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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:
17 November 2003 to 15 December 2003
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Full GLP method to recognised test guidelines; all results documented.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
GLP compliance:
yes (incl. certificate)

Test material

Test material form:
solid: crystalline
light brownish
Details on test material:
- Name of test material (as cited in study report): 4,4’-Oxydianiline
- Molecular formula (if other than submission substance): Not specified
- Molecular weight (if other than submission substance): Not specified.
- Smiles notation (if other than submission substance): Not specified
- InChl (if other than submission substance): Not specified
- Structural formula attached as image file (if other than submission substance): Not included
- Substance type: Organic
- Physical state: Crystalline powder / light brownish
- Analytical purity: >99.8%
- Impurities (identity and concentrations): Not specified.
- Composition of test material, percentage of components: Not specified.
- Isomers composition: Not specified.
- Purity test date: Not specified.
- Lot/batch No.: 47-279
- Expiration date of the lot/batch: Nov 2008
- Radiochemical purity (if radiolabelling): Not applicable.
- Specific activity (if radiolabelling): Not applicable.
- Locations of the label (if radiolabelling): Not applicable.
- Expiration date of radiochemical substance (if radiolabelling): Not applicable.
- Stability under test conditions: Stable in water. Light sensitive.
- Storage condition of test material: Room temperature in the dark. Keep container tightly closed.
- Other:
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material :
Not applicable.

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Secondary activated sludge from Cecil County Fletchwood Wastewater Treatment Plant.
- Laboratory culture: None
- Method of cultivation: None
- Storage conditions: Kept under aerobic conditions in the period between sampling and application
- Storage length: Not specified.
- Preparation of inoculum for exposure: To prepare the inoculum, the sewage effluent was allowed to settle for one hour and the decanted effluent was kept aerobic until used.
- Pretreatment: None
- Concentration of sludge: Not specified.
- Initial cell/biomass concentration: Not specified.
- Water filtered: No
- Type and size of filter used, if any: Not applicable.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
as Biological Oxygen Demand
Details on study design:
- Composition of medium:
An appropriate number of glass carboys (20 L) was filled to make up the BOD media with deionized water. The contents of the BOD Nutrient Buffer Pillows were then added, 1 pillow for each 3,000 mL needed. All volumes were made in increments of 3 L. Each 20 L flask (containing medium) was aerated with organic-free compressed air for at least 1 hour. The 20 L carboy (containing medium) was allowed to stand overnight at the test temperature. The concentration of dissolved oxygen was determined for control purposes. All transfer and filling operations of the air-saturated water were conducted bubble-free by siphon or dispenser. Each BOD bottle was filled to about 2/3 of their volume with the Standard Dilution Water. The carboy used for filling was recorded.
Next, the respective Test or Control Substance was added in such amounts that final concentrations of 2 mg/L were attained. Subsequently, the microbial inoculum was added to the Inoculation Blank (no test substance), Control (sodium acetate) test vessels and Test Substance vessels.
Since the toxicity of the test substance was unknown, another series of bottles (toxicity control) were prepared with Test and Control substances together with microbial inoculum at the same concentrations as those in the previous bottles.
Finally, the solutions were made up to volume using the 20 L glass carboy with a dispenser. All the BOD bottles were filled with aerated Standard Dilution Water from the corresponding Stock Solution Flask. The Stock Flask solution was used to fill the Inoculum Blank BOD bottles, the Test Substance BOD bottles, and the Control Substance BOD bottles. Gently tap the BOD bottle to remove any trapped air bubbles.

- Additional substrate: None used
- Solubilising agent (type and concentration if used): None used.
- Test temperature: 22 ± 3°C
- pH: Not measured
- pH adjusted: Not applicable
- CEC (meq/100 g): Not applicable.
- Aeration of dilution water: Aerated with organic-free compressed air for at least 1 hour
- Suspended solids concentration: Aerated with organic-free compressed air for at least 1 hour
- Continuous darkness: No. Specified as in the dark or low light
- Other:

- Culturing apparatus: 300 mL BOD bottles with glass stoppers
- Number of culture flasks/concentration: 1
- Method used to create aerobic conditions: As above
- Method used to create anaerobic conditions: Not applicable
- Measuring equipment: Dissolved Oxygen meter
- Test performed in closed vessels due to significant volatility of test substance: No
- Test performed in open system: No
- Details of trap for CO2 and volatile organics if used: Not applicable
- Other:

