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Diss Factsheets

Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
other information
Study period:
2013-02-14 to 2013-03-15
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
3-({3-[(2-methylnonyl)oxy]propyl}amino)propanenitrile
EC Number:
919-218-0
Cas Number:
72162-47-1
Molecular formula:
C16H32N2O
IUPAC Name:
3-({3-[(2-methylnonyl)oxy]propyl}amino)propanenitrile

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Municipal sewage treatment plant, D-31137 Hildesheim, Germany
- Pretreatment/Concentration of sludge: The activated sludge was washed twice with tap water. After the second washing the settled sludge was resuspended in mineral salts medium and was maintained in an aerobic condition by aeration for 2 hours. Thereafter the sludge was homogenized with a blender. The supernatant was decanted and maintained in an aerobic condition by aeration with CO2 free air until test start. 10 mL/L of this mixture were used to initiate inoculation.
- Initial cell/biomass concentration: approx. 4.0 x 109 CFU/L, corresponding to approx. 4.0 x 107 CFU/L in the test vessel.


Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
15 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral salts medium acc. to OECD 301 B / CO2 Evolution Test
- Test temperature: 22 +/- 2°C (21.5 - 23.0 °C)
- pH adjusted: no
- Continuous darkness: low light conditions (brown glass bottles)



TEST SYSTEM
- Culturing apparatus: 5000 mL brown glass flasks
- Number of culture flasks/concentration: 1 for the reference item, 1 for toxicity control (test and reference item), 2 for the control, 2 for the test item
- Method used to create aerobic conditions: Aeration with 30 - 100 mL/min
- Measuring equipment: Visual check of aeration twice per day
- Details of trap for CO2 and volatile organics if used: CO2 adsorption vessels were connected to the air outlets of the incubation vessels via a series of 3 gas wash bottles, each containing 100 mL of a 0.0125 mol/L Ba(OH)2 solution.
- Course of the study: The following incubation vessels were prepared:

- two for the test item concentration (P1, P2)
- one for the functional control (R1)
- two for the inoculum control (C1, C2)
- two for the toxicity control (T1, T2)
The necessary amounts of bidistilled water, mineral salts medium and inoculum were placed in each of the incubation vessels. The vessels were aerated for 24 hours with CO2 free air. After 24 hours the CO2 adsorption vessels were connected to the air outlets of the incubation vessels via a series of 3 gas wash bottles, each containing 100 mL of a 0.0125 mol/L Ba(OH)2 solution.
Test and reference item were weighed out for each replicate. A defined volume of bidistilled water was added to the test item and it was treated with ultrasound. The test item dispersions and the reference item were transferred into the incubation vessels; the vessels were made up to 3 L with bidistilled water and connected to the system for the production of CO2 free air.
On day 28, 1 mL 37 % HCl was added to each of the vessels. Aeration was continued for further 24 hours and the quantity of CO2 released was determined.



SAMPLING
- Sampling frequency: Backtitration of the residual Ba(OH)2 with 0.05 N HCL was carried out three times a week during the first ten days and thereafter twice weekly.
- Sampling method: For each titration the first gas wash bottle was removed and a new bottle was connected to the last one.



CONTROL AND BLANK SYSTEM
- Inoculum blank: Test medium without test and reference item
- Abiotic sterile control: No
- Toxicity control: Test item and reference item in test concentration



STATISTICAL METHODS:
The theoretical production of carbon dioxide (ThCO2) of the test item and functional control is calculated by the carbon content and the molecular formula, respectively.
The produced CO2 was calculated by: 1 mL HCl (c = 0.05 mol/L) = 1.1 mg CO2
The net amount of CO2 produced is calculated by correcting the results of the test item and functional control for endogenous CO2 production of the inoculum controls.
The biodegradation is calculated from the ratio theoretical CO2 production to net CO2 production.
Reference substance
Reference substance:
benzoic acid, sodium salt
Remarks:
20 mg/L

Results and discussion

Preliminary study:
Not performed
% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
39
Sampling time:
28 d
Details on results:
The adaptation phase of the functional control changed after 2 days into the degradation phase (degradation  10 %). The course of the degradation was rapid and the functional control reached the pass level of 60 % within 6 days and a biodegradation of 82 % after 28 days. The validity criterion degradation  60 % after 14 days is fulfilled.
The biodegradation of the test item is shown in comparison to the readily degradable functional control and the toxicity control.
The mean 10 % level (beginning of biodegradation) was reached after 10 days. The biodegradation progressed steadily, but the 60 % pass level was not reached. The mean biodegradation after 28 days was 39 %.

Under the test conditons the test item is not readily biodegradable within 28 days.

In the inoculum control the total CO2 production was 27.7 mg CO2/L after 28 days.

The mean 10 % level (beginning of biodegradation) as well as the 60 % pass level was not reached within the 28 day test duration. The mean biodegradation after 28 days was 4 %.

Under the test conditions the test item is not readily biodegradable within 28 days.

BOD5 / COD results

Results with reference substance:
In the toxicity control containing both test and reference item a biodegradation of 45 % was determined within 14 days and it came to 53 % after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
other: not ready biodegradable
Conclusions:
Under the test conditions the test item is not readily biodegradable within 28 days.
Executive summary:

The ready biodegradability of the test item 3-[[3-(Isodecyloxy)propyl]amino]-propanenitrile (batch no.: F-212289) was determined with a non-adapted activated sludge over a test period of 28 days in the Modified Sturm Test. The study was conducted from 2013-02-14to 2013-03-15according to OECD 301 B at Dr.U.Noack-Laboratorien. The test item was tested at a concentration of 15 mg/L with 2 replicates, corresponding to a carbon content (TOC) of 10.6 mg C/L in the test vessels. The test vessels were incubated at low light conditions and at a temperature of 21.5 – 23 °C.

The biodegradation of the test item was followed by titrimetric analysis of the quantity of CO2produced by the respiration of bacteria. The degradation was stopped on day 28 by acidification of the test solutions. The last titration was made on day 29, after residual CO2 had been purged from the test solutions over a period of 24 hours. The percentage CO2 production was calculated in relation to the theoretical CO2 production (ThCO2) of the test item. The biodegradation was calculated for each titration time.

To check the activity of the test system, sodium benzoate was used as functional control. The percentage degradation of the functional control reached the pass level of 60 % within 6 days and a biodegradation of 82 % after 28 days.

In the toxicity controlcontaining both test and reference item a biodegradation of  45 % was determined within 14 days and it came to 53 % after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.

The biodegradation of the test item is shown in comparison to the readily degradable functional control and the toxicity control. The mean 10 % level (beginning of biodegradation) was reached after 10 days. The biodegradation progressed steadily, but the 60 % pass level was not reached under the above described conditions after 28 days. The mean biodegradation after 28 days was 39 %.

Under the test conditions the test item is
not readily biodegradable
within 28 days.

Biodegradation of the Test Item 3-[[3-(Isodecyloxy)propyl]amino]-propanenitrile in Comparison to the Functional Control and Toxicity Control

Biodegradation [%]

Study Day [d]

6

14

21

28

Test Item, 1st Replicate

5

14

21

36

Test Item, 2nd Replicate

7

17

26

41

Functional Control

61

80

82

82

Toxicity Control
test item + reference item

32

45

48

53