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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2010-07-12 to 2010-08-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD 310 Guideline and under GLP with all validity criteria fulfilled.
Qualifier:
according to
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
GLP compliance:
yes (incl. certificate)
Remarks:
2009-08-05
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Municipal Sewage Treatment Plant (Darmsadt, Germany)
- Laboratory culture: no data
- Method of cultivation: The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was de-canted. The solid material was re-suspended in tap water and again centrifuged. This procedure was repeated up to three times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight determined.
- Storage conditions: no data
- Storage length: no data
- Preparation of inoculum for exposure: The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in tap water and again centrifuged. This procedure was repeated up to three times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight determined.
Based on this ratio, calculated aliquots of washed sludge suspension were mixed with test water corresponding to 4 g dry material per litre and then aerated using CO2-free air until use.
The dry matter of the sludge suspension was determined before test start and appropriate amounts of the sludge suspension were mixed with test medium to achieve a final concentration of 4 mg dry material per litre.
- Pretreatment: no data
- Concentration of sludge: 4 mg/L of dry material
- Initial cell/biomass concentration: no data
- Water filtered: yes
- Type and size of filter used, if any: no data
Duration of test (contact time):
ca. 28 d
Initial conc.:
ca. 83 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium:
- Additional substrate:
- Solubilising agent (type and concentration if used):
- Test temperature:
- pH:
- pH adjusted: yes/no
- CEC (meq/100 g):
- Aeration of dilution water:
- Suspended solids concentration:
- Continuous darkness: yes/no
- Other:

TEST SYSTEM
- Culturing apparatus:
- Number of culture flasks/concentration:
- Method used to create aerobic conditions:
- Method used to create anaerobic conditions:
- Measuring equipment:
- Test performed in closed vessels due to significant volatility of test substance:
- Test performed in open system:
- Details of trap for CO2 and volatile organics if used:
- Other:

SAMPLING
- Sampling frequency:
- Sampling method:
- Sterility check if applicable:
- Sample storage before analysis:
- Other:

CONTROL AND BLANK SYSTEM
- Inoculum blank:
- Abiotic sterile control:
- Toxicity control:
- Other:

STATISTICAL METHODS:
Reference substance:
benzoic acid, sodium salt
Preliminary study:
not applicable
Test performance:
No deviation.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. -0.5
Sampling time:
28 d
Details on results:
- Percentage Biodegradation: The mean percentage biodegradation at the end of the 28-day exposure period was -0.5%. The results are represented in Table 1.
- Conclusion: The percentage biodegradation did not exceed 60% after 28 days of incubation. The test item can therefore be considered not to be readily biodegradable.
Results with reference substance:
- Percentage Biodegradation: The reference item sodium benzoate was degraded to 162% after 14 days and to 146% after 28 days of incubation. Degradation rates above 100% result from a complete degradation of the reference item and also from an additional utilisation of retained resources. The sludge bacteria were very active so that after five days the reference item was more than completely degraded. In case of a lack of nutrition the bacteria began to degrade themselves.
- Conclusion: The percentage biodegradation of the reference item confirms the suitability of the used aerobic activated sludge inoculum.

Table1: Inorganic Carbon produced in Test Flasks During the Test Period of 28 Days

