 10 ml of solution (A),
1 ml of solutions (B) to (D) and Milli-RO water.

Barium hydroxide: 0.0125 M Ba(OH)2 (Boom, Meppel, The Netherlands and Merck, Darmstadt, Germany), stored in a sealed vessel to prevent absorption of CO2 from the air.

Synthetic air (CO2 < 1 ppm): A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. The synthetic air was sparged through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 ml/min).

Illumination: The test media were excluded from light.

Preparation of bottles
Pre-incubation medium: The day before the start of the test (day -1) mineral components, Milli-RO water (ca. 80% of final volume) and inoculum (1% of final volume) were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.

Type and number of bottles: 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).

Preparation: At the start of the test (day 0), test and reference substance were added to the bottles containing the microbial organisms and mineral components. The volumes of suspensions were made up to 2 litres with Milli-RO water, resulting in the mineral medium described before. Three CO2-absorbers (bottles filled with 100 ml 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle.
Reference substance
Reference substance:
acetic acid, sodium salt
Remarks:
Sodium acetate

Results and discussion

% Degradationopen allclose all
Parameter:
% degradation (CO2 evolution)
Value:
11
Sampling time:
29 d
Remarks on result:
other: Bottle A
Parameter:
% degradation (CO2 evolution)
Value:
19
Sampling time:
29 d
Remarks on result:
other: Bottle B
Details on results:
The relative biodegradation values calculated from the measurements performed during the test period revealed 11% and 19% biodegradation of V173520 (based on ThCO2), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Since all criteria for acceptability of the test were met, this study was considered to be valid.
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The relative biodegradation values calculated from the measurements performed during the test period revealed 11% and 19% biodegradation of V173520 (based on ThCO2), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.