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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:
2010-03-08 to 2010-05-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study performed according OECD guidline and GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Diethylenglykoldimethylether
- Molecular formula: C6H14O3
- Molecular weight: 134.18 g/mol
- Physical state: Liquid, colourless
- Analytical purity: 99.9 % (Bis(2-methoxyethyl)ether)
- Storage condition of test material: Room temperature, protected from moisture and light.

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 municipal sewage treatment plant, 31137 Hildesheim, Germany, comprising mostly municipal sewage and hardly industrial chemical waste
- Preparation of inoculum for exposure: The activated sludge was washed twice with autoclaved tap water. The dry sludge concentration was determined and an appropriate volume of inoculum was chosen to get a dry sludge concentration of 0.2 - 1.0 g/L in the test vessels and a ratio of DOC / inoculum in the range of 1:2.5 - 1:4.
- Dry sludge concentration of inoculums: 4.09 g/L
- Water filtered: no
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
95 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: 20-24 °C
- pH: please refer to the respective table
- pH adjusted: no
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: Closed 5000 mL brown glass flasks with a cartridge at the air outlet
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Permanent aeration
- Measuring equipment: The inherent biodegradation was monitored by determination of DOC according to Guideline DIN EN 1484. Samples were taken at the beginning of the test (0h, 3h) and after 3, 7, 14, 21, 28 and 36 days. The primary biodegradation was determined by SPME-GCMS. Samples were taken at the beginning of the test and after 7, 16, 21 and 28 days.



SAMPLING
- Sampling frequency: Samples were taken at the beginning of the test (0h, 3h) and after 3, 7, 14, 21, 28 and 36 days.


CONTROL AND BLANK SYSTEM
- Inoculum blank: Test item in test concentration without inoculum, poisoned with
10 mL/L of 10 g/L HgCl2 solution.
- Abiotic sterile control: No
- Toxicity control: Test item and reference item in test concentrations



STATISTICAL METHODS:
The inherent biodegradation was monitored by determination DOC at eight sampling points.
The ratio of eliminated DOC, corrected for the control at each time interval to the initial DOC (0h) value is expressed as the percentage biodegradation at the sampling time.
Reference substance
Reference substance:
diethylene glycol

Results and discussion

Preliminary study:
No preliminary study
Test performance:
The test concentration of the test item was calculated based on the DOC of 0.54 mgC/mg test item.
Appropriate volumes of nutrient media and inoculum were filled into the incubation vessels for the test item and test item GC replicates and were made up to 2 L with aqua demin. The test item and the test item stock solution, respectively were injected directly into the solution (below the surface) of each replicate.

For the sterile control appropriate volumes of nutrient media and HgCl2 solution were filled into the incubation vessel and were made up to 2 L with aqua demin. 201 µL of the test item stock solution was injected directly into the solution (below the surface).

For the sterile control GC-MS replicates appropriate volumes of nutrient media, inoculum and HgCl2 solution were filled into the incubation vessels and were made up to 2 L with aqua demin. Appropriate vol-umes of the test item stock solution were injected directly into the solution (below the surface) of each replicate.

For the functional control replicates a stock solution of the reference item was prepared. Appropriate volumes of the stock solution, nutrient media and inoculum were filled into the incubation vessels and were made up to 2 L with aqua demin.

For the inoculum control replicates appropriate volumes of nutrient media and inoculum were filled into the incubation vessel and made up to 2 L with aqua demin.

The incubation vessels were aerated and stirred continuously on a magnetic stirrer.

Adjusting of pH values to 6.5 to 8 was not necessary.

