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
Study period:
2009-03-09 to 2009-06-26
Reliability:
1 (reliable without restriction)
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
1998
Deviations:
no
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Aldimine 2
- Molecular formula (if other than submission substance): C21H41NO4
- Molecular weight (if other than submission substance): 371.55 g/mol
- Physical state: liquid, faint yellow
- Analytical purity: 96.7 %
- Lot/batch No.: UB2.398B /11
- Expiration date of the lot/batch: 04 August 2011
- Stability under test conditions: stable for at least 3 years
- Storage condition of test material: room temperature, closed bottle
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
NA

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Test System
Species: Activated sludge, microorganisms from a domestic waste water treatment plant.
Origin: The activated sludge was supplied from the sewage plant for domestic sewage in Veszprém, Hungary.
Conditioning: The activated sludge used for this study was washed by centrifugation (10 min) and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution and again centrifuged (10 min). This procedure was repeated three times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 4 g dry material per litre were mixed with test water and then aerated until use. Before use the sludge was filtered through cotton wool.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
2.3 mg/L
Based on:
other: ThODNO3
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Test Units
Type and Size: BOD bottles of 300 mL volume with special neck and glass stoppers.
Identification: Each of the test flasks were uniquely identified with study code, test group, days of measurement and replicate number.
Test Conditions
Surrounding Type: Incubator and controlled environment room (during the formulation and oxygen measuring)
Temperature: 21.0 – 23.2°C
Light Conditions: Darkness
pH-Value of Reconstituted Water: 7.32 (measured without adjustment at the start of the test)
Oxygen conc. of Reconstituted Water: Recording: 9.0 mg O2/L (measured at the start of the test) Test conditions were measured with suitable instruments and documented in the raw data.
Reconstituted Water:
In deionised water analytical grade salts were added to prepare the following stock solutions:
a) 2.125 g KH2PO4, 5.4375 g K2HPO4, 16.79 g Na2HPO4 x 12H2O, 0.125 g NH4Cl filled up with deionised water to 250 mL volume
b) 5.625 g MgSO4 x 7H2O filled up with deionised water to 250 mL volume
c) 9.10 g CaCl2 x 2H2O filled up with deionised water to 250 mL volume
d) 0.25 g FeCl3 x 6H2O filled up with deionised water to 1000 mL volume (stock solution was applied on the day of preparation)
Ratio of ingredients: 1 mL of the stock solutions a) - d) were combined and filled to a final volume of 1000 mL with deionised water. The test medium was aerated for 20 minutes and allowed to stand for about 20 hours at the test temperature. The dissolved oxygen concentration was about 9.0 mg/L at about 20 °C.
Equipments:
Large glass tanks with valve,
Large glass bottles with valve,
Narrow necked,
300 mL BOD bottles with glass stoppers,
Funnels and coarse filter papers,
Moisture Content Analyzer,
Centrifuge, Balance,
Self stirring O2 electrode,
Oxygen and pH meter,
Aeration system,
Incubator with thermometer
Study Design
Preparation of the Test Solutions
The components were applied in the following ratio in the test flasks:
The preparation of the respective test solutions with Sika Hardener LI was performed according to the followings: The respective amount of Sika Hardener LI was weighed in directly to reach the required test item concentration of 2.0 mg/L. During the performance of the test the test solutions were ultrasonicated under continuous cooling for < 10 min. to ensure a good dispersion. The chosen test item concentration was based on the theoretical oxygen demand (ThODNO3): 2.96 mg O2/mg test item and on the performed 14-d preliminary test.
Test Item (flasks 1a and 1b): Based on the theoretical oxygen demand (ThODNO3) of 2.96 mg O2/mg test item, 6.9 mg of Sika Hardener LI was thoroughly mixed into 3.46 litres of aqueous test medium (corresponding to 2.0 mg/L test item, respectively a ThODNO3 of about 5.92 mg O2/L).
Procedure Control: Sodium benzoate (flasks 2a and 2b): Based on the theoretical oxygen demand (ThODNH4) of Sodium benzoate (1.67 mg O2 per mg) (details on calculation are given in the guidelines), stock solution* corresponding to 12.096 mg of Sodium benzoate was mixed into 3.36 litres of aqueous test medium (corresponding to 3.6 mg/L reference item, respectively a ThODNH4 of about 6.012 mg O2/L). * the concentration of the stock solution was 360 mg/L
Inoculum Control (flasks 3a and 3b): Only filtered inoculum was added to 3.40 litres of aqueous test medium.
Toxicity Control (flasks 4a and 4b): 6.9 mg of Sika Hardener LI and reference item stock solution* (34.6 mL) were mixed into 3.46 litres of aqueous test medium corresponding to 2 mg/L test item (ThODNO3 of 5.92 mg O2/L) and 3.6 mg/L reference item (ThODNH4 of 6.012 mg O2/L). * the concentration of the stock solution was 360 mg/L
General: Microbial inoculum (2.0 mL per litre) was added to each preparation bottle.
Course of the Test
Preparation of Test Flasks
Sufficient number of BOD flasks was cleaned with 5-10 mL of a wash liquid (2.5 g iodine and 12.5 g potassium iodide per litre of 1 % w/v sulphuric acid) by shaking well to coat the bottle walls. After allowing to stand for 15 minutes, the wash liquid was poured off, and the bottles were thoroughly rinsed with tap water and deionised water. Then, the previously described test solutions were filled into the bottles bubble-free until the bottles were completely filled. Then they were tightly stoppered.
The following bottles were prepared:
10 bottles containing the test item and inoculum
10 bottles containing the reference item and inoculum (procedure control)
10 bottles containing only inoculum (inoculum control)
10 bottles containing the test item, reference item and inoculum (toxicity control)
Test Parameters
Measurement of Oxygen: The oxygen concentrations were measured with oxygen meter with a stirring O2 electrode. Oxygen measurements were performed in duplicate on days 0, 7, 14, 21 and 28.
Measurement of Temperature: Temperature was measured continuously and registered on weekdays.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Preliminary study:
No preliminary study
Test performance:
The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum.
% Degradation
Parameter:
% degradation (O2 consumption)
Value:
61.2
Sampling time:
28 d
Details on results:
Under the test conditions the percentage biodegradation of Sika Hardener LG was already 29.0 % on day 7, it had been increasing and reached a mean of 61.2 % after 28 days based on theoretical oxygen demand (ThODN03) of the test item.

