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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:
1998-11-10 to 1998-12-09
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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)
according to
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
according to
EPA OPPTS 835.3110 (Ready Biodegradability)
GLP compliance:
yes (incl. certificate)

Test material

Test material form:
Details on test material:
- Appearance: Off-white powder
- Storage condition of test material: At room temperature protected from light
Specific details on test material used for the study:
- Source and lot/batch No.of test material: Sponsor and 002

- Storage condition of test material: Room temperature in darkness
- Solubility and stability of the test substance in the solvent/vehicle:

- Treatment of test material prior to testing: The test material was prepared by direct dispersion in culture medium

FORM AS APPLIED IN THE TEST (if different from that of starting material)
14.6 mg test subtance/L, equivalent ot 10 mg carbon/L

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A mixed population of activated sewage sludge micro-organisms was obtained on 9 November 1998 from the aeration stage of the Severn Trent Water Pic sewage treatment plant at Belper, Derbyshire, UK, which treats predominantly domestic sewage.
- Storage conditions: Aerated continuously
- Storage length: Not specified
- Preparation of inoculum for exposure: Washed 3 times by settlement and resuspension in culture mediuim to remove any excessive amounts of dissolved organic carbon (DOC)
- Concentration of sludge: 30 mg suspended solids / L
- Initial cell/biomass concentration:
- Water filtered: Water was purified using reverse osmosis and arrived onsite deionized
Duration of test (contact time):
ca. 29 d
Initial test substance concentration
Initial conc.:
ca. 14.6 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium: The culture medium used in this study was that recommended in the OECD guidelines
- Test temperature: 21
- pH: 7.4
- pH adjusted: no
- Aeration of dilution water: Yes with oil free air
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes

- Culturing apparatus: 5 L glass culture vessels each containing 3 liters of test solution.
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Aeration by CO2 free air
- Measuring equipment: The samples were analysed for CO2 using an Ionics 1555B TOC analyser and a Dohrmann DC-190 TOC analyser.
- Test performed in closed vessels
- Details of trap for CO2 and volatile organics if used:

- Sampling frequency: Samples were taken from the first CO2 absorber vessel on days 0, 1,2, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, 28 and 29. The second absorber vessel was sampled on days 0 and 29
- Sampling method: 2 ml samples were removed from the test solution.
- Sample storage before analysis: If not analyzed immediately, they were stored deep frozen at -20 C prior to analysis.

- Inoculum blank: Yes
- Toxicity control: Yes
- Other: A reference compound control was used as well.

Evaluation of Data:
Calculation of carbon content:
The theoretical amount present as test material for BMS 233101-01 (C32H42N4O5) was calculated as follows:
(No of C atoms)*(mol wt of C)/(mol wt of test material)*100
Thus for a concentration of 10 mg C/l (a total of 43.8 mg) the total organiccarbon present was 30 mg C.
Thus for a 10 mg C/l test concentration (a total of 51.4 mg) the total organic carbon present for sodium benzoate was 30 mg C.

Percentage degradation:
The percentage degradation or percentage of Theoretical Amount of Carbon Dioxide (ThCO2) produced is calculated by substituting the inorganic carbon
values given in Table 1 in the following equation:
The values of Replicates R1 and R2 are meaned for the control, test and standard materials before substitution in the equation.
% ThCO2 (= % degredation) = ((mg IC in test flask) - (mg IC in control)) / (mg TOC as test material) * 100

The total CO2 evolution In the control vessels at the end of the test is calculated from the equation below. The inorganic carbon values for replicates R^ and R2
on day 28 are meaned before substitution into the equation.

Total CO2 evolution = (mg IC in control) * (100 / (%C of CO2)) * ( 1 / (test volume)) = (mg IC in control) * (100 / (27.29)) * ( 1 / (3))

Validation criteria
The results of the degradation test are considered valid if in the same test the standard material yields >=60% degradation by day 14.
The test material may be considered to be readily biodegradable if >=60% degradation is attained after 28 days. This level of degradation must be reached
within 10 days of biodegradation exceeding 10%.
The toxicity control (BMS 233101-01 and sodium benzoate) should attain >=25%degradation by day 14 for the test material to be considered as non-inhibitory.
The test is considered valid if the difference of the extremes of replicate values of production of CO2 at the end of the test is less than 20%.
The total CO2 evolution in the control vessels at the end of the test should not normally exceed 40 mg/l medium (== 120 mg/3l).
The IC content of the test material in the mineral medium at the beginning of the test must be <5% of the TC.

Results and discussion

Preliminary study:
No preliminary study was conducted.
Test performance:
The TC/IC ratio of the test material dispersions was in excess of the recommended level of 5% given in the Test Guidelines (see Table 3). This is considered to be
due to the low TC concentration in the test medium and hence the IC contribution is relatively large. This is not considered to affect the integrity of the study or the
results obtained given the low level of IC in the test media and that the C02 evolution in the control vessels did not exceed the upper limit of 70 mg/l given in the OECD Guidelines.

The results of the inorganic carbon analysis of samples from the first absorber vessels on day 29 showed an increase in all replicate vessels. These increases are considered to be due to CO2 present in solution being driven off by the addition of hydrochloric acid on day 28 and resulted in an increase in the percentage
degradation value for the test material from 16% on day 28 to 20% on day 29.

The increase in inorganic carbon in the first absorber vessels on day 29 resulted in an increase in the percentage degradation value for the standard material from 85% on day 28 to 89% on day 29.
% Degradation
Key result
% degradation (CO2 evolution)
ca. 16
Sampling time:
28 d
Details on results:
The results of the inorganic carbon analysis of samples from both absorber vessels on day 29 confirmed that no significant amounts of CO2 were present in solution in the culture vessels as inorganic carbonate.

The toxicity control attained 44% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study. The increase in inorganic carbon in the first absorber vessels on day 29 resulted in an increase in the percentage degradation value for the toxicity control
from 44% on day 28 to 45% on day 29.

BOD5 / COD results

Results with reference substance:
Sodium benzoate, the reference substance, attained 85% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
This substance was determined by this study to be not readily biodegradeable according to the specified guidelines.