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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:
25 Mar - 23 Apr 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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)
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):
Not adapted activated sludge from the aeration tank of the ARA Werdhölzli (8048 Zürich, Switzerland), a municipal biological waste water treatment plant.
- Sampling: 23 March 2010, 11:00 a.m.
- Pretreatment: The activated sludge was used after sampling from the treatment plant without adaptation. However, the sludge was pre-conditioned for 2 days (aerated but not fed) to reduce the amount of CO2 produced by the blank controls.
Prior to the test the sludge was washed twice with tap water. After centrifugation the sludge, at a tenfold concentration of the final concentration to be achieved for the test, was suspended in test medium as described in Table 1 (see below).
- Concentration of sludge: 30 mg/l dry matter in the final mixture
Duration of test (contact time):
28 d
Initial conc.:
34 mg/L
Based on:
test mat.
Initial conc.:
20 mg/L
Based on:
other: TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: according to OECD guideline
- Additional substrate: none
- Solubilising agent (type and concentration if used): none
- Test temperature: 22± 2 °C
- pH: 7.4± 0.2
- pH adjusted: yes (at beginning of test, with NaOH or HCl, if necessary)
- CEC (meq/100 g): no data
- Aeration of dilution water: aerated with CO2-free air
- Suspended solids concentration: 30 mg/l dry matter in the final mixture
- Continuous darkness: yes
- Other: Concentration of test substance and reference material: The test substance and reference material were applied by direct addition to give a final test concentration of about 20 mg/l with respect to the total organic carbon (TOC).

TEST SYSTEM
- Culturing apparatus: 2500 ml closed gas bottle
- Number of culture flasks/concentration: For each test series the following number of test flasks was set up: Test suspension containing inoculum, test medium and test substance (three replicates)
- Method used to create aerobic conditions: The test vessels were stirred thoroughly and aerated with synthetic CO2-free air
- Test performed in closed vessels due to significant volatility of test substance: -
- Test performed in open system: yes
- Details of trap for CO2 and volatile organics if used: The air leaving the individual vessels was passed through gas-absorption bottles filled with KOH (125 ml of 0.13 M KOH).
- Other: -

SAMPLING
- Sampling frequency: The biodegradation of the test material was followed by CO2 measurements at frequent intervals to allow the assessment of the 10-d window (after 0, 1, 4, 7, 12, 14, 18, 21, 26, 28 days).
- Sampling method: not specified
- Sterility check if applicable: -
- Sample storage before analysis: -
- Other: -

CONTROL AND BLANK SYSTEM
- Inoculum blank: Inoculum blank containing inoculum and test medium (three replicates)
- Abiotic sterile control: Abiotic sterile control: containing test substance, test medium and 0.2 mM HgCl2 as sterilizing agent to prevent microbial decomposition (one replicate)
- Toxicity control: Toxicity control: containing inoculum + test medium + test substance + sodium benzoate as ready biodegradable reference compound (one replicate). In the toxicity control about 20 mg/l test substance and about 20 mg/l reference substance with respect to the total organic carbon (TOC) were tested as a mixture.
- Other: Procedure control: Procedure control containing inoculum, test medium and sodium benzoate as ready biodegradable reference compound (three replicates)

STATISTICAL METHODS: -
Reference substance:
benzoic acid, sodium salt
Remarks:
100% pure
Preliminary study:
not conducted.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
58 - 61
Sampling time:
28 d
Remarks on result:
other: range of 2 replicates
Details on results:
Based on the data of the individual inorganic carbon determinations, the mean biodegradability in the CO2 Evolution Test of the test substance was calculated to be 60% (Table 2, Figure 1) after 28 days. The biodegradation of the test substance reached 43% at the end of the 10-d window. Biodegradation of the test substance was observed without any significant lag phase.
Results with reference substance:
The positive control, sodium benzoate, reached 100% biodegradation after 14 days, thus confirming suitability of inoculum and test conditions (Table 3, Figure 2).

Table 2: CO2produced by the test units, the inoculum blank and the corresponding degradation data.

  Inoculum Test unit no. 1 Test unit no. 2  
  blank * containing test material  containing test material  
             
  Total CO2 Total CO2   Total CO2   Mean
Time release release Degradation release Degradation Degradation
(days) in test in test (%) ** in test (%) ** of no. 1+2
  sample sample   sample   (%)
  (mg IC/l)  (mg IC/l)    (mg IC/l)     
             
             
0 0.0 0.0 0 0.0 0 0
1 -0.1 0.4 3 0.9 5 4
4 2.5 3.7 6 4.4 10 8
7 4.8 7.3 13 7.9 16 14
12 7.3 14.5 36 12.9 28 32
14 7.6 16.7 45 14.9 37 41
18 8.7 18.8 50 17.8 46 48
21 9.3 19.7 52 19.2 49 51
26 9.5 21.1 58 20.9 57 57
28 9.8 21.4 58 22.0 61 60
* Mean of two replicates
** The calculation is based on an organic carbon concentration
in mg/l of 20.0 (Test unit no. 1)
20.0 (Test unit no. 2)

Table 3: CO2produced by the procedure control, the inoculum blank and the corresponding degradation data.

  Inoculum Procedure control Toxicity control (test Abiotic
  blank * (sodium benzoate) material + sodium benzoate) control
             
  Total CO2 Total CO2   Total CO2   Total CO2
Time release release Degradation release Degradation release
(days) in test in test (%) ** in test (%) *** in test
  sample sample   sample   sample
  (mg IC/l)  (mg IC/l)    (mg IC/l)    (mg IC/l) 
             
             
0 0.0 0.0 0 0.0 0 0.0
1 -0.1 2.7 14 1.4 4 0.0
4 2.5 12.5 50 16.4 35 0.4
7 4.8 18.3 68 21.8 42 0.7
12 7.3 25.6 91 33.0 64 1.4
14 7.6 27.9 101 36.1 71 1.5
18 8.7 29.3 103 40.3 79 1.5
21 9.3 29.9 103 42.2 82 1.4
26 9.5 30.7 106 45.5 90 0.7
28 9.8 30.9 105 46.2 91 0.6
* Mean of two replicates
** The calculation is based on an organic carbon concentration in mg/l of 20.0
*** The calculation is based on an organic carbon concentration in mg/l of 40.0
(sum of test substance and procedure control)

Results of additional controls:

Toxicity control

At the applied initial test concentration Photomer 4149 F showed no significant toxic effect on the microbial population, since the biodegradation of the mixture (test substance + reference compound sodium benzoate) was within the expected theoretical value during thewhole test period (Table 3, Figure 2). The test was considered valid, since more than 25 % degradation occurred within 14 days.

Abiotic sterile control

Photomer 4149 F was not abiotically degraded (by processes producing CO2) during thewhole test period of 28 days in the absence of microorganisms as confirmed by the determinations of the inorganic carbon concentrations

Further remarks:

At the end of the test the pH value of both inoculum blanks and the procedure control was 7.4, respectively. The pH values of the test units and the toxicity control were 7.3 and 7.5, respectively.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test substance did not reach the pass level of 60% for ready biodegradability in the CO2 Evolution Test within the 10-d window. However, the pass level was reached after 28 days of incubation. As the test substance is aan UVCB, the 10-day window criterion does not apply. Therefore, the test substance is considered readily biodegradable.
Executive summary:

The biodegradability of the test substance exposed to microorganisms derived from the activated sludge of a municipal sewage treatment plant was investigated under aerobic static exposure conditions.

The biodegradability - based on CO2evolution - of the test substance

was calculated to be 60% of the theoretical value (ThCO2) after an incubation time of 28 days.

The biodegradation of the test substance reached 43% at the end of the 10-d window.

Biodegradation of the test substance was observed without any significant lag phase.

The positive control, sodium benzoate, reached 100% biodegradation after 14 days, thus confirming suitability of inoculum and test conditions.

As the test substance is a mixture of homologues, the 10-day window does not apply. Therefore, the test substance is considered readily biodegradable.

Description of key information

In a study according to OECD test guideline 301B, the test substance was biodegraded to 60% after 28 days. As the test substance is an UVCB, the 10 -day window criterion does not apply. Hence, the test substance is considered readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

The biodegradability of the test substance exposed to microorganisms derived from the activated sludge of a municipal sewage treatment plant was investigated under aerobic static exposure conditions. The biodegradability - based on COevolution - of the test substance was calculated to be 60% of the theoretical value (ThCO2) after an incubation time of 28 days.


As the test substance is an UVCB, the 10-day window criterion does not apply. Hence, the test substance is considered readily biodegradable.