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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-12-07 to 2018-02-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Version / remarks:
March 2006, corrected September 2014
Deviations:
no
Principles of method if other than guideline:
The study has been conducted to examine the biodegradability in an enhanced ready biodegradability test over an extended period of 60 days. The rate of biodegradation has been determined by measurement of the total inorganic carbon (TIC). Additionally, the primary degradation of the test item has been analysed by GC-MS. To analysed the complex mixture the components were grouped according to their degree of branching by an increment system (described by Kovats). Enhancements to the standard test guideline has been done in accordance with the ECHA guidance document Chapter R.7b (version 4.0; 2017) and a publication on behalf of the German Environment Agency (Assessment of environmental persistence, Texte 10/2017, Jan 2017).
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- C16 fraction of Tetrabutan
- TOC of test item: 0.84 mg C/ mg test item
- Stock solution: 14.16 g/l in silicone oil.
- The test item was applied via a stock solution in silicone oil.
- Pipetting of the stock solution: 250 µL / 200 mL test solution (corresponding to 3.54 mg test item / test vessel)
- Carbon content in the vessel: 14.9 mg C/L. Test concentration: 17.7 mg test substance/L.


Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Remarks:
Inoculum of the aqueous phase of non-adapted activated sludge
Details on inoculum:
- Test system: Inoculum
Source of inoculum/activated sludge: Municipal sewage treatment plant, Germany (Hildesheim)
- Reasons for the selection: Activated sludge from the sewage plant at Hildesheim is well suited as it receives predominantly municipal sewage and hardly any industrial chemical waste.
- Preparation of inoculum for exposure/ Pretreatment: The activated sludge was washed twice with chlorine free tap water. Then the settled sludge was resuspended in mineral salts medium and was maintained in an aerobic condition by aeration with CO2 free air until test start (1 day).
- Initial cell/biomass concentration: The amount of inoculum used to initiate inoculation was 5.9 mL/L (approx. 29.85 mg/L dw).
- CFUof the inoculum at test start: 1.03*10^9 CFU/L, corresponds to 1.03*10^7 CFU/L per test vessel (determined by standard dilution plate count).
Duration of test (contact time):
60 d
Initial conc.:
17.7 mg/L
Based on:
TOC
Remarks:
The test substance concentration corresponds to 14.9 mg C/L carbon content in the vessel
Parameter followed for biodegradation estimation:
CO2 evolution
Parameter followed for biodegradation estimation:
test mat. analysis
Remarks:
Primary degradation was examined by GC-MS analysis.
Details on study design:
TEST CONDITIONS
- Composition of medium: mineral salts medium acc. to OECD310 (200 mL volume per test vessel)
- Test temperature: 20 ± 1°C, actual 19.0-21 °C
- Suspended solids concentration: 1.03 * 10^9 CFU/L, corresponding to 1.03 * 10^7 CFU/L per test vessel.
- Continuous darkness: low lights conditions
- Other: Appliciation (of test item/reference item/silicone oil) once at start. Silicone oil was used to improve the bioavailability of the test item.

TEST SYSTEM
- Culturing apparatus: Headspace flasks (Volume 250 mL nominal, total volume 300, Headspace ratio 1:2, closed test vessels)
- Number of culture flasks/concentration: Three replicates for test item concentration and each control according to sampling shedule. 5 replicates after 28 days and at test end.
- Method used to create aerobic conditions: The inoculum was maintained in aerobic conditions by aeration with CO2 free air before test start. During the test, agitation on shaker (150-200 rpm).
- Measuring equipment: CO2 evolution was determined by IC analysis with a carbon analyser (according to DIN EN 1484). The total amount of CO2 produced in 28 days was analysed by TIC measurements at 7 sampling times.

SAMPLING
- Sampling frequency: Sampling was carried out at test start (day 0) of all replicates and on 11 further sampling points of the test item replicates and the inoculum control replicates. The functional control replicates were sampled on 3 further sampling points and the toxicity control replicates were sampled on 2 further sampling points.
- Sampling method: Sodium hydroxide solution (0.74 mL of 7 mol/L solution to 80 mL medium) was injected to each vessel sampled. The TIC of the sodium hydroxide solution (blind value) was determined at each sampling time.
- Other: Sampling according to sampling shedule.