The biodegradation was monitored by determination of DOC and test item concentration at regular time intervals.
% Degradation
Parameter:
% degradation (DOC removal)
Value:
99
Sampling time:
36 d
Details on results:
The test item concentration was determined by SPME GC/MS at five sampling times over a period of 28 days. The test item was tested at a concentration of 600µg/L.
The physico-chemical elimination (volatilisation) of the test item was monitored in sterile controls (with inoculum and poisoned with HgCl2) at 300 µg/L and 600 µg/L.
No physico-chemical elimination occurred in the sterile control after 28 days.
The primary degradation started after an adaptation phase of 16 days. The biodegradation was fast and on day 23 the pass level of 70 % was reached. After 28 days the primary degradation came to 95 %.
The test item is classified as primary biodegradable after 23 days.
The physico-chemical elimination (volatilisation) of the test item was monitored in the sterile control with the test item concentration of 95 mg/L but without inoculum and poisoned with HgCl2. No physico-chemical elimination (volatilisation) occurred in the sterile control until day 36.


The inherent degradation started after a long lasting adaption phase of 21 days. The biodegradation was fast and the biodegradation reached the 70 % pass level after 29 days. After 36 days a biodeg-radation of 99 % was reached.
The test item is classified as inherent biodegradable after 29 days.

BOD5 / COD results

Results with reference substance:
The adaptation phase of the functional control changes after 3 days into the degradation phase (degradation  10 %). The course of the degradation phase is rapid and reaches a degradation rate > 70 % after 7 days. After 14 days a degradation rate 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:
inherently biodegradable
Conclusions:
The test item is classified as primary biodegradable after 23 days and inherent biodegradable after 29 days.
Executive summary:

The primary and inherent biodegradability were determined in the Zahn-Wellens-Test / EMPA Test with a non adapted activated sludge for the test item Diethylenglykoldimethylether (batch number DEG4071791) over a period up to 36 days. The study was conducted from 2010-03-08 to 2010-05-19 with the definitive exposure phase oft the main test from 2010-04-13 to 2010-05-19 according to OECD 302 B at Dr.U.Noack-Laboratorien.

For the determination of the primary biodegradation the test item was tested at a concentration of 600 µg/L in duplicates. The primary biodegradation was determined by SPME GC/MS analysis of the test item. For the determination of the inherent biodegradation the test item was tested at a concentration of 95 mg/L in duplicates, corresponding to a DOC of 51.3 mg C/L in the test vessel. The inherent biodegradation of the test item was followed by determination of DOC. The ratio of eliminated DOC, corrected for the control at each time interval 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 in a concentration of 120 mg/L was used as functional control. After 14 days a degradation rate of 100 % was reached.

The physico-chemical elimination (volatilisation) of the test item was monitored in separate sterile controls. At the test item concentration of 95 mg/L a sterile control without inoculum and poisoned with HgCl2 was used. For determination of the primary biodegradation sterile controls (with inoculum and poisoned with HgCl2) with a test item concentration of 300 µg/L and 600 µg/L were tested. No physico-chemical elimination (volatilistion) occurred in the sterile controls until test end.

The primary degradation started after an adaptation phase of 16 days. The biodegradation was fast and on day 23 the pass level of 70 % was reached. After 28 days the primary degradation came to 95 %.

The inherent degradation started after a long lasting adaption phase of 21 days. The biodegradation was fast and the biodegradation reached the 70 % pass level after 29 days. After 36 days a biodegradation of 99 % was reached.

The test item is classified as primary biodegradable after 23 days
and inherent biodegradableafter 29 days.


Table 1:          Primary and Inherent Biodegradability of the Test Item Diethylenglykoldimethylether in Comparison to the Functional Control and the Sterile Control

Inherent Biodegradation / Elimination [%]

Day

7

14

21

28

36

test item
95 mg/L

0

3

4

67

99

functional control
120 mg/L

97

100

100

95

95

Sterile control*
95 mg/L test item

0

0

0

0

0

Primary Biodegradation / Elimination [%]

Test item
600 µg/L

9

01)

60

95

Sterile control*
600 µg/L test item

4

01)

0

2

Sterile control*
300 µg/L test item

0

01)

0

7