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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:
25 August 2010 to 3 december 2010
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:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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): inoculum was collected from a water treatment plant containing effluent from a predominantly domestic origin.

- Storage conditions: 22 +/- 2°C

- Preparation of inoculum for exposure: the inoculum was prepared by initially decanting and sieving the sludge to concentrate the suspended matter and remove the biggest particles. The sludge was centrifuged for 5 minutes, the supernatant was discarded and the pellet resuspended in the mineral medium (see § "Acclimatation period"). The sludge was then centrifuged again and the pellet resuspended in the mineral medium (first washing). In order to reduce the carbon organic content of the sludge, it was preconditioned under the environmental conditions of the test during 6 days
(see § "Acclimation period"). After this preconditioning period, the inoculum was centrifuged again and the pellet resuspended in the mineral medium (second washing). Finally, the inoculum was aerated until use for the test.

- Pretreatment: the inoculum was prepared by initially decanting and sieving the sludge to concentrate the suspended matter and remove the biggest particles. The sludge was centrifuged for 5 minutes, the supernatant was discarded and the pellet resuspended in the mineral medium (see § "Acclimatation period").

- Concentration of sludge: 2.06 g/L (dry weight/v).
Duration of test (contact time):
28 d
Initial conc.:
10 mg/L
Based on:
other: TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Test temperature: 20-22°C
- pH: 7.55 measured in the mineral medium before the start of the test; 7.47 to 7.71 measured in the test flasks at the end of the test
- pH adjusted: yes - with HCL 1N
- CEC (meq/100 g):
- Aeration of dilution water: test flasks were aerated overnight to drive off the remaining carbon dioxide
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus:
- Number of culture flasks/concentration: 2 (test item), 2 (control), 1 (positive control)
- Method used to create aerobic conditions: bubbled with CO2 scrubbed air
- Test performed in closed vessels due to significant volatility of test substance: no
- Details of trap for CO2 and volatile organics if used: three wash bottles filled with 100 mL of 0.0125 M barium hydroxide solution

SAMPLING
- Sampling frequency: days 2, 5, 7, 9, 12, 15, 19, 23, 26 and 30 (day 1 corresponding to the first day of treatment).

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Toxicity control: no
Reference substance:
other: sodim acetate
Preliminary study:
not applicable
Parameter:
% degradation (CO2 evolution)
Value:
18
Sampling time:
28 d
Remarks on result:
other: duplicates with 11% and 25% degradation
Details on results:
The 10-day window of the test item started only on the 26th day (defined graphically). Biodegradation of the test item reached only 18% (mean of duplicates with 11% and 25% degradation) at the end of the test.
Results with reference substance:
biodegradation in the reference test was 83.82% after 14 days: valid
Validity criteria fulfilled:
no
Remarks:
only 3rd criterion not fulfilled, no impact, see executive summary
Interpretation of results:
not readily biodegradable
Conclusions:
Due to a low biodegradation rate of the test item, the 10-day window (period of 10 days immediately following the attainment of 10% biodegradation) was not closed within the study period and at the end of the 28-day test the biodegradation of BAB was largely below 60% in both duplicates.
In order to classify a test item as readily biodegradable, a biodegradation of 60% has to be reached within the 10-day window and within the 28-day period of the test.
Therefore, under the experimental conditions of this study, the test item BAB was not readily biodegradable in the 28-day modified Sturm test.
 
One validity criterion was not fulfilled (variability between test item duplicates was high), however based on the low degradation recorded in both duplicates, it was concluded that this had no impact on the reliability of the above conclusion.
Executive summary:

The ready biodegradability of the test item, BAB, was evaluated in a 28-day modified Sturm test according to Commission Regulation (EC) No. 440/2008 Annex, Part C, C.4, 30 May 2008 and OECD guideline No. 301B (17th July 1992).

 

Methods

Sewage sludge, i.e. inoculum, was sampled from the aeration tank of a sewage treatment plant and aerated for 6 days. A sludge suspension was subsequently prepared intofiveflasks to obtain a final concentration of 30 mg/L (dry weight/v) in reconstituted water (OECD mineral medium):

.           two flasks were left unexposed to any item (blank control),

.           one flask was exposed to the reference item sodium acetate at 10.0 mg/L of total organic carbon (TOC) (procedure control),

.           two flasks were exposed to the test item at 10.0 mg/L of TOC (test solution).

 

The test suspensions were bubbled with CO2scrubbed air for 28 days at room temperature (to). After 28 days, pH ranged from 7.47 to these suspensions.

Evolved CO2 from each suspension was measured at regular intervals and the % of biodegradation of the test and reference items was determined as a % of the theoretical CO2 (ThCO2) that may be produced from the complete degradation of the known total carbon content of these items.

 

Results

Validity Criteria

The first two of the three study validity criteria were fulfilled:

.           the blank value (a measurement of CO2evolved uniquely from the breakdown of organic matter in the inoculum and mineral medium) was <= 70 mg of CO2evolved per liter of suspension at the end of the test (average of the two controls),

.           biodegradation in the reference test was at least 60% after 14 days,

.           biodegradation values of test item replicates deviated by more than 20% at the end of the test.

The fact that the third validity criterion was not fulfilled was considered to have no impact on the qualitative conclusion of the study (absence of ready biodegradation) because biodegradation in each duplicate stayed markedly below the pass level of 60%.

 

Test item biodegradation

The 10-day window of the test item started only on the 26th day (defined graphically). Therefore the 28-day test finished before the end of the 10-day window.

Biodegradation of the test item reached only 18% (mean of duplicates with 11% and 25% degradation) at the end of the test.

Description of key information

An experiment concluded that BAB was not readily biodegradable in the 28-day modified Sturm test, with 18% degraded in 4 weeks (test according to Council regulation n°440/208, Annex, Part C, C 4.30 (30/05/2008) and OECD guideline n°301B (17/07/92).

According to literature search and with a category approach the substance is intended to be not easily biodegradable.
(Q)SAR estimation with BIOWIN models with different components of the substance also indicate the probability of a low biodegradation of the substance.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable, not fulfilling specific criteria
Type of water:
freshwater

Additional information

Not readily biodegradable. Inherently biodegradable but not fulfilling ECHA Guidance R7b criteria (less than 20% degraded).

Based on these data, the substance fulfils the biodegradability criterion for aquatic environment chronic hazard:

According to CLP: Aquatic Chronic 4/H413: May cause long lasting harmful effects to aquatic life.

This classification is confirmed by the estimated log Kow > 4 and BCF > 500.