- Sampling frequency: days 0, 7, 14, 21 and 28
- Sampling method: Dissolved Oxygen meter inserted into test system.
- Sterility check if applicable: Not applicable.
- Sample storage before analysis: Room temperature in the dark
- Other:

- Inoculum blank: Yes, Stock Solution only
- Abiotic sterile control: None
- Toxicity control: Yes, Test and Control substances together with microbial inoculum at the same concentrations
- Other:


Biodegradability was calculated as follows:
O2 depletion (mg BOD/L) after x days:
BODx (mg/L) = mibx - mtcx
x = # of Days
mibx = Mean DO Value of Inoculation Blank after x days
mtcx = Mean DO Value of Test Substance after x days
The calculation for "% biodegradability" is as follows:
% Biodegradability = [BOD (mg/L) / ThOD x SubstanceTest] x 100
The theoretical oxygen demand (ThOD) was calculated from the elemental composition as follows:
For the compound: Cc Hh Clcl Nn Na na Oo Pp Ss
ThODNH3 =( [16[2c + 1/2(h - cl - 3n) + 3s + 5/2p + 1/2na - o] (mg/mg)] / MW) x 100
Where MW = molecular weight

For C6H13N
ThODNH3 = [16[2(6) + ½ (13-0-3)+3(0)+5/2(0) + ½(0)-(0) (mg/mg)] / 99.18 x 100
ThODNH3 =2.74
Theoretical oxygen demand corrected for percent composition (CThOD) may be calculated using the following equation:
CThODNH3 = (ThODNH3 x Percent Composition) / 100
CThODNH3 = (2.74 x 99) / 100 = 2.72

Validity Criteria of the Study
The biodegradability of control substance, sodium acetate, must exceed 60% within 14 days for the test to be valid.
Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

Preliminary study:
Not conducted.
Test performance:
No unusual observations or deviations were observed during the study.
% Degradation
Key result
% degradation (O2 consumption)
Sampling time:
28 d
Details on results:
The results of the 28-day Closed Bottle Test (Table 2 below and Figure 1 attached under “background information, below) indicated the following:

Test Substance:
4,4’-Oxydianiline reached a maximum biodegradability of 7.6% by Day 28.

Toxicity Control:
The biodegradability of 4,4’-Oxydianiline with sodium acetate did not exceed 25% within 14 days, demonstrating that the test chemical was inhibitory to the microorganisms at the test concentration of 2 mg/L.

Additional information:
The test substance had no lag phase. The lag phase of the control substance occurred within the first sevens days of the test. The test substance had no degradation phase. The degradation phase of the control substance was about from about day 3 to 4 to between day 21 and 28 when greater than 90% biodegradability had been attained (Fig. 1, attached under “background information, below).

The ThOD of the test and control substances was determined to be 2.71 and 0.78 mg O2/mg substance, respectively (Table 1, below). These values were determined using the molecular formulas and the purity of the substances because the compositions of both substances were known.

BOD5 / COD results

Results with reference substance:
Control Substance:
The Theoretical Oxygen Demand (ThOD) of sodium acetate (Control Substance - CS) was calculated as 0.78 mg O2 per mg of active substance (AS). The biodegradability of sodium acetate exceeded 60% within 14 days.

Any other information on results incl. tables

Table1 -Descriptions Of Test And Control Substances



EMSE Sample No.     

Test Concentration

ThOD* of Substances



mg AS**perL

mg O2/mg AS**

4,4'-Oxydianiline – test substance




Sodium acetate – control substance




* Theoretical Oxygen Demand

** Active substance


Table2 -Biodegradation Of Test And ControlSubstances






Sodium acetate


Toxicity Control



% Biodegradability
















Not determined




Not determined


Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
under test conditions no biodegradation observed
1) “Ready Biodegradation“ of the test substance 4,4’-Oxydianiline was not demonstrated in this test.
2) The test substance can be assumed to be inhibitory to the microbial inoculum used in this test.
Executive summary:

1) “Ready Biodegradable“ of the test substance 4,4’-Oxydianiline was not demonstrated in this test.

2) The test substance can be assumed to be inhibitory to the microbial inoculum used in this test.