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Registration Dossier
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EC number: 213-561-3 | CAS number: 980-26-7
Biodegradation in water: screening tests
Administrative data
Link to relevant study record(s)
- Endpoint:
- biodegradation in water: ready biodegradability
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 7 June 2006 to 6 December 2006
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- GLP according to German "Chemikaliengesetz", EC and OECD Guideline principles
- Oxygen conditions:
- aerobic
- Inoculum or test system:
- activated sludge, domestic, non-adapted
- Details on inoculum:
- - Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge from the sewage plant in Hildesheim, Germany, comprising mostly municipal sewage (industrial chemical waste: 30-33%, referring to BOD)
- Pretreatment: The activated sludge was washed twice with autoclaved tap water. After the second washing the settled sludge was filled up with mineral salts medium and was maintained in an aerobic condition by aeration with CO2-free air for 4 days. 10 mL/L were used to initiate inoculation.
- Concentration of sludge: 10 ml/L
- Initial cell/biomass concentration: Colony forming units in the test vessels: 10E07-10E08 CFU/L - Duration of test (contact time):
- 28 d
- Initial conc.:
- 40 mg/L
- Based on:
- ThOD/L
- Parameter followed for biodegradation estimation:
- O2 consumption
- Details on study design:
- TEST CONDITIONS
- Composition of medium: Mineral salts medium according to OECD 301 F
- Test temperature: 19.5 - 22.0°C
- pH:
Start End
Inoculum Control 7.59 1) 7.56
2) 7.54
Functional Control 7.59 7.73
Test Item 1) 7.56 1) 7.51
2) 7.53 2) 7.53
Toxicity Control 7.50 7.72
- Continuous darkness: yes
TEST SYSTEM
- Culturing apparatus: brown glass bottles (volume 500 mL)
- Number of culture flasks/concentration: two replicates
- Measuring equipment: The oxygen consumption was determined continuously from the pressure in the incubation vessels by OxiTop(R) measuring system at 360 measuring points (every 112 minutes) during the 28 d incubation period.
- Test performed in closed vessels: closed with OxiTop(R) device (see above)
SAMPLING
The temperature in the incubator was documented continuously by a thermohygrograph.
At the beginning of the test the pH-values in the brown glass bottles (test item, toxicity control) and in the measuring flasks (inoculum control before dividing, functional control) were determined. At the end of the test the pH-values of the test solutions in the brown glass bottles were determined.
CONTROL AND BLANK SYSTEM
- Inoculum blank: mineral salts medium
- Toxicity control: test item and reference substance in test concentrations
- Functional control: benzoic acid, sodium salt - Reference substance:
- benzoic acid, sodium salt
- Parameter:
- % degradation (O2 consumption)
- Value:
- 0
- Sampling time:
- 28 d
- Remarks on result:
- other: 0% biodegradation after 7, 14, 21 and 28 d
- Details on results:
- Test item was tested in two replicates (see Table 1 below for detailed results)
pH at the end of all tests (test item, controls) was in the range of 6.0-8.5 (validity criterion), for detailed values, see above
Toxicity control showed 39% biodegradation after 14 d and 29% biodegradation after 28 d
Inoculum control showed oxygen depletion of 33.8 mg/l after 28 d - Results with reference substance:
- Functional control (reference substance: 45 mg/l sodium benzoate) showed 94% biodegradation after 14 d and 92% biodegradation after 28 d
- Validity criteria fulfilled:
- yes
- Remarks:
- see above for details
- Interpretation of results:
- under test conditions no biodegradation observed
- Conclusions:
- Not readily biodegradable in the 10-d window and after 28 days
- Executive summary:
The ready biodegradability in the Manometric Respirometry Test was determined with a non adapted activated sludge for the test item over a period of 28 days. The study was conducted from 2006-06-20 to 2006-07-18 according to OECD guideline 301 F at DR.U.NOACK-LABORATORIEN, D-31157 Sarstedt. The test item concentration selected as appropriate was 40 mg/L, corresponding to a ThOD of 87.2 mgO2/L in the test vessel. The oxygen was depleted by the respiration of bacteria and the degradation was followed by measuring the oxygen concentration. The biodegradation is therefore expressed as the percentage BOD and was calculated for each study day.
The mean oxygen depletion in the inoculum control came to 33.8 mgO2/L after 28 days. In order to check the activity of the test system sodium benzoate was used as functional control. The pass level > 60 % was reached after 3 days. The biodegradation rate came to 92 % after 28 days.