CONTROL AND BLANK SYSTEM
- Inoculum control: Mineral salts medium + inoculum and 250 µL silicon oil without test item, 3 replicates (5 replicates after 28 days and at test end).
- Abiotic sterile control:
- Abiotic test item control: Same as the test item replicates, but incl. 50 mg/L HgCl2. 3 replicates (5 replicates after 28 days and at test end).
- Abiotic inoculum control: Mineral salts medium + inoculum and 250 µL silicon oil without test item, but incl. 50 mg/L HgCl2. 3 replicates (5 replicates after 28 days and at test end).
- Toxicity control: Mineral salts medium + inoculum and test item (incl. silicon oil) and reference item in test concentration. 3 replicates (5 replicates after 28 days)
- Functional Control: Mineral salts medium + inoculum and reference item incl silicone oil (250µL / 200 mL)

STATISTICAL METHODS: The confidence intervall was calculated using the software SigmaPlot.
Reference substance:
benzoic acid, sodium salt
Remarks:
30 mg/L corresponding to 17.5 mg C/L carbon content in the vessel
Preliminary study:
In a non-GLP preliminary study the optimal procedure for the test item has been examined. The test design was practically identical to the GLP-Study (Test vessels: 250 mL headspace vials (250 ml nominal volume with 200 mL test solution), sludge concentration of approx. 30 mg dry weight/L (according to guideline) and silicone oil. In the preliminary study with three sampling times (days 0,14, abd 26), a biodegradation rate (mean of two replicates) of 49% after 14 days and 73% after 26 days has been observed.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
59
Sampling time:
60 d
Remarks on result:
other:
Remarks:
95% confidence intervall 53 - 63 %
Parameter:
% degradation (test mat. analysis)
Value:
65
Sampling time:
60 d
Remarks on result:
other:
Remarks:
The level of primary biodegradation is about 65% for Group 1 at test end.
Parameter:
% degradation (test mat. analysis)
Value:
106
Sampling time:
60 d
Remarks on result:
other:
Remarks:
The level of primary biodegradation is about 106% for Group 2 at test end.
Details on results:
The test item was tested at a concentration of 17.7 mg/L, corresponding to a carbon content of 14.9 mg C/L. The mean amount of TIC present in the inoculum controls on day 28 is 5.93 mg C/L and 7.15 mg C/L at test end. Colony forming units (CFU) of the inoculum at test start was 1.03 * 10^9 CFU/L, corresponding to 1.03 * 10^7 CFU/L in the test vessel.

The adaptation phase of the functional control changed within 7 days to the degradation phase (degradation ~ 10%). The course of the degradation phase was rapid and the pass level of 60 % was reached within 7 days, too. The biodegradation came to a maximum of 95% after 28 days. The validity criterion degradation > 60% after 14 d was fulfilled. The 95% confidence interval on day 28 was 93 - 97%. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.

The test item replicates reached the 10% level (beginning of biodegradation) within 14 days. The 95 % confidence interval on day 28 was 39 – 44%. The biodegradation came to 59% after 60 days. The 95% confidence interval on day 60 was 55 – 63%. The test item is classified as not readily biodegradable within the 60 day period of the study. Nevertheless. the test item shows a very good potential for biodegradation with 3 out of 5 replicates with at least 60% biodegradation after 60 days.

The primary degradation (or elimination, respectively) was calculated in relation to the mean value of the respective group at test start corrected for the background. The mean value of all inoculum control replicates (biotic and abiotic) of all sampling times was considered as background. In Group 3 (no or one branch) the concentration of species with no or only one branching was throughout the study in the range of the background values. Accordingly, an evaluation of the primary biodegradation (or primary elimination for the abiotic replicates, respectively) is prone to deliver unrealistic values. Therefore, the values has not been reported. The level of primary degradation / elimination for Group 1 (3 or 4 branches) is about 65% at test end and about 106% for Group 2 (2 or 3 branches) at test end.
Results with reference substance:
The functional control reached the pass level of 60% degradation within 7 days, with a maximum of 95% degradation on day 28 (95% confidence interval 93 – 97%).
In the toxicity control, a biodegradation of 56% was determined after 14 days and 63% after 28 days (confidence interval 62-65%). The biodegradation of the reference item was not inhibited by the test item in the toxicity control.

