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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
Study period:
from 2014-01-23 to 2014-03-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
[(3S,4R,4aR,6S,6aS,12R,12aS,12bS)-3-(cyclopropanecarbonyloxy)-6,12-dihydroxy-4,6a,12btrimethyl-11-oxo-9-(pyridin-3-yl)-1,2,3,4,4a,5,6,6a,12a,12b-decahydro-11H,12Hbenzo[f]pyrano[4,3-b]chromen-4-yl]methylcyclopropanecarboxylate
Cas Number:
915972-17-7
Molecular formula:
C33H39NO9
IUPAC Name:
[(3S,4R,4aR,6S,6aS,12R,12aS,12bS)-3-(cyclopropanecarbonyloxy)-6,12-dihydroxy-4,6a,12btrimethyl-11-oxo-9-(pyridin-3-yl)-1,2,3,4,4a,5,6,6a,12a,12b-decahydro-11H,12Hbenzo[f]pyrano[4,3-b]chromen-4-yl]methylcyclopropanecarboxylate
Test material form:
solid
Details on test material:
- Analytical purity: >94 %
Specific details on test material used for the study:
- Batch identification: 080722
- Expiry date: 2015.11.01

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum: Activated sludge from a municipal sewage plant (Municipal waste water treatment plant Mannheim, Germany)
- Preparation of inoculum for exposure: At the day of exposure the suspension was washed one time with drinking water. Therefore the aeration was stopped and the sludge was allowed to settle. After settling the supernatant was discarded and the remaining sludge suspension was filled up with drinking water. To reduce the content of inorganic carbon in the blank controls the activated sludge was aerated with carbon dioxide free air for about 48 hours at 22 ± 2 °C.
- Pretreatment: Not performed
- Concentration of sludge: 6.0 g/L dry weight. Aliquots of 7.5 mL were added to the test vessels to obtain an activated sludge concentration of 30 mg/L dry weight.
- Filtration: Yes
- Type and size of filter used: The inoculum was sieved by a finely woven mesh with a mesh size about 1 mm.
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
20 mg/L
Based on:
TOC
Initial conc.:
30 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: The used mineral medium complies with the test guideline OECD 301 B (1992)
- Additional substrate: No
- Solubilising agent: No
- Test temperature: 22 ± 2 °C
- pH: 7.3 - 7.5
- pH adjusted: Yes. The pH value was adjusted to 7.4.
- Aeration of dilution water: Yes.

TEST SYSTEM
- Culturing apparatus: 2 L incubation bottles filled up to a volume of 1.5 L.
- Number of culture flasks/concentration: 2
- Number of blank control flasks: 2
- Number inhibition control flasks: 1
- Number of reference substance flasks: 1
- Method used to create aerobic conditions: At begin of the exposure phase the test vessels were connected with an aeration unit and the bubble aeration with carbon dioxide free air was started after connecting the several test vessels with the absorption units. The aeration was continued for about 24 hours and the released carbon dioxide amounts in both traps of each test vessel were determined and added to the calculated amount of the previous day. The incubation bottles were stirred on magnetic stirrers; the aeration was performed with carbon dioxide free air at a flow of approximately 800 mL per hour.
- Details of trap for CO2 and volatile organics if used: For stripping of carbon dioxide, dissolved in the test medium, each test vessel was acidified by adding 2 mL of concentrated hydrochloric acid. The concentration of dissolved organic carbon in the blank controls and reference substance assays were determined. Since the test substance was insufficiently soluble in water, no DOC-measurements could be performed from the test assay of the inhibition control and from the test substance test assays.
- Test performed in closed vessels: The bottles were connected to two serial scrubbing bottles (total volume 250 mL) filled with 100 mL 0.05 sodium hydroxide solution for the adsorption of carbon dioxide from degradation processes. Usually twice a week the Total Inorganic Carbon (TIC) values of the adsorption solutions of the first trap were determined and used for the calculation of the produced carbon dioxide. After each sampling the second trap was moved forward and the new trap with fresh sodium hydroxide solution was placed into the second position. Each trap was analyzed separately.

SAMPLING
- Sampling frequency: The samples were analyzed daily.
- Sampling method: The samples for TIC analysis (absorption solution) were measured without further treatment. For determination of the decrease of dissolved organic carbon (DOC) samples were taken from the test vessels of the blank control and from the test vessel of the reference substance control
- Sample storage before analysis: Room temperature
- Centrifugation: Yes. The samples for the DOC-analysis were centrifuged for about 15 minutes at 4000 rpm.

ANALYSIS
- Measuring equipment: The TIC- and DOC-analyses were performed as repeat determination, using a TOCanalyzer equipped with an auto sampler (Shimadzu TOC-5000A and/or TOC-L CSH/CSN). The sys tem works with a combustion/non-disperse infrared gas analysis method. For calibration of the TOC-Analyzer, standard samples were measured before start of measurements to prove the conformity with the calibration curve. The samples for TICanalysis (absorption solution) were measured without further treatment.
- Centrifugation: The samples for the DOC-analysis were centrifuged for about 15 minutes at 4000 rpm. The samples were analyzed on the day of sampling. The TIC-value of the freshly prepared sodium hydroxide solution was determined and considered by the calculation of biogenic produced carbon dioxide amount. The incubation bottles were stirred on magnetic stirrers; the aeration was performed with carbon dioxide free air at a flow of approximately 800 mL per hour.

The DOC was calculated using the following formula: TC – IC = TOC*
TC = total carbon
IC = inorganic carbon
TOC = total organic carbon
*Since samples were centrifuged TOC = DOC

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Toxicity control: Yes
- Positive control: Yes
Reference substance
Reference substance:
aniline

Results and discussion

Preliminary study:
- The Total Organic Carbon (TOC) of the test substance was determined to be 669 mg/g.
Test performance:
- The measured DIC-concentrations in the blank controls at begin of the exposure was determined to be 0.7 mg/L (mean value).
- The amount of produced CO2 in the blank controls at the end of exposure was determined to be 34.9 mg/L (mean value).
- The deviation of the degree of biodegradation of the test substance in the plateau phase was < 20 %.
- The degree of biodegradation of the reference substance was > 60% CO2/ThCO2 after 14 days.
- The degree of biodegradation in the inhibition control was > 25 % CO2/ThCO2 after 14 days.
- The content of DIC in the blank control at start of exposure at the test concentration of 20 mg/L TOC was <1 mg/L.
- The amount of produced CO2 in the inoculum blank (“blank controls”) at the end of exposure (mean value) was < 70 mg/L
% Degradation
Key result
Parameter:
% degradation (CO2 evolution)
Value:
< 10
Sampling time:
28 d
Details on results:
The degree of biodegradation in the inhibition control after 14 days was determined to be 32 % CO2/ThCO2. The degree of biodegradation of the test substance after an exposure period of 28 days was determined to be <10 % CO2/ThCO2. Based on the rate of biodegradation at the end of exposure the test substance can be evaluated as not ready biodegradable in this test. The validity criteria were met.

BOD5 / COD results

Results with reference substance:
The degree of biodegradation of the reference substance after 14 days was determined to be 63 % CO2/ThCO2. The validity criteria was met.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable