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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23th April to 31th May, 2004
2 (reliable with restrictions)

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
not specified
GLP compliance:

Test material

Constituent 1
Reference substance name:
Ortho-Diethoxy Benzene
Ortho-Diethoxy Benzene
Constituent 2
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Constituent 3
Reference substance name:
Test material form:
other: colorless solid
Details on test material:
- Name of test material (as cited in study report): ODEB or Ortho-Diethoxy Benzene
- For more details, see below the "confidential details on test material"

Study design

Oxygen conditions:
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): Collected from the aeration tank of a full-sclae wastewater treatment plant which treats predominantly domestic sewage (ETE Jundiai-SP/Brazil)
- Preparation of inoculum for exposure: The activated sludge was treated by allowing to settle three times for 30 minutes. For each settling the supernatant was rejected and the solids was redispersed in the mineral medium. In order to lower carbon organic content and to reduce the blank CO2 evolution, the activated sludge was aerated overnight.
- Concentration of sludge: The final concentration of the treated activated sludge was adjusted to 1000 mg/L dry solids.
- Water filtered: no data
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
20 mg/L
Based on:
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
The test substance, normally at 20 mgC/l, as the sole source of carbon and energy, is incubated in a buffer-mineral salts medium which has been inoculated with a mixed population of microorganisms. The test is performed in sealed vessels with a headspace of air, which provides a reservoir of oxygen for aerobic biodegradation. Carbon dioxide (CO2) evolution resulting from the ultimate aerobic biodegradation of the test substance is determinated by measuring the inorganic carbon (IC) produced in the test vessels in excess of that produced in blank vessels containing inoculated medium only. The extent of biodegradation is expressed as a percentage of the theoretical maximum IC production (ThIC), based on the quantity of the test substance (as organic carbon) added initially.

Test System description:
The test was conducted in serum bottles of 120 mL capacity sealed with butyl rubber septa and aluminium crimp seals.
Forty six serum bottles were used for the test and were designated as follows:
• Twelve blank controls: containing only the inoculated test medium;
• Fitteen test vessels: containing the test substance (at 20 mg/L of TOC) and the inoculated test medium;
• Fifteen reference vessels: containing sodium benzoate (at 20 mg/L of TOC) and the inoculated test medium;
• Four inhibition controls: containing both the test substance and sodium benzoate at the same concentrations (at 20 mg/L of TOC) and the inoculated test medium;
The blank controls and the test vessels were prepared by adding 80 mL of inoculated medium, which was kept adequately mixed to ensure that it is uniformly distributed to the bottles.
The reference vessels and the inhibition controls were prepared by adding 80 ml of reference substance solution, which was kept adequately mixed to ensure that it is uniformly distributed to the bottles.
The recommended headspace to liquid volume ratio of 1:2 (80 mL liquid volume to 120 mL-capacity bottles) was used.
Bottles were sealed with butyl rubber stoppers and crimped aluminium caps and incubated at 22 °C in the dark.

Sampling and Inorganic Carbon (IC) Analysis:
Bottles were sacrificed for inorganic carbon (IC) analysis on the following times : days 3, 5, 7, 14, 21 and 28.
Assay for CO2 production was conducted for duplicate bottles on days 3, 5, 7, 14 and 21 and five replicate bottles on day 28.
The samples were prepared by injecting 1.0 mL of sodium hydroxide solution through the septum of each bottle sampled, shaking on the incubation rotary shaker for 1 h at the test temperature, removing the bottles from the shaker and allowing to settle. Suitable volume of the liquid phase were sampled and injected into a Shimadzu Model TOC 5000 inorganic carbon analyser, which was calibrated using appropriate standards, and the concentrations of IC were recorded.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradation
% degradation (CO2 evolution)
St. dev.:
Sampling time:
28 d

BOD5 / COD results

Results with reference substance:
The mean cumulative CO2 evolved from the sodium benzoate procedural control was 95.22% of the theoretical amount in 28 days (for more details, see below the table of resutlts). In addition, the reference substance, sodium benzoate, passed the OECD "10-Day window" criterion. This rapid degradation of the reference material confirmed the presence of an acceptable microbial community and confirmed system integrity.

Any other information on results incl. tables

                  % Biodegradation
VesselsContents   3 days 5 days  7 days  14 days  21 days  28 days 
 Test  -  -  - 69.60 
  Test  -  - 79.55 
  Test   -  -  - 78.25 
  Test  2.35   0.40 1.75  28.10  52.70   85.50
  Test 2.15   0.15 1.25  27.75   54.05  72.15
 Mean  2.25  0.28 1.50  27.93   53.38  77.01
  Reference  -  -  93.95
  Reference  -  -  95.15
  Reference   94.20
  Reference  68.75  83.70  86.70  97.00  93.95 96.25 
 Reference  71.35  82.05   88.10 94.05  95.50  96.55 
 Mean  70.00  82.88  87.40  95.53  94.73 95.22 

Cumulative (mg/L) Inorganic Carbon Evolved                
 VesselsContents 3 days  5 days  7 days  14 days  21 days  28 days 
Test  - 16.93 
 Test   - - 18.92 
 Test   - 18.66
Test  2.73 3.03  3.09  8.44 13.56  20.11 
 Test   2.69 2.98 2.99   8.37 13.83  17.44 
Mean  2.71  3.01  3.04   8.41 13.70  18.41
 Reference 21.80 
 Reference  - 22.04 
 Reference  - - 21.85 
 Reference  15.99 19.69  20.08  21.68  21.81  22.26 
 Reference  16.53  19.36 20.36  21.63  22.12  22.32 
Mean  16.26  19.53   20.22 21.66   21.97 22.05 
 Blank  -
 Blank  - -
 Blank  -
 Blank 2.32  3.13   2.69 2.73  2.78  2.97 
Blank  2.19  2.76  2.79 2.91  3.25 3.05 
Mean  2.26 2.95 2.74  2.82  3.02  3.01 
Inhibition   18.07  -  -  37.93
Inhibition   18.13  -  -  38.41
Mean  18.10 -  38.17

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
readily biodegradable, but failing 10-day window
According to the classification, Ortho-Diethoxy Benzene cannot be classifed as readily biodegradable.
Executive summary:

During 28 days, the biodegradation of Ortho-Diethoxy Benzene at an initial concentration of 20mg/L (TOC concentration) was studied according to OECD guideline 310 and ISO 14953.

Inoculum used in the test was an activated sludge at a concentration of 4mg/L (activated sludge dry solids) obtained with domestic sample from ETE Jundiai-SP (Brazil).

The corresponding percentage of biodegradation obtained after 28 days was 77.01% (ThCO2). The pass level of 60% ThCO2 production is attained without meeting the 10 -days window criterion, therefore the substance cannot be considered as readily biodegradable, but can be classified as rapidly biodegradable.

Reference material (sodium acetate) validates the test since its biodegradation was greater than 60% ThCO2 at 14 days (95.5%).

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