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Environmental fate & pathways

Biodegradation in water: screening tests

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biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 June-29 July 2011
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 B (Ready Biodegradability: CO2 Evolution Test)
see below
Principles of method if other than guideline:
According to the OECD 301B, the concentration of the test substance should correspond to 10-20 mg TOC/L. The applied test substance concentration of 1 mg/L corresponded to 0.29 mg TOC/L. This is acceptable as the analytical method with LSC is very sensitive.
GLP compliance:

Test material

Constituent 1
Reference substance name:
Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): NXL-104
- Chemical name: Sulfuric acid, mono[(1R,2S,5R)-2-(aminocarbonyl)-7-oxo-1,6-diazabicyclo[3.2.1]oct-6-yl] ester, sodium salt (1:1)
- Molecular formula (if other than submission substance): C7H10N3O6S1 (+ Na)
- Molecular weight (if other than submission substance): ca. 288.23
- Substance type: salt
- Physical state: slightly yellow crystalline powder
- Analytical purity: 99.7%
- Impurities (identity and concentrations): not indicated
- Composition of test material, percentage of components: not indicated
- Purity test date: not indicated
- Lot/batch No.: AFCH005151
- Expiration date of the lot/batch: not indicated
- Stability under test conditions: not indicated
- Storage condition of test material: The sample was stored in a fridge.

Test substance [14C] NXL104
- Physical state: green brown solid
- Lot CFQ40927
- Radiochemical purity (if radiolabelling): 99.6%
- Locations of the label (if radiolabelling): see attached picture
- Expiration date of radiochemical substance (if radiolabelling): not indicated

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: Activated sludge was obtained from Totnes Sewage Treatment Works, Devon, UK, on 22 June 2011. This works treats sewage of predominantly domestic origin. At the laboratory, the activated sludge was kept aerated at room temperature.
- Concentration of sludge: On the study start day the activated sludge solids concentration was determined. This sludge was then diluted in test medium to give a sludge solids concentration of 33 mg/L. The diluted sludge was then added to the test vessels to give a final sludge solids concentration of 30 mg/L.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
1 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
radiochem. meas.
([14C]CO2 evolution)
Details on study design:
- Composition of medium: according to OECD Guidelines
- Test temperature: 22 ± 2°C
- Suspended solids concentration: 30 mg/L
- Test vessels containing [14C]NXL 104 or [14C]benzoic acid were set up. Air drawn from the laboratory was bubbled through sodium hydroxide to remove any atmospheric carbon dioxide and then humidified before it entered the test vessel. Air leaving the vessel passed through two Orbo(TM) tubes to trap any volatile organic compounds evolved, followed by two traps containing 2 M sodium hydroxide solution to trap [14]CO2. Empty vessels prevented liquid being siphoned into the wrong vessel. Test vessels were incubated at 22 ± 2°C, under red light.

- Number of culture flasks/concentration: 4, containing [14C]NXL 104 mixed with non-labelled NXL 104, at a nominal concentration of 1.0 mg/L with test medium.
- Measuring equipment: LC-MS

- Test vessel analysis: all vessels were opened on days 0, 14 and 28 and subsamples removed for specific analysis, via liquid chromatography mass spectrometry (LC-MS)
- Carbon dioxide trap analysis: the gas traps containing 2 M sodium hydroxide were analysed on days 2, 5, 7, 9, 14, 19, 23 and 28 by LSC. The radioactivity measured in the sodium hydroxide was attributed to evolved [14]CO2.
- Volatile trap analysis: at the end of the test the volatile traps (Orbo(TM) tubes) were removed and back washed with 10 ml methanol into vials, which were then analysed by LSC
- Sludge solids combustion: The filter papers holding the sludge solids from each test vessel were combusted using a Packard 307 sample oxidiser. Any 14C in the sample was oxidised to 14CO2, which was trapped and mixed with scintillator. The vial containing the trapped 14CO2 was then analysed by LSC.

- Inoculum blank: one vessel containing test medium and water.
- Positive control: 2 vessels containing [14C]benzoic acid, mixed with non-labelled benzoic acid (reference substance) at a nominal concentration of 1.0 mg/L
and test medium.
- Abiotic control: two autoclaved test vessels were prepared containing [14C]NXL 104 at a nominal concentration of 1.0 mg/L without the addition of inoculum
- Toxicity control: two vessels, containing test medium, [14C]NXL 104 and [14C]benzoic acid, to assess for toxicity
Reference substance
Reference substance:
other: Benzoic acid, purity 100% and [14C] Benzoic acid, radiochemical purity of 99%

Results and discussion

% Degradation
% degradation (CO2 evolution)
Sampling time:
28 d
Remarks on result:
other: [14]CO2 evolved as % of applied radioactivity
Details on results:
Mass balance: Recoveries in all the test and reference vessels were 94-97% of the applied radioactivity.
Distribution of radioactivity: In the [14C]benzoic acid vessels mineralisation accounted for a mean of 78.1% of the applied radioactivity, while a mean of 12.5% was bound to the sludge solids. In the inoculated [14C]NXL 104 vessels, a mean 82.8% of the applied radioactivity remained in the filtrate, while a mean of 10.4% had mineralised, and less than 3.2% was bound to the sludge solids.
Toxicity controls: At day 14, the radioactivity measured in the sodium hydroxide traps from the toxicity control vessels were similar to the radioactivity measured in the traps associated with the two individual components, indicating inhibition had not occurred.
Difficulties: The specific analysis (LC-MS) indicated variable and inconsistent results from day 0, 14 and the day 28 re-analysed samples. The inconsistencies in the filtrate samples are thought to be due to storage and are therefore not deemed reliable. The Day 28 results were the only samples analysed immediately and indicated high concentrations of NXL 104 remaining in the filtrate, on average 0.92 mg/L. This roughly complies with the LSC results from Day 28, which showed an average of 82.8% radioactivity in the filtrate, indicating no primary degradation to have occurred; however caution should still be applied to the day 28 LC-MS as factors other than storage may have influenced the analysis.

As the conclusion of the study is based only on evolved [14C]carbon dioxide in % of applied radioactivity, this is not deemed to invalidate the study

BOD5 / COD results

Results with reference substance:
In the [14C]benzoic acid vessels greater than 60% degradation was achieved within the 10 day window, as expected for a biodegradable substance, thus confirming that the activated sludge contained viable organisms. By Day 28 approximately 78% of the applied radioactivity was measured as [14C]carbon dioxide.

Any other information on results incl. tables

Validity criteria:

-The difference of extremes of replicate values of the test chemical removal at the end of the test was < 20% (i.e. 1%).

- The % degradation of the reference compound has reached the pass level by day 14.

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
under test conditions no biodegradation observed
NXL 104 is not readily biodegradable under the conditions of OECD 301B tested with radiolabeled test material.