In the toxicity control containing both test and reference item 39 % degradation occurred within 14 days. The degradation of the reference item was not inhibited by the test item.
The biodegradation of the test item is shown in Table 1 and graphically in Figure 1 in comparison to the readily degradable functional control. The 10 % level (beginning of biodegradation) was not reached until 28 days. The mean biodegradation remained at 0 %.
The validity criteria of the guideline are fulfilled.
The test item must be regarded as not readily biodegradable in the 10-d-window and after 28 days.
- Endpoint:
- biodegradation in water: ready biodegradability
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2004-10-21 - 2004-12-21
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: The test was performed according to relevant guidelines and compliant to GLP. The results are plausible and well documented.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Swiss Ordinance relating to Good Laboratory Practice, adopted February 2'1, 2000 [RS 813.016.5]. based on OECD-GLP 1997 (C(97) 186/Final).
- Specific details on test material used for the study:
- Details on properties of test surrogate or analogue material (migrated information):
not applicable - Oxygen conditions:
- aerobic
- Inoculum or test system:
- activated sludge, domestic, non-adapted
- Details on inoculum:
- The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf, Switzerland) treating predominantly domestic wastewater. The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.
Based on this ratio, calculated amounts of wet sludge were suspended in test water to obtain a concentration equivalent to 4 g (±10%) dry material per liter. During holding, the sludge was aerated at room temperature until use. Prior to use, the sludge was diluted with test water to a concentration of 1 g per liter (dry weight basis). Defined volumes of this diluted activated sludge were added to test water to obtain a final concentration of 30 mg dry material per liter. - Duration of test (contact time):
- 29 d
- Initial conc.:
- ca. 20 mg/L
- Based on:
- test mat.
- Initial conc.:
- ca. 15.7 mg/L
- Based on:
- other: TOC (total organic carbon)
- Parameter followed for biodegradation estimation:
- CO2 evolution
- Details on study design:
- Preparation of Test Flasks
One day before test start (Day -1), between 2400 and 3000 mL of untreated test medium was filled into 5-liter flasks (amber glass). To each flask (except for the abiotic control and the abiotic control blank), 90 mL of activated sludge inoculum was added.
For the abiotic control and the abiotic control blank, the untreated test medium was poisoned with mercury dichloride at a concentration of 10 mg/L (3 mL of a stock solution containing 10 g HgCl2/L filled up to 3 liters with test medium).
The test media were aerated ovemight with CO2-free air to purge the system of carbon dioxide.
On the following day (Day 0), defined amounts of the test item were directly weighed into the test flasks. No emulsifiers or solvents were used.
The reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. A stock solution containing 770 mg sodium benzoate per 100 mL test water (purged with CO2-free air) was prepared. From this, 10 mL aliquots were added to the corresponding test flasks.
The test flasks were made up to a volume of three liters with test water (purged with CO2-free air). Two absorber flasks, the first one containing 300 mL of 0.05 M NaOH, and the second one containing 200 mL of 0.05 M NaOH, were connected in series to the exit air line of each test flask. - Reference substance:
- benzoic acid, sodium salt
- Preliminary study:
- not applicable
- Test performance:
- No special observations
- Parameter:
- % degradation (inorg. C analysis)
- Value:
- 3.2
- Sampling time:
- 28 d
- Remarks on result:
- other: mean of 3.8 and 2.7
- Details on results:
- The percent biodegradation of the test item was calculated based on a total carbon content (TOC) of 0.78 mg C/mg test item.
The CO2 production of the test item in the test media was in the range of the inoculum controls.
Consequently, the test item was found not biodegradable under the test conditions within 28 days.
Abiotic control:
No degradation of the test item occurred in the abiotic control under the test conditions. - Results with reference substance:
- The percent biodegradation of the reference item was calculated based on a total carbon content (TOC) of 0.58 mg C/mg sodium benzoate.
In the procedure controls, the reference item degraded by an average of 79% by exposure Day 14, thus confirming the suitability of the activated sludge (>60% degradation by Day 14). By the end of the test (Day 28), the reference item was completely degraded.
BIODEGRADATION IN THE TOXICITY CONTROL
The percent biodegradation in the toxicity control, containing both the test item and the reference item, was calculated based on the sum of the total carbon content (TOC) of the test item and the reference item.