Treatment C-content (mg/L) Replicate TIC [mg/L] day TIC corrected [mg/L] day % biodegradation [net measured TIC/initial C x 100]
0 2 5 7 9 14 21 28 0 2 5 7 9 14 21 28 0 2 5 7 9 14 21 28
Control - 1 0.31 1.89 0.33 1.04 0.95 2.94 1.41 2.81                                
2 -0.25 0.94 0.73 1.12 0.7 1.98 1.35 3.14      
3 * 0.92 0.33 0.32 0.57 1.45 0.72 2.37      
4   1.96      
5   2.67      
mean 0.03 1.25 0.46 0.82 0.74 2.13 1.16 2.59      
CV % 1359 44 49 54 26 36 33 17                                
Na-Benzoate 19.824 1 0.33 - 35.08 - - 32.69 - 36.55 0.3 - 34.62 - - 30.57 - 33.96 1.5 - 174.6 - - 154.2 - 171.3
2 0.28 - 27.86 - - 35.94 - 35.99 0.25 - 27.39 - - 33.81 - 33.4 1.3 - 138.2 - - 170.6 - 168.5
3 0.36 - 21.82 - - * - 31.46 0.33 - 21.36 - - - 28.87 1.7 - 107.7 - - - 145.6
4   27.28   24.69 124.6
5   25.99   23.4 118
mean 0.32 - 28.25 - - 34.31 - 31.45 0.29   27.79     32.19   28.86 1.5   140.2     162.4   145.6
CV % 13 - 24 - - 7 - 15 15   24     7   17 13   24     7   17
Trifluoroethanol 19.871 1 0.54 1.19 0.33 1.5 0.5 1.07 1.14 2.43 0.51 -0.06 -0.14 0.67 -0.24 -1.06 -0.03 -0.16 2.6 -0.3 -0.7 3.4 -1.2 -5.3 -0.1 -0.8
2 0.22 0.76 0.78 1.31 1.09 0.72 0.53 2.23 0.19 -0.49 0.31 0.49 0.35 -1.41 -0.63 -0.36 0.9 -2.5 1.6 2.4 1.8 -7.1 -3.2 -1.8
3 0.61 0.95 0.45 1.8 0.2 1.42 1.14 3.06 0.58 -0.3 -0.01 0.98 -0.54 -0.71 -0.02 0.47 2.9 -1.5 -0.1 4.9 -2.7 -3.6 -0.1 2.4
4       -0.26 -1.3
5       -0.2 -1
mean 0.45 0.97 0.52 1.54 0.6 1.07 0.94 2.49 0.42 -0.28 0.05 0.71 -0.14 -1.06 -0.22 -0.1 2.1 -1.4 0.3 3.6 -0.7 -5.3 -1.1 -0.5
CV % 46 23 44 16 76 33 37 13 49 -78 461 35 -325 -33 -159 -320 51 -79 398 35 -327 -33 -163 -333
Toxicity control 19.824 1 0.7 - 26.77 - - 34.09 - 10.84 0.67 - 26.3 - - 31.96 - 8.25 3.4   132.7     161.2   41.6
2 0.65 - 28.36 - - 30.5 - 30.59 0.62 - 27.9 - - 28.37 - 28 3.1 140.7 143.1 141.2
3 * - 25.56 - - 34.75 - 29.06   - 25.09 - - 32.62 - 26.47 126.6 164.6 133.5
4   36.18   33.59 169.4
5   33.06   30.47 153.7
mean 0.67   26.89     33.11   27.94 0.64   26.43     30.99   25.35 3.3   133.3     156.3   127.9
CV % 6   5     7   36 6   5     7   39 6   5     7   39
% Inhibition in the toxicity control at day 28                                               12
Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The percentage biodegradation did not exceed 60% after 28 days of incubation. The test item can therefore be considered not to be readily biodegradable.
Executive summary:

A ready biodegradability test was performed, according to OECD Guideline 310, which allows the biodegradability to be measured in an aerobic aqueous medium. Microorganisms (obtained from the municipal sewage treatment plant of Darmstadt, Germany) are inoculated into a chemically defined liquid medium containing only the test substance (Trifluoroethanol at C-content 19.871 mg/L) or only the reference substance (Sodium benzoate at 19.824 mg/L) or the test substance with the reference substance (toxicity control) under aerobic conditions for a period of 28 days. The temperature ranged from 20°C. The biodegradation was determined by following the CO2 evolution of the test item in the incubation flasks during exposure. The percentages biodegradation after 28 days were -0.5 % for Trifluoroethanol, 146 % for the reference item and 123% for the toxicity control. Degradation rates above 100% result from from a complete degradation of the reference item and also from additional utilisation of retained resources. The sludge bacteria were very active so that after five days the reference item was completely degraded. In the toxicity control the inhibition value was 12 %, and therefore clearly below the 25 % criterion. Thus, the test item can be assumed to be not inhibitory to the aerobic activated sludge micro organisms.

