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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Experimental result using guidelines.
Qualifier:
according to guideline
Guideline:
other: EEC, L 251 Vol. 27 (19.09.84) 84/449/EEC,C.5
Deviations:
yes
Remarks:
The volume of the test solution was reduced from 3.01 to 1.51.
Principles of method if other than guideline:
Biodegradation study was conducted according to EEC, L 251 Vol. 27 (19.09.84) 84/449/EEC,C.5 guideline for evaluating the percentage biodegradability of test substance .
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Bacteria collected from activated sludge of the sewage treatment plant of CH-4106 Therwil on 16/10/89 The preparation was carried out according to the method described in the guideline.
Duration of test (contact time):
28 d
Initial conc.:
10.3 mg/L
Based on:
test mat.
Initial conc.:
20.3 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Vessels : 2 Liter flasks (dark brown glass) equipped with gas inlet and magnetic stirrer.
- Water : The test medium was prepared according to the method described in the guideline
- Temperature : 22 ± 2 deg C
- Duration : 28 days
- Air : = 25 ml/min purified from carbon dioxide.
- Blank: Water as specified in the guideline containing 0.5 ml of the Nonylphenol 10EO5PO solution.

STATISTICAL METHODS: The biodegradation was calculated on the basis of the theoretical carbon content of the test substance and the cumulative quantities of carbon dioxide determined on the days of measurements. For the calculation the formula given in the guideline was used.
Reference substance:
aniline
Remarks:
MERCK No, : 1261; Batch : 12/09/89; Stability : 12/03/90; Storage : room temperature; concentrations : 20 mg/l
Key result
Parameter:
% degradation (CO2 evolution)
Value:
34
Sampling time:
28 d
Remarks on result:
other: 10.3 mg test substance/L
Key result
Parameter:
% degradation (CO2 evolution)
Value:
7
Sampling time:
28 d
Remarks on result:
other: 20.3 mg test substance/L
Details on results:
Test substance undergoes 34% and 7% after 28 days at a chemical conc. of 10.3 & 20.3 mg/l, respectively.

Values

Theoretical carbon dioxide evolution

Test substance: 10.3 mg/l = 39.87 mg CO2/I.5 liter,

Test substance: 20.3 mg/l = 78.21 mg COa/l.S liter.

This calculation is based on the chemical formula: C28H31CIN2O3

Validity criteria fulfilled:
not specified
Interpretation of results:
other: primary inherently biodegradable
Conclusions:
The test chemical undergoes 34% and 7% after 28 days at a chemical conc. of 10.3 & 20.3 mg/l, respectively in the test condition. Thus, the test item was considered to be primary inherently biodegradable.
Executive summary:

28-days biodegradation study was conducted following the EEC, L 251 Vol. 27 (19.09.84) 84/449/EEC,C.5 guideline to determine the ready biodegradability of the test chemical. The study was performed at a temperature of 22±2°C. The test system included control, test item and reference item. Bacteria was used as a test inoculum for the study which was collected from activated sludge of the sewage treatment plant of CH-4106 Therwil on 16/10/89 The preparation was carried out according to the method described in the guideline. Test chemical conc. used for the study was 10.3 and 20.3 mg/l, respectively while that of reference substance was 20 mg/l conc. Aniline was used as a reference substance for the study. Flasks (dark brown glass) of 2 lit equipped with gas inlet and magnetic stirrer was used as a test vessel for the study.The test medium was prepared according to the method described in the guideline. Blank test system was setup during the study containing only water as specified in the guideline containing 0.5 ml of the Nonylphenol 10EO5PO solution.1200 ml of the mineral solution with the inoculum were aerated for 24 hours in the test vessel. In 300 ml mineral solution 0.5 ml Nonylphenol 10EO5PO (solution of 30 mg in 100 ml bidist. water) and 15.5 rsp. 30.4 mg of test substance were added and homogenized. This solution was given to the test vessel which was immediately connected to the CO2 traps. Determination of the initial CO2 of the 0.05 N sodium hydroxide and the CO2, absorbed in the absorbers filled with 200 ml 0.05 N sodium hydroxide on the days 3, 7, 10, 13, 17, 20, 24, 27 and 28.The biodegradation was calculated on the basis of the theoretical carbon content of the test substance and the cumulative quantities of carbon dioxide determined on the days of measurements. Accordingly, the % degradation of the test item after 28 days of incubation at 22 ± 2°C according to test was determined to be 34% and 7% after 28 days at a chemical conc. of 10.3 & 20.3 mg/l, respectively. Based on the results, the test item, under the test conditions, was considered to be primary inherently biodegradable in nature.