Biodegradation in the toxicity control showed a similar course over the 28-day exposure period, when compared to the two procedural controls, containing the reference item only. Within 14 days of exposure, biodegradation amounted to 36%.
Thus, according to the test guidelines, the test item had no inhibitory effect on activated sludge microorganisms because biodegradation in the toxicity control was >25% within 14 days of incubation. - Validity criteria fulfilled:
- yes
- Interpretation of results:
- under test conditions no biodegradation observed
- Conclusions:
- In a ready biodegradability test based upon CO2-evolution (reliability category 1, compliant to GLP) no biodegradation could be observed at the end of the test period (28 days). The reference substance (sodium benzoate) was thoroughly degraded at day 28 and to 79% at day 14 proving the suitability of the activated sludge used. On the other hand, lack of biodegradation was not caused by toxicity of the test item as the toxicity control with both, the test item and the reference substance, showed no inhibition compared to the procedural controls with the reference substance only.
- Executive summary:
The test item was investigated for its ready biodegradability in a 28-Day CO2 Evolution (Modified Sturm) Test according to EU Commission Directive 92/69/EEC C.4-C (1992) and OECD Guideline for Testing of Chemicals, No. 301 B (1992). The test item was found to be not biodegradable under the test conditions within 28 days. In the abiotic control, containing the test item and poisoned test medium, no degradation was noted at the end of the 28-day exposure period. In the toxicity control, containing both the test item and the reference item sodium benzoate, no inhibitory effect on activated sludge microorganisms was observed. In the procedure controls, the reference item degraded by an average of 79% by exposure day 14, thus confirming suitability of the activated sludge (>60% degradation by Day 14). By the end of the test (Day 28), the reference item was completely degraded.
- Endpoint:
- biodegradation in water: ready biodegradability
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 2004-10-21 - 2004-12-21
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: The test was performed according to relevant guidelines and compliant to GLP. The results are plausible and well documented.
- Justification for type of information:
- see Rationale and Justification for the Analogue Read-Across Approach used for the Registration of Pigment Red 122 (Chapter 13)
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Swiss Ordinance relating to Good Laboratory Practice, adopted February 2'1, 2000 [RS 813.016.5]. based on OECD-GLP 1997 (C(97) 186/Final).
- Specific details on test material used for the study:
- Details on properties of test surrogate or analogue material (migrated information):
not applicable - Oxygen conditions:
- aerobic
- Inoculum or test system:
- activated sludge, domestic, non-adapted
- Details on inoculum:
- The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf, Switzerland) treating predominantly domestic wastewater. The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.
Based on this ratio, calculated amounts of wet sludge were suspended in test water to obtain a concentration equivalent to 4 g (±10%) dry material per liter. During holding, the sludge was aerated at room temperature until use. Prior to use, the sludge was diluted with test water to a concentration of 1 g per liter (dry weight basis). Defined volumes of this diluted activated sludge were added to test water to obtain a final concentration of 30 mg dry material per liter. - Duration of test (contact time):
- 29 d
- Initial conc.:
- ca. 20 mg/L
- Based on:
- test mat.
- Initial conc.:
- ca. 15.7 mg/L
- Based on:
- other: TOC (total organic carbon)
- Parameter followed for biodegradation estimation:
- CO2 evolution
- Details on study design:
- Preparation of Test Flasks
One day before test start (Day -1), between 2400 and 3000 mL of untreated test medium was filled into 5-liter flasks (amber glass). To each flask (except for the abiotic control and the abiotic control blank), 90 mL of activated sludge inoculum was added.
For the abiotic control and the abiotic control blank, the untreated test medium was poisoned with mercury dichloride at a concentration of 10 mg/L (3 mL of a stock solution containing 10 g HgCl2/L filled up to 3 liters with test medium).
The test media were aerated ovemight with CO2-free air to purge the system of carbon dioxide.
On the following day (Day 0), defined amounts of the test item were directly weighed into the test flasks. No emulsifiers or solvents were used.
The reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. A stock solution containing 770 mg sodium benzoate per 100 mL test water (purged with CO2-free air) was prepared. From this, 10 mL aliquots were added to the corresponding test flasks.