Under the test conditions, Trifluoroethanol was not biodegraded and should therefore not be classified as readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
A biodegradation screening test of TFEA was conducted according to Japanese MITI guideline but the english report available is not well documented (summary) and the methodology is not adapted to the test substance (volatilization from test bottles). However, the results are in line with the key study.
Principles of method if other than guideline:
Test was conducted according to Japanese guideline “Test method on new chemical substances” (S 49 ENV5, MH615, 49MITI392).
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
Urban drainage return sludge was used for this study
Duration of test (contact time):
28 d
Details on study design:
Test system
No.1 positive control aniline + sludge + incubation medium
No.2 Control sludge + incubation medium
No. 3 & 4. Test test substance + sludge + incubation medium

Test volume 300 ml

Temperature 25 +/- 1°C

Concentrations
Standard sample (aniline) and test sample 100 mg/L
Sludge 30 mg/L
Preliminary study:
Test results after 28 days
Items Test sample No*) Theoretical
1 2
BOD, mg -0.8 -1.0 21.6
DOC, mg/l 14.6 15.4 24.0

*) values after Calibrated by control system.
Test performance:
No data
Key result
Parameter:
other: BOD
Value:
0
Sampling time:
28 d
Parameter:
% degradation (DOC removal)
Value:
38
Sampling time:
28 d
Details on results:
Degradability derived from BOD gave negative values.
Results with reference substance:
No data
Validity criteria fulfilled:
not specified
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
Average degradability of TFEA after 28 days derived from BOD was 0% and that derived from DOC was 38 %. Thus the biodegradability of TFEA seemed to be non-biodegradable.
Because of low boiling point and high vapor pressure of TFEA, TFEA volatilized from test bottles seemed to give apparent biodegradability derived from COD measurement.
Executive summary:

A biodegradation screening test of TFEA was conducted according to Japanese MITI guideline “Test method on new chemical substances” (S 49 ENV5, MH615, 49MITI392). Average degradability of TFEA after 28 days derived from BOD was 0% and that derived from DOC was 38 %. Thus the biodegradability of TFEA seemed to be non-biodegradable. However the english report available is not well documented enough (summary) to assess the study reliability and the methodology is not adapted to the test substance. Because of low boiling point and high vapor pressure of TFEA, TFEA volatilized from test bottles seemed to give apparent biodegradability derived from COD measurement.The results ae considered as sshow that trifluoroethanol is not ready biodegradable which is in line with the key study.

Description of key information

A ready biodegradability test was performed, according to EU Determination of the "Ready" Biodegradability - Dissolved Organic Carbon (DOC) Die-Away Test, which allows the biodegradability to be measured in an aerobic aqueous medium. The percentages biodegradation of Trifluoroethanol were 52 and 75% in the replicates after 35 days of exposure and the control abiotic degradation was 64%. Trifluoroethanol was considered not to be biodegraded and should therefore not be classified as readily biodegradable. However, these results were invalided because the differences of extremes of replicate values of the removal of the test chemical at the end of the test are > 20%.

Another biodegradation screening test of Trifluoroethanol was conducted according to Japanese MITI guideline “Test method on new chemical substances” (S 49 ENV5, MH615, 49MITI392). Average degradability after 28 days derived from BOD was 0% and that derived from DOC was 38 %. However the english report available is not well documented enough (summary) to assess the study reliability and the methodology was not adapted to the test substance. Indeed, it is reported that apparent biodegradability derived from COD measurement is probably due to Trifluoroethanol volatilization from test bottles.

Based on these results, a new ready biodegradability in a CO2 headspace test was performed, according to OECD Guideline 310. Microorganisms from a municipal sewage treatment plant were inoculated into medium containing only the test substance or only the reference substance or the test substance with the reference substance (toxicity control) under aerobic conditions for a period of 28 days. The biodegradation was determined by the CO2 evolution in the incubation flasks during exposure. The percentages biodegradation after 28 days were -0.5% forTrifluoroethanol, 146 % for the reference item and 123% for the toxicity control. It was postulated that degradation rates above 100% resulted from a complete degradation of the reference item and also from additional utilisation of retained resources. Under these test conditions, Trifluoroethanol was not biodegraded and should therefore not be classified as readily biodegradable. This result is supported by the two others studies.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information