Biodegradation and Confidence Interval of the Test Substance in Comparison to the Functional Control and the Toxicity Control after 28 days

 

Biodegradation [%]
on Day 28

Confidence
Interval on Day 28

Replicates

1

2

3

4

5

P = 95 %

Test Item

38

42

43

41

42

39 – 44

Functional Control

96

93

96

97

94

93 - 97

Toxicity Control,
Test Item + Reference Item

64

64

64

62

63

62 - 65

Biodegradation and Confidence Interval of the Test Substance after 60 days

Biodegradation [%]
on Day 60

Confidence
Interval on Day 60

Replicates

1

2

3

4

5

P = 95 %

Test Item

54

61

63

60

57

55 - 63

Validity criteria fulfilled:
yes
Remarks:
The degradation of the functional control reached the pass level of 60% within 7 days.
Interpretation of results:
other: The test item is classified as not readily biodegradable within the 60 day period of the study. Nevertheless, the test item shows a very good potential for biodegradation with 3 out of 5 replicates with at least 60% biodegradation after 60 days.
Conclusions:
The test substance replicates reached the 10% level (beginning of biodegradation) within 14 days. The 95% confidence interval on day 28 was 39 - 44%. The biodegradation came to 59% after 60 days. The 95% confidence interval on day 60 was 55 - 63%. In 3 out of 5 replicates at least 60% biodegradation has been observed after 60 days. The test substance is classified as not readily biodegradable within the 60 day period of the study. Nevertheless, the test substance shows a very good potential for biodegradation with 3 out of 5 replicates with at least 60% biodegradation after 60 days.

This is supported by the analysis of the primary degradation using GC/MS. The level of primary degradation/ elimination for group 1 is about 65% and about 106% for group 2 at test end, indicating that also the higher branched isomers are susceptible for biodegradation. In group 3 the values were in the range of the background and therefore not evaluated.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04. Nov. 1993 - 10. Dec. 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
ISO Draft (BOD Test for Insoluble Substances)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: activated sludge taken from a municipal waste water treatment plant (Marl-Ost, Germany)
- Preparation of inoculum for exposure: The activated sludge from WTP Marl-Ost, Germany was washed several times with culture medium and then the dry matter content was determined
- Concentration of sludge: 3.2 g/L
- pH: 7.6
- Stabilisation phase: test flasks with Inoculum and culture medium (Inoculum 10 mL/L) were aerated and incubated for 1 week on a shaking incubator. after 4 days flasks were aerated again.
Duration of test (contact time):
28 d
Initial conc.:
23.2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: culture medium according to guideline
- Test temperature: 19.3 - 21.7 °C
- Suspended solids concentration: 3.2 g/L


TEST SYSTEM
- Culturing apparatus: closed glas bottles filled 2/3 with culture medium and 1/3 with air
- Number of culture flasks/concentration: 3
- Method used to create aerobic conditions: closed flasks with air space

SAMPLING
- Sampling frequency: once a week
- Sampling method: oxygen measurement
Reference substance:
diethylene glycol
Parameter:
% degradation (O2 consumption)
Value:
32
Sampling time:
28 d
Details on results:
For Tetrabutan, dest. the following biodegradtaion values were obtained:
after 7 days: 9.6 %
after 14 days: 19.3 %
after 21 days: 28.9 %
after 28 days: 32.3 %
Results with reference substance:
The reference substance diethylene glycol reached a biodegradation value of 84 % after 28 days thus confirming the suitability of the used activated sludge inoculum.

 

 

Tetrabutan, dest.

Diethylene glycol

Sampling day

7

14

21

28

7

14

21

28

Biodegradation

in %

10.48

19.24

28.00

33.24

60.32

75.41

82.10

83.76

8.39

18.42

28.46

31.83

61.69

80.36

84.68

84.68

10.04

20.08

30.12

31.83

43.02

74.20

80.12

84.57

 

 

 

 

 

 

 

 

 

 

Mean

9.64

19.25

28.86

32.30

55.01

76.66

82.30

84.34

 

 

 

 

 

 

 

 

 

 

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
For Tetrabutan, dest. a biodegradation value of 32 % of the corresponding ThoD was recorded after 28 days in a BODIS test. Thus, the test substance is not readily biodegradable (> 60 % of ThoD after 28 days within the 10 day window ) but can be considered to have a potential for biodegradation.
Executive summary:

Tetrabutan, dest. was tested for its biodegradation potential in a BODIS test according to ISO Draft (BOD Test for Insoluble Substances). The activated sludge inoculum obtained from a domestic sewage treatment plant (Marl-Ost, Germany) proved its suitability, the reference substance diethylene glycol reached biodegradation value of 84,3 % after 28 days. With a biodegradation value of 32.3 % of the corresponding Theoretical Oxygen Demand (ThOD) after 28 days, readily biodegradability could not be shown for Tetrabutan, dest..