BOD5 / COD results

Results with reference substance:
The reference item Sodium benzoate was sufficiently degraded to a mean of 76.7 % after 14 days, and to a mean of 91.7 % after 28 days of incubation, based on ThODNH4, thus confirming the suitability of the used activated sludge inoculum.

Any other information on results incl. tables

Biodegradation of reference item:

The reference item Sodium benzoate was sufficiently degraded to a mean of 76.7 % after 14 days, and to a mean of 91.7 % after 28 days of incubation, based on ThODNH4, thus confirming the suitability of the used activated sludge inoculum.

Biodegradation of toxicity control:

In the toxicity control containing both, the test item and the reference item, a mean of 49.7 % biodegradation was noted within 14 days and 55.7 % biodegradation after 28 days of incubation.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable, but failing 10-day window
Conclusions:
The test item can be considered to be not ready biodegradable. According to the test guidelines the pass level for ready biodegradability, 60 % of ThODN03 has to be reached in a 10-day window within the 28-d period of the test.
Executive summary:

Sika Hardener LG was assessed in a closed bottle test over a period of 28 days, according the OECD guideline 301 D and the EU-guideline. Under the test conditions the percentage biodegradation of Sika Hardener LG reached a mean of 61.2 % after 28 days based on the ThODNO3. The pass level, 60 % of ThODNO3 was reached in a 10-day window, within the 28-d period of the test. Therefore the test item was considered ready biodegradable, but failing the 10-day window.