The test flasks were made up to a volume of three liters with test water (purged with CO2-free air). Two absorber flasks, the first one containing 300 mL of 0.05 M NaOH, and the second one containing 200 mL of 0.05 M NaOH, were connected in series to the exit air line of each test flask. - Reference substance:
- benzoic acid, sodium salt
- Preliminary study:
- not applicable
- Test performance:
- No special observations
- Parameter:
- % degradation (inorg. C analysis)
- Value:
- 3.2
- Sampling time:
- 28 d
- Remarks on result:
- other: mean of 3.8 and 2.7
- Details on results:
- The percent biodegradation of the test item was calculated based on a total carbon content (TOC) of 0.78 mg C/mg test item.
The CO2 production of the test item in the test media was in the range of the inoculum controls.
Consequently, the test item was found not biodegradable under the test conditions within 28 days.
Abiotic control:
No degradation of the test item occurred in the abiotic control under the test conditions. - Results with reference substance:
- The percent biodegradation of the reference item was calculated based on a total carbon content (TOC) of 0.58 mg C/mg sodium benzoate.
In the procedure controls, the reference item degraded by an average of 79% by exposure Day 14, thus confirming the suitability of the activated sludge (>60% degradation by Day 14). By the end of the test (Day 28), the reference item was completely degraded.
BIODEGRADATION IN THE TOXICITY CONTROL
The percent biodegradation in the toxicity control, containing both the test item and the reference item, was calculated based on the sum of the total carbon content (TOC) of the test item and the reference item.
Biodegradation in the toxicity control showed a similar course over the 28-day exposure period, when compared to the two procedural controls, containing the reference item only. Within 14 days of exposure, biodegradation amounted to 36%.
Thus, according to the test guidelines, the test item had no inhibitory effect on activated sludge microorganisms because biodegradation in the toxicity control was >25% within 14 days of incubation. - Validity criteria fulfilled:
- yes
- Interpretation of results:
- under test conditions no biodegradation observed
- Conclusions:
- In a ready biodegradability test based upon CO2-evolution (reliability category 1, compliant to GLP) no biodegradation could be observed at the end of the test period (28 days). The reference substance (sodium benzoate) was thoroughly degraded at day 28 and to 79% at day 14 proving the suitability of the activated sludge used. On the other hand, lack of biodegradation was not caused by toxicity of the test item as the toxicity control with both, the test item and the reference substance, showed no inhibition compared to the procedural controls with the reference substance only.
- Executive summary:
The test item was investigated for its ready biodegradability in a 28-Day CO2 Evolution (Modified Sturm) Test according to EU Commission Directive 92/69/EEC C.4-C (1992) and OECD Guideline for Testing of Chemicals, No. 301 B (1992). The test item was found to be not biodegradable under the test conditions within 28 days. In the abiotic control, containing the test item and poisoned test medium, no degradation was noted at the end of the 28-day exposure period. In the toxicity control, containing both the test item and the reference item sodium benzoate, no inhibitory effect on activated sludge microorganisms was observed. In the procedure controls, the reference item degraded by an average of 79% by exposure day 14, thus confirming suitability of the activated sludge (>60% degradation by Day 14). By the end of the test (Day 28), the reference item was completely degraded.
Referenceopen allclose all
Table 1: Mean Biodegradation of the Test Item in Comparison to the Functional Control and Toxicity Control
|
Biodegradation [%] |
||||
|
[d] |
||||
|
Replicate |
7 |
14 |
21 |
28 |
Test Item 40 mg/L
|
1 |
0 |
0 |
0 |
0 |
2 |
0 |