 

Description of key information

28-days biodegradation study was conducted following the EEC, L 251 Vol. 27 (19.09.84) 84/449/EEC,C.5 guideline to determine the ready biodegradability of the test chemical. The study was performed at a temperature of 22±2°C. The test system included control, test item and reference item. Bacteria was used as a test inoculum for the study which was collected from activated sludge of the sewage treatment plant of CH-4106 Therwil on 16/10/89 The preparation was carried out according to the method described in the guideline. Test chemical conc. used for the study was 10.3 and 20.3 mg/l, respectively while that of reference substance was 20 mg/l conc. Aniline was used as a reference substance for the study. Flasks (dark brown glass) of 2 lit equipped with gas inlet and magnetic stirrer was used as a test vessel for the study.The test medium was prepared according to the method described in the guideline. Blank test system was setup during the study containing only water as specified in the guideline containing 0.5 ml of the Nonylphenol 10EO5PO solution.1200 ml of the mineral solution with the inoculum were aerated for 24 hours in the test vessel. In 300 ml mineral solution 0.5 ml Nonylphenol 10EO5PO (solution of 30 mg in 100 ml bidist. water) and 15.5 rsp. 30.4 mg of test substance were added and homogenized. This solution was given to the test vessel which was immediately connected to the CO2 traps. Determination of the initial CO2 of the 0.05 N sodium hydroxide and the CO2, absorbed in the absorbers filled with 200 ml 0.05 N sodium hydroxide on the days 3, 7, 10, 13, 17, 20, 24, 27 and 28.The biodegradation was calculated on the basis of the theoretical carbon content of the test substance and the cumulative quantities of carbon dioxide determined on the days of measurements. Accordingly, the % degradation of the test item after 28 days of incubation at 22 ± 2°C according to test was determined to be 34% and 7% after 28 days at a chemical conc. of 10.3 & 20.3 mg/l, respectively. Based on the results, the test item, under the test conditions, was considered to be primary inherently biodegradable in nature.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable

Additional information

Experimental studies for the target chemical were reviewed for the biodegradation end point and their results are summarized as below:

In an experimental key study from study report (1989), 28-days biodegradation study was conducted following the EEC, L 251 Vol. 27 (19.09.84) 84/449/EEC,C.5 guideline to determine the ready biodegradability of the test chemical. The study was performed at a temperature of 22±2°C. The test system included control, test item and reference item. Bacteria was used as a test inoculum for the study which was collected from activated sludge of the sewage treatment plant of CH-4106.The preparation was carried out according to the method described in the guideline. Test chemical conc. used for the study was 10.3 and 20.3 mg/l, respectively while that of reference substance was 20 mg/l conc. Aniline was used as a reference substance for the study. Flasks (dark brown glass) of 2 lit equipped with gas inlet and magnetic stirrer was used as a test vessel for the study. The test medium was prepared according to the method described in the guideline. Blank test system was setup during the study containing only water as specified in the guideline containing 0.5 ml of the Nonylphenol 10EO5PO solution.1200 ml of the mineral solution with the inoculum were aerated for 24 hours in the test vessel. In 300 ml mineral solution 0.5 ml Nonylphenol 10EO5PO (solution of 30 mg in 100 ml bidist. water) and 15.5 rsp and 30.4 mg of test substance were added and homogenized. This solution was given to the test vessel which was immediately connected to the CO2 traps. Determination of the initial CO2 of the 0.05 N sodium hydroxide and the CO2, absorbed in the absorbers filled with 200 ml 0.05 N sodium hydroxide on the days 3, 7, 10, 13, 17, 20, 24, 27 and 28.The biodegradation was calculated on the basis of the theoretical carbon content of the test substance and the cumulative quantities of carbon dioxide determined on the days of measurements. Accordingly, the % degradation of the test item after 28 days of incubation at 22 ± 2°C according to test was determined to be 34% and 7% after 28 days at a chemical conc. of 10.3 & 20.3 mg/l, respectively. Based on the results, the test item, under the test conditions, was considered to be primary inherently biodegradable in nature.

Another biodegradation study was conducted following the EEC L251, Vol. 27 (19.09.84), 84/449/EEC, C.9 guideline to determine the ready biodegradability of the test chemical. The study was performed at a temperature of 148 ± 3°C. For the preparation of the stock solution of test substance (TK 11484 ), 9.1 mg were dissolved and filled up to 100 ml with silver sulfate in sulfuric acid. The preparation of the stock solution of the reagents was carried out according the method described in the guideline. Potassium hydrogen phthalate was used as a reference substance for the study. Round bottom flasks of 250 ml equipped with a condenser was used as a test vessel for the study. Test system include the following test solutions i.e, Potassium dichromate solution, Mercury sulfate solution ,Silver sulfate solution and Sulfuric acid d=1.84 ,respectively. Bidistilled Water was used as a blank. Titration of the unreacted potassium dichromate with ammonium iron-II-sulfate was done on a METTLER Memotitrator. Titration solution used for the study was ammonium iron-II-sulfate 0.12 mol/l. The chemical oxygen demand was calculated on the basis of the amount of test substance and consumption of potassium dichromate. COD of the reference substance was determined to be 193.7 mg COD/l (limit: 192 – 208 mg COD/l) (n = 3, Srel.= 0.00%) and COD of the test chemical salts was determined to be 1.26 g O2/g test mat. after 2 hrs in the test condition. Based on the results, the test item, under the test conditions, was considered to be biodegradable in nature.

In a supporting weight of evidence study, biodegradation experiment was conducted for 35-days following the OECD guideline 301 D for determining the ready biodegradability of the test chemical. The study was performed at a temperature of 20°C under aerobic conditions. Aerobic conditions was provided by means of mineral media which is aerated for 20 hours prior to start of the experiment. The test system included control, test chemical and reference substance. Polyseed capsule (mixed culture) was used as a test inoculum for the study. Test inoculum polyseed capsule was composed of blend of specialized microbial cultures and food grade gelatin made by International Laboratory Supply (InterLab), LTD. No pretreatment / preconditioning was given to the test inoculum as the polyseed capsule requires only one hour of stirring to activate it. 1 Polyseed capsule was added in 500 mL DI water and then stirred for 1 hour for proper mixing and functioning of inoculum & settled for 15 minutes to eliminate the bran. Decanted polyseed solution was used as mixed inoculum. Thus, concentration of test inoculum used for the study was 32 ml/l which corresponds to 10E7 to 10E8 CFU/ml. The concentration of test and reference substance (Sodium Benzoate) chosen for both the study was 4 mg/L. OECD mineral medium was used for the study. ThOD (Theoretical oxygen demand) of test and reference chemical was determined by calculation. % degradation was calculated using the values of BOD and ThOD for test chemical and reference substance. The % degradation of procedure control (reference substance) was also calculated using BOD & ThOD and was determined to be 70.48%. Degradation of Sodium Benzoate exceeds 66.26% on 7 days & 72.28 % on 14th day. The activity of the inoculum is thus verified and the test can be considered as valid.The BOD42 value of test chemical was observed to be 1.15 mgO2/mg. ThOD was calculated as 4.54 mgO2/mg. Accordingly, the % degradation of the test chemical after 42 days of incubation at 20 ± 1°C according to Closed Bottle test was determined to be 25.33%. Based on the results, the test chemical under the test conditions, was considered to be inherently biodegradable in nature.

On the basis of above results for test chemical, it can be concluded that the test chemical was considered to be inherently biodegradable in nature.