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Environmental fate & pathways

Biodegradation in water: screening tests

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Administrative data

Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-08-20 to 2012-09-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study, no deficiencies

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)
Deviations:
yes
Principles of method if other than guideline:
The ratio of test compound to inoculum dry weight was 1:7 instead of 1:2.5 to 1:4.

GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): (Pentapropylensuccinimido)-capronic acid
- Physical state: Liquid, brown viscous
- Analytical purity: 95.0 % (w/w) (100 % minus by-products)
- Purity test date: 2011-11-15
- Lot/batch No.: ESD0009272
- Expiration date of the lot/batch: 2014-04-26
- Stability under test conditions: Not specified
- Storage condition of test material: Room temperature, protected from light, in original container

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): Activated sludge from the sewage plant at 31137 Hildesheim is well suited as it receives predominantly municipal sewage and hardly any industrial chemical waste.

- Preparation of inoculum for exposure: The activated sludge was washed twice with chlorine free tap water. After the second washing the settled sludge was resuspended in mineral salts medium. The inoculum was maintained in an aerobic condition by aeration with CO2 free air until test start. The dry sludge concentration was determined and an appropriate volume of inoculum was chosen to get a dry sludge concentration of approx. 0.2 g/L.
- Dry sludge concentration of inoculum: 1.53 g/L
- Water filtered: no
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
40 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral salts medium acc. to OECD 302 B
- Additional substrate: No
- Test temperature: 20-25 °C
- pH: please refer to the respective table
- pH adjusted: no
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: 2000 mL test vessels
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Permanent aeration
- Measuring equipment: The biodegradation was monitored by determination of DOC in filtered samples taken at 6 sampling points.
The DOC of every replicate was determined at the beginning of the test, on day 1 and on days 7, 14, 21 and 28.
The pH was measured after every sampling point. The temperature was measured at test start in one replicate.



SAMPLING
- Sampling frequency:The DOC of every replicate was determined at the beginning of the test, on day 1 and on days 7, 14, 21 and 28.


CONTROL AND BLANK SYSTEM
- Inoculum blank: Mineral salts medium and inoculum
- Abiotic sterile control: Test item in test concentration without inoculum, poisoned with 10 mL/L HgCl2 solution (10 g/L).
- Functional control: Diethylene glycole 120 mg/L



STATISTICAL METHODS:
The biodegradation was monitored by determination of DOC in filtered samples taken at six sampling points.
The ratio of eliminated DOC, corrected for the inoculum control at each time interval to the initial DOC (0h) value is expressed as the percentage biodegradation at the sampling time. The percentage biodegradation is plotted against time to give the biodegradation curve.
Reference substance
Reference substance:
diethylene glycol

Results and discussion

Preliminary study:
No preliminary study
Test performance:
The required amount of test item was given in the test vessel filled with 1000 mL ultrapure water and stirred overnight. The test item was added at a test concentration of 40 mg/L, corresponding to a carbon content of 28.3 mg C/L in the test vessels.

The following incubation vessels were prepared:
- two for the test item (P1, P2)
- one for the functional control (R1)
- two for the inoculum control (C1, C2)
- one for the sterile control (B1)

The DOC of every replicate was determined at the beginning of the test, on day 1 and on days 7, 14, 21 and 28.

The temperature was measured at test start in one replicate. The room temperature was recorded continously by a thermohygrograph.The pH was measured once at test start in each replicate.
% Degradation
Parameter:
% degradation (DOC removal)
Value:
22
Sampling time:
28 d
Details on results:
The physico-chemical elimination of the test item was checked in the sterile control at the test item concentration of 40 mg/L (without inoculum and poisoned with HgCl2). No physico-chemical elimination occurred in the sterile control after 28 days.

The biodegradation/elimination reached the 10 % level after 3 days. The biodegradation/elimination remained in the range of 22-35 % and the level of 70% was not reached within the test duration of 28 days. After 28 days a biodegradation/elimination of 22 % was determined.

According to the criteria of the OECD 302B guideline the test item is not inherently biodegradable within 28 days. But the results indicate that the test item is inherently, primary biodegradable.

BOD5 / COD results

Results with reference substance:
The adaptation phase of the functional control changed after 2 days into the degradation phase (degradation  10 %). The course of the degradation phase was rapid and the pass level of 70 % was reached within 5 days. After 7 days a biodegradation of 100 % was reached. The validity criterion degradation  70 % after 14 d is fulfilled.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
not inherently biodegradable
Conclusions:
According to the criteria of the OECD 302B guideline the test item is not inherently, ultimately biodegradable within 28 days. But the results indicate the the test item is inherently, primary biodegradable.

Executive summary:

The inherent biodegradability/elimination of the test item (Pentapropylensuccinimido)-capronic acid (batch number ESD0009272) was determined in the Zahn-Wellens-Test / EMPA Test with a non adapted activated sludge over a period of 28 days. The definitive study was conducted from 2012-08 -20 to 2012-09-19 according to OECD guideline 302 B at Dr.U.Noack-Laboratorien,D-31157 Sarstedt.
The test item was tested at a concentration of
40 mg/L in duplicates, corresponding to a DOC of 28.3 mgC/L in the test vessel. The biodegradation/elimination of the test item was followed by determination of DOC in filtered samples. The ratio DOC to activated sludge dry weight at the beginning was 1:7. The ratio of eliminated DOC, corrected for the inoculum control to the initial DOC value is expressed as the percentage biodegradation at each sampling date.

In order to check the activity of the test system diethylene glycole at a concentration of 120 mg/L was used as functional control. After 7 days a biodegradation rate of 100 % was reached.

The physico-chemical elimination of the test item was checked in the sterile control (without inoculum and poisoned with HgCl2) at the test item concentration of 40 mg/L. At test start only 67 % of the nominal concentration was determined in the test solution. Until day 7 the concentration increased to 77 % of the nominal concentration and from day 14 until test end the concentration was ≥ 100 % of the nominal concentration. It is assumed that the lower concentration until day 7 is due to incomplete dissolution of the test item in the test solution. The increase of the test concentration indicated that the test item was completely dissolved after 14 days. No physico-chemical elimination occurred in the sterile control after 28 days.

The biodegradation/elimination reached the 10 % level after 3 days. The biodegradation/elimination remained in the range of 22 -35 % and the level of 70 % was not reached within the test duration of 28 days. After 28 days a biodegradation of 22 % was determined.

According to the criteria of the OECD 302B guideline the test item is
not inherently, ultimately biodegradable within 28 days. But the results indicate that the test item is inherently, primary biodegradable.

Inherent Biodegradability/Elimination of the Test Item (Pentapropylensuccinimido)-capronic acid in
                   Comparison to the Functional Control and Sterile Control

Inherent Biodegradation / Elimination [%]

Study Day [d]

0

1

7

14

21

28

Test Item

42#

5

21

35

28

22

Functional Control

0

8

100

99

100

100

Sterile Control*

33

27

23

0

0

0

# assumed due to incomplete dissolution of the test item

* physico-chemical elimination