0 |
0 |
0 |
|
Functional Control 45 mg/L |
1 |
82 |
94 |
90 |
92 |
Toxicity Control 40 + 45 mg/L |
1 |
36 |
39 |
36 |
29 |
Biodegradation of test and reference item:
Time |
% Degradation |
|||||||
Test item |
Na-benzoate |
Toxicity |
Abiotic control |
|||||
|
Flask No. |
Flask No. |
Flask No. |
|||||
|
1 |
2 |
Mean |
5 |
6 |
Mean |
4 |
3 |
2 |
0.6 |
0.0 |
0.3 |
39.0 |
38.9 |
38.9 |
17.6 |
0.0 |
5 |
0.3 |
0.2 |
0.3 |
59.1 |
64.1 |
61.6 |
28.6 |
-0.4 |
7 |
0.4 |
0.8 |
0.6 |
63.0 |
69.1 |
66.0 |
29.8 |
-1.2 |
9 |
-1.0 |
-0.5 |
-0.8 |
67.3 |
73.1 |
70.2 |
31.4 |
-1.4 |
12 |
-0.4 |
-0.2 |
-0.3 |
74.7 |
79.0 |
76.8 |
34.3 |
-2.0 |
14 |
-2.0 |
-1.3 |
-1.7 |
75.8 |
81.8 |
78.8 |
36.4 |
-3.1 |
19 |
0.9 |
0.7 |
0.8 |
86.4 |
88.6 |
87.5 |
39.5 |
-4.6 |
23 |
2.5 |
1.4 |
2.0 |
98.4 |
97.1 |
97.8 |
44.3 |
-6.0 |
27 |
1.8 |
1.7 |
1.8 |
99.4 |
100.3 |
99.8 |
45.2 |
-6.8 |
28 |
3.8 |
2.7 |
3.2 |
98.3 |
100.7 |
99.5 |
45.7 |
-7.0 |
Biodegradation of test and reference item:
Time |
% Degradation |
|||||||
Test item |
Na-benzoate |
Toxicity |
Abiotic control |
|||||
|
Flask No. |
Flask No. |
Flask No. |
|||||
|
1 |
2 |
Mean |
5 |
6 |
Mean |
4 |
3 |
2 |
0.6 |
0.0 |
0.3 |
39.0 |
38.9 |
38.9 |
17.6 |
0.0 |
5 |
0.3 |
0.2 |
0.3 |
59.1 |
64.1 |
61.6 |
28.6 |
-0.4 |
7 |
0.4 |
0.8 |
0.6 |
63.0 |
69.1 |
66.0 |
29.8 |
-1.2 |
9 |
-1.0 |
-0.5 |
-0.8 |
67.3 |
73.1 |
70.2 |
31.4 |
-1.4 |
12 |
-0.4 |
-0.2 |
-0.3 |
74.7 |
79.0 |
76.8 |
34.3 |
-2.0 |
14 |
-2.0 |
-1.3 |
-1.7 |
75.8 |
81.8 |
78.8 |
36.4 |
-3.1 |
19 |
0.9 |
0.7 |
0.8 |
86.4 |
88.6 |
87.5 |
39.5 |
-4.6 |
23 |
2.5 |
1.4 |
2.0 |
98.4 |
97.1 |
97.8 |
44.3 |
-6.0 |
27 |
1.8 |
1.7 |
1.8 |
99.4 |
100.3 |
99.8 |
45.2 |
-6.8 |
28 |
3.8 |
2.7 |
3.2 |
98.3 |
100.7 |
99.5 |
45.7 |
-7.0 |
Description of key information
The ready biodegradability in the Manometric Respirometry Test was determined with a non adapted activated sludge for the test item over a period of 28 days. The study was conducted from 2006-06-20 to 2006-07-18 according to OECD guideline 301 F at DR.U.NOACK-LABORATORIEN, D-31157 Sarstedt. The test item concentration selected as appropriate was 40 mg/L, corresponding to a ThOD of 87.2 mgO2/L in the test vessel. The oxygen was depleted by the respiration of bacteria and the degradation was followed by measuring the oxygen concentration. The biodegradation is therefore expressed as the percentage BOD and was calculated for each study day.
The mean oxygen depletion in the inoculum control came to 33.8 mgO2/L after 28 days. In order to check the activity of the test system sodium benzoate was used as functional control. The pass level > 60 % was reached after 3 days. The biodegradation rate came to 92 % after 28 days.
In the toxicity control containing both test and reference item 39 % degradation occurred within 14 days. The degradation of the reference item was not inhibited by the test item.
The 10 % level (beginning of biodegradation) was not reached until 28 days. The mean biodegradation remained at 0 %.
The validity criteria of the guideline are fulfilled.
The test item must be regarded as not readily biodegradable in the 10-d-window and after 28 days.
In addition, a biodegradation test with a read across substance is available with the same result.
In a ready biodegradability test based upon CO2-evolution (reliability category 1, compliant to GLP) no biodegradation could be observed at the end of the test period (28 days). The reference substance (sodium benzoate) was thoroughly degraded at day 28 and to 79% at day 14 proving the suitability of the activated sludge used. On the other hand, lack of biodegradation was not caused by toxicity of the test item as the toxicity control with both, the test item and the reference substance, showed no inhibition compared to the procedural controls with the reference substance only.
Key value for chemical safety assessment
- Biodegradation in water:
- not biodegradable
- Type of water:
- freshwater
Additional information
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