Description of key information

Tetrabutan, dest. reached a biodegradation value of 32.3 % after 28 days in a BODIS test. Therefore, the test substance is considered not readily biodegradable after 28 days. However, an enhanced ready biodegradation test according to test guideline OECD 310 demonstrated a good potential for biodegradation and reveals a biodegradation rate of 59% after 60 days. The primary degradation was further investigate by GC-MS analysis. Primary degradation of alkanes with different branching has been observed although the analytical analysis has been overlayed with abiotic elimination processes.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable
Type of water:
freshwater

Additional information

Biodegradation of Tetrabutane or fractions of Tetrabutane has been examined in two ready biodegradation test. Tetrabutane, dest. was tested for its ready biodegradation in a BODIS test according to ISO Draft (BOD Test for Insoluble Substances). With a biodegradation value of 32.3 % of the corresponding Theoretical Oxygen Demand (ThOD) after 28 days, readily biodegradability could not be shown for Tetrabutane, dest. by this test.

Additionally, biodegradation has been examined in an enhanced ready biodegradability study. According to the ECHA document chapter R.7b, the enhanced ready biodegradability test can used to confirm the potential for biodegradation. The enhanced ready biodegradability test has been performed with non-adapted activated sludge over a test period of 60 days in a Headspace test according to the test guideline OECD 310. A test item concentration of 17.7 mg/L (corresponding to 14.9 mg C/L carbon content in the test vessel) has been tested and the CO2 evolution was determined by TIC analysis during the test period.

The test item replicates reached the 10% level (beginning of biodegradation) within 14 days. The 95% confidence interval on day 28 was 39 – 44%. The biodegradation came to 59% after 60 days (confidence interval 55-63%). Overall, the test item is classified as not readily biodegradable within the study period. Nevertheless, the test item shows a very good potential for biodegradation with 3 out of 5 replicates with at least 60% biodegradation after 60 days.

The functional control reached the pass level of 60% within 7 days and the biodegradation of the reference item was not inhibited by the test item in the toxicity control. The chosen enhancements (like the extended test period or larger test vessels) were in accordance to the guidance document chapter R.7b (2017).

 

Furthermore, primary biodegradation has been examined with GC-MS analysis. To analyze the complex mixture of branched and linear alkanes (C16) in the test substance, the components were grouped according to their degree of branching identified by their Kovats Retention Indices (KRI). Three groups were defined: Group 1: Grade of branching 3 + 4; Group 2: Grade of branching 3 + 2 and Group 3: Grade of branching 0 + 1. The limits of the groups were defined by their specific range of their Kovats Retentions Indices (KRI).

The test item replicates from Group 1 and Group 2 show a significant decrease in concentration to approx. the background level from the abiotic inoculum control samples until day 28. The corresponding abiotic test item replicates of these two groups show also a decrease in concentration over the whole 60 days. For Group 2, a linear decrease was observed as expected from abiotic adsorption processes as opposed to a fast decrease in the beginning and levelling out after 28 days to 60 days. After correction for the background, the primary degradation / elimination in relation to the mean value at test start shows a similar course with a plateau approximately after day 28. The level of primary degradation / elimination for Group 1 has been about 65% at test end and about 106% for Group 2 at test end.

The concentration of species of group 3 with no or one branching was in the range of the background values. Accordingly, an evaluation of the primary biodegradation was prone to deliver unrealistic values and, therefore, detailed values have not been reported. A linear increase of primary elimination in the abiotic test item replicates has been observed and might be caused by adsorption processes, e.g. to the sludge. As the mineralization data proves, this elimination or adsorption does not inhibit the biodegradation processes.

 

Overall, the enhance biodegradation test showed a good potential for biodegradation of the tested fraction of registered substance. The mineralization data demonstrates that the main part of the test substance has been degraded. This suggests that also branched alkanes must have been degraded to reach the biodegradation rate of 59% after 60 days. Based on the course of the biodegradation rate during to study period, a tiered biodegradation process of the species with different branching can be assumed. However, due to abiotic elimination processes, the analytical method has been limited to support this hypothesis.