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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:
8 September 2003 to 7 October 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
yes
Remarks:
(see below)
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
yes
Remarks:
(see below)
Principles of method if other than guideline:
Section IV.A of Protocol EN-105-632021-A states that DOC concentrations of the test substance solutions will be determined at the start and end of the test as a verification measure of biodegradability. The decision to determine DOC concentrations was based on information from the MSDS which states the test substance was completely soluble in water. It was observed, after the addition of the test substance to the test vessels at test start, that the test substance was not soluble in the test media, therefore DOC concentrations were not determined. This deviation did not adversely affect the study.

Section XIII.1 of Protocol EN-105-632021-A and Section 5.6 of SOP EN-1032 state that as one of the criterion of a valid test, the positive control should yield => 60 % biodegradation by day 14. The Positive Control reached 54 % degradation on day 14. The final suspended solids level of the inoculum in the test vessels was 22.4 mg/L, less than the maximum concentration of 30 mg/L suspended solids for this test. This lower concentration of suspended solids by the inoculum may have been a contributing factor of the failure of the positive control to meet the pass level. By the end of the test, the Positive Control reached 62 % biodegradation. This deviation did not affect the interpretation of the test substance data.
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
PREPARATION OF THE INOCULUM
On arrival at the laboratory, the activated sludge was aerated before use. A sample of the mixed liquor was homogenised for two minutes with a mechanical blender. It was allowed to settle for approximately 60 minutes. The supernatant was pipetted to provide sufficient volume of inoculum for each carboy. Viability of the supernatant was confirmed by using an Easicult® TTC dip slide to estimate microbe numbers.
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST PROCEDURE
The CO2 scrubbing apparatus was set up to remove CO2 at a constant rate from the air supplied to the carboy solutions. This was done by diverting the air through a drying column (containing Drierite®), a CO2 absorption column (containing Ascarite II®) and flow meters before being bubbled through the test carboys. The air passes through the system at a rate of approximately 50-100 mL/min (one to two bubbles per second) although this was not measured. All test carboys were covered with aluminium foil for the duration of the study to protect the solutions from the light.

Five inoculated carboys were required for the testing of the test substance: two for the Inoculum Blank, one for the Positive Control and two for the test substance. Basal Salts Medium (BSM) was prepared first. The carboys were filled with 2290 mL of BSM. Next, 210 mL of the prepared inoculum supernatant was added to each carboy. The mixture was aerated with CO2-free air for approximately 24 hours to purge the system of CO2.

After the aeration period, the test substance was directly added to two of the carboys with 500 mL purged BSM to begin the test period. The substance was tested at 30.5 mg/L (theoretically 20 mg DOC/L). Purged BSM (500 mL) was added to each of the carboys used as Inoculum Blank controls (containing no test substance). The Positive Control Stock Solution (20 mg DOC/L) was added to the carboy used for the Positive Control. The final volume in each carboy was 3000 mL. Each carboy was agitated with a magnetic stir bar. Three CO2 absorber bottles were connected in series to the exit airline of each carboy. Each absorber bottle contained 100 mL of 0.0125M Ba(OH)2.

At test start, the solutions are aerated with CO2-free air. The CO2 produced in each carboy reacts with the Ba(OH)2 in the absorber bottles where it is precipitated as barium carbonate; the amount of CO2 produced was determined by titrating the Ba(OH)2 remaining in solution with 0.05 N standardised HCl. The course of degradation was followed by performing titrations over the 28 day test period.

Periodically, the CO2 absorber bottle nearest the carboy was removed for titration. The remaining two absorber bottles were moved one place closer to the carboy and a new absorber bottle containing 100 mL of fresh 0.0125M Ba(OH)2 was placed at the far end of the series.

On day 27, titrations were performed as usual. An aliquot of test solution was removed from each test vessel to measure pH. Next, 1 mL of concentrated HCl was added to each carboy to drive off inorganic carbonate. The carboys were aerated overnight. Final titrations were performed on day 28.

ADDITIONAL CONTROLS
An additional set of 3 absorber bottles containing 0.0125M Ba(OH)2 was connected directly to the scrubbed airline and titrated in the same manner as the test. These served as the airline control. The BA(OH)2 was used throughout the study was also monitored by titrating the stored solution when first prepared and at weekly intervals. The two sets of titration data were compared in order to demonstrate that the air supply was free of CO2.

MICROBIOLOGY
An Easicult® TTC dip slide was used to estimate microbial numbers in the supernatant used to inoculate the test. The microbe count of the supernatant was 10E+6 orga/mL. The suspended solids level in the test vessels was 22.4 mg/L.
Reference substance:
benzoic acid, sodium salt
Remarks:
CAS 523-32-1; weight % purity 103 %
Test performance:
The pH of the Basal Salts Medium at the start of the test was 7.578. No unusual variation on pH was noted from day 0 to day 27 in any of the test carboys. The pH ranged from 7.407 to 7.586 on day 27. The Inoculum Blanks released an average of 63.8 mg CO2 (21.3 mg CO2/L) over the test period. The Ba(OH)2 stock solution needed 48.1 ± 0.2 mL of titrant per titration compared to 47.6 ± 0.4 mL for the airline control. This indicates that the airline did not contain CO2 after scrubbing. The air temperature during the test period ranged from 21-22 ºC.
Key result
Parameter:
% degradation (DOC removal)
Value:
4
Sampling time:
28 d
Remarks on result:
other: Test vessel #1
Key result
Parameter:
% degradation (DOC removal)
Value:
-1
Sampling time:
28 d
Remarks on result:
other: Test vessel #2
Details on results:
At the end of the test, cumulative biodegradation of the test substance reached 4 and -1 % in test vessels # 1 and 2, respectively. Less CO2 was evolved from test vessel #2 than the blank resulting in a negative value after subtraction of the blank (this procedure corrects for background CO2 evolution). The graph showing percent biodegradation for both test vessels and the Positive Control is provided in Figure 1.
Results with reference substance:
The Positive Control, sodium benzoate, yielded 62 % of the theoretical CO2 possible over the course of the test.

Table 1: CO2 evolution titration data worksheet

Day  Blank average  Blank cumulative CO2  Positive control  Control cumulative % biodegradation  Test #1  Test #1 cumulative % biodegradation  Test #2  Test #2 cumulative % biodegradation
 0    0.0    0    0    0
 1  47.9  5.6  33.8  4  42.8  0  43.08  -1
 3  46.7  17.7  2.6  21  33.5  1  36.0  -1
 6  47.0  31.1  5.8  36  33.2  2  35.1  -1
 8  47.8  39.6  16.3  47  38.3  3  39.6  -1
 10  47.9  44.7  34.2  52  42.8  3  43.1  0
 14  47.6  49.6  38.2  54  43.3  3  43.5  -1
 17  47.8  53.7  40.8  56  44.0  3  44.3  -1
 20  47.4  57.2  40.8  58  44.5  3  44.4  -1
 23  47.7  59.6  43.0  59  45.4  3  45.5  -1
 27  47.4  62.0  41.8  61  44.6  3  45.4  -1
 28  48.0  63.8  44.4  62  45.7  4  46.6  -1
Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
Under the conditions of the test, the test substance was determined to be not readily biodegradable.
Executive summary:

In a GLP compliant study conducted in line with OECD Guideline 301B and EU Method C.4, the ready biodegradability of butanoic acid, 4-[[4-[7-chloro-6-(1,1-dimethyethyl)-3H-pyrazolo[1,5-b][1,2,4]triazol-2-yl]phenyl]amino]-4-oxo, tetradecyl ester was investigated. The test substance was determined to be not readily biodegradable under the conditions of the test.

Description of key information

Key study:- Moulton (2003) 'Determination of ready biodegradability (biotic degradation) using the CO2 evolution test (modified sturm)' conducted in line with OECD Guideline 301B and EU Method C.4. The test substance was determined to be not readily biodegradable under the conditions of the test.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

The key study (Moulton, 2003) was performed in compliance with GLP and to OECD Guideline 301B and EU Method C.4 with a sufficient level of detail to assess the quality of the presented data. The study was performed to a good standard in line with accepted, standardised guidelines and was assigned a reliability score of 1 using the principles for assessing data quality as set out in Klimisch et al. (1997). The test substance was determined to be not readily biodegradable under the conditions of the test.

The supporting study (Foley, 2004c) was performed in compliance with GLP and EU Method C.6 with a sufficient level of detail to assess the quality of the presented data. The study was performed to a good standard in line with an accepted, standardised guideline but there is limited information on the methodology and the results should be used with caution due to the applicability of the test method with this test substance. The study was therefore assigned a reliability score of 2 using the principles for assessing data quality as set out in Klimisch et al. (1997). The COD of the test substance was determined to be 2 g COD per gram of test substance.

The disregarded study (Foley, 2004b) could not be performed due to the low water solubility